1
EVOLUTION AND CELLULAR BIOLOGY OF THE UTERINE SERPINS
By
MARIA B. PADUA
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2009
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© Maria B. Padua
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I dedicate this work to my mother Carmen, uncle Adán, nanny Zenaida
and siblings Carolina and Fernando
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ACKNOWLEDGMENTS
I would like to thank my advisor Dr. Peter J. Hansen for his guidance, support and
challenge through my PhD program. I am grateful to him for encouraging me as a scientist
during all these years. Also, I would like to thank my committee members Dr. Nasser Chegini,
Dr. David Julian, Dr. Phyllip LuValle, Dr. Karen Moore for their contributions and suggestions
to improve my research projects and academic training. I am also grateful to Dr. William
Thatcher for his continuous support and participation.
I also thank my lab mates, Luciano and Aline Bonilla, Barbara Loureiro, Lilian Oliveira,
Justin Fear, Silvia Carambula and Jim Moss and my old lab mates, Dr. Jeremy Block, Dr. Dean
Jousan, Moisés Franco, Dr. Luiz Augusto de Castro and Amber Brad for their great help through
many different ways. Likewise, I want to thank my international friends Dr. Yaser Al-Katanani,
Dr. Fabiola Paula-Lopes, Dr. Zvi Roth, Dr. Paolete Soto, and Dr. Olga Ocon for their friendship
and additional contributions.
Thanks are also extended to all personnel in the Department of Animal Sciences,
especially to Rick Wenzel for making the cell culture room a more functional place to work.
Likewise, I want to thank the International Student Center staff, especially to Debra Anderson
and Maud Fraser for their kindness and assistance to all international students. I am also grateful
to Neal Bensen and Steve McClellan from the Flow Cytometry Core Laboratory, Dr. Gigi
Ostrow from the Gene Expression Core Laboratory, Dr. Savita Shanker and Xiao Hui Zhou from
the DNA Sequencing Core Laboratory and to the University of Florida Diagnostic Referral
Laboratories of the Interdisciplinary Center for Biotechnology Research-Cancer Genetics
Research Complex at the University of Florida for their assistance to perform many of the
experiments presented in this dissertation.
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Gratitude is also extended to the following University of Florida people for providing
access to laboratory equipment: Dr. Owen Rae and Shelley Lanhart from the Department of
Large Animal Clinical Sciences, Dr. Pushpa Kalra from the Deparment of Physiology and
Functional Genomics, Dr. Lori Warren, Jan Kivilpeto, Dr. Lokenga Badinga and Dr. Alan Ealy
from the Department of Animal Sciences, University of Florida.
Likewise, I am grateful to the following people for their invaluable assistance in providing
tissue samples for some of the experiments presented in this dissertation: Dr. Dan C. Sharp, Dr.
Luciano Silva and Dr. Claudia Klein from the Animal Sciences Department, University of
Florida, Dr. Ellis Greiner from the college of Veterinary Medicine, University of Florida and
Mirian Y. Cañas from the Laboratorio de Embriologia and Endocrinologia Molecular, Decanato
de Agronomia, Universidad Centroccidental Lisandro Alvarado, Venezuela, Dr. Douglas C.
Antczak and Christina Costa from the Baker Institute for Animal Health, College of Veterinary
Medicine, Cornell University, Dr. John Verstegen from the College of Veterinary Medicine,
University of Florida, Dr. Fernanda Agreste from the Surgery Department, Faculdade de
Medicina Veterinária e Zootecnia, Universidade de São Paulo, Brazil and Dr. Stephen Shores
and Debi Gibson from the Animal Shores Hospital in Gainesville.
I am also grateful to Dean Leed for his care and support. Special thanks go to Dr. Andrés
Kowalski, Mónica Prado-Cooper, José Cristobal Nieto, Marília and José Trujillo, Armando Luiz
and Claudia García for their sincere friendship for many years. Also, I am grateful to my very
good friends in Gainesville Domenicchella Dean, Cristina Caldari-Torres and Milerky Perdomo
for their friendship and support in many different ways. I really appreciate it.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS.................................................................................................................... 4
LIST OF TABLES................................................................................................................................ 9
LIST OF FIGURES ............................................................................................................................ 10
LIST OF ABBREVIATIONS ............................................................................................................ 12
ABSTRACT ........................................................................................................................................ 14
CHAPTER
1 LITERATURE REVIEW ........................................................................................................... 16
Introduction ................................................................................................................................. 16
Serine Proteinase Inhibitor Superfamily .................................................................................... 17
Mechanism of Action .......................................................................................................... 17
Non-Inhibitory Serpins ........................................................................................................ 20
Functions of Inhibitory Serpins Distinct from Proteinase Inhibition ............................... 21
Serpinopathies ...................................................................................................................... 22
Receptor Mediated Internalization of the Serpin-Proteinase Complex ............................ 23
Evolution of Serpins and Their Functions ......................................................................... 26
Phylogeny of the Serpin Superfamily................................................................................. 31
Positive Selection in Serpins ............................................................................................... 33
Novel Serpins and their Functions ...................................................................................... 35
Uterine Serpins ............................................................................................................................ 37
Ovine Uterine Serpin ........................................................................................................... 39
Endometrial secretion. ................................................................................................. 40
Biological function. ...................................................................................................... 41
Porcine Uterine Serpin ........................................................................................................ 44
Endometrial secretion. ................................................................................................. 44
Biological function. ...................................................................................................... 45
Bovine Uterine Serpin ......................................................................................................... 46
Endometrial secretion. ................................................................................................. 47
Biological function. ...................................................................................................... 47
Caprine Uterine Serpin ........................................................................................................ 48
Evolution and Phylogeny of Uterine Serpins ..................................................................... 48
Synopsis and Objectives...................................................................................................... 50
2 MOLECULAR PHYLOGENY OF UTERINE SERPINS AND ITS RELATIONSHIP
TO EVOLUTION OF PLACENTATION ................................................................................ 51
Introduction ................................................................................................................................. 51
Materials and Methods ................................................................................................................ 53
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Data Base Queries to Identify Uterine Serpin Genes ........................................................ 53
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)........................................ 53
Identification of cDNA for Equine Uterine Serpin Gene .................................................. 54
Sequencing of Amplicons ................................................................................................... 56
Detection of Equine Uterine Serpin by Western Blotting ................................................. 57
Amino Acid Sequence Alignments and Analysis of Phylogenetic Tree .......................... 57
Results .......................................................................................................................................... 59
Identification of Coding Sequences of Known and New Uterine Serpins Using
Blastn ................................................................................................................................ 59
Uterine Serpin Gene Organization in the Bovine and Canine .......................................... 62
Characteristics of CanUS and Expression in Tissues ........................................................ 62
Uterine Expression and Amino Acid Sequence of EqUS ................................................. 62
Endometrial Secretion of EqUS .......................................................................................... 66
Lack of Expression of the Uterine Serpin Gene in the Pregnant Cat ............................... 67
Amino Acid Sequence Conservation.................................................................................. 67
Identification of the Putative Hinge Region and P1-P1’ Site of the RCL........................ 70
Phylogenetic Analysis of Uterine Serpins.......................................................................... 72
Positive Selection of the Uterine Serpin Gene ................................................................... 72
Discussion .................................................................................................................................... 78
3 COMPARISON OF THE NATIVE AND RECOMBINANT FORMS OF OVINE
UTERINE SERPIN FOR INHIBITION OF CELL PROLIFERATION ................................ 85
Introduction ................................................................................................................................. 85
Materials and Methods ................................................................................................................ 86
Materials ............................................................................................................................... 86
Collection of Uterine Fluid and Purification of Native OvUS ......................................... 87
[3H]thymidine Incorporation by D17 and PC-3 Cells ....................................................... 87
Induction of Apoptosis in D17 and PC-3 Cells ................................................................. 88
Purification of His-Tagged rOvUS from Conditioned Medium ....................................... 89
Expression and Purification of β-Galactosidase ................................................................ 90
Proliferation of P388D1 and PC-3 Cells ............................................................................ 90
Statistical Analysis............................................................................................................... 91
Results .......................................................................................................................................... 92
Inhibition of Proliferation and Induction of Apoptosis in D17 and PC-3 Cells .............. 92
Antiproliferative Actions on P388D1 and PC-3 Cell Lines: Comparison of the
Native and Recombinant Forms of OvUS ...................................................................... 92
Discussion .................................................................................................................................... 95
4 REGULATION OF DNA SYNTHESIS AND THE CELL CYCLE IN HUMAN
PROSTATE CANCER CELLS AND LYMPHOCYTES BY OVINE UTERINE
SERPIN ........................................................................................................................................ 97
Introduction ................................................................................................................................. 97
Materials and Methods ................................................................................................................ 99
Materials ............................................................................................................................... 99
Purification of rOvUS.......................................................................................................... 99
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PC-3 Cell Culture .............................................................................................................. 100
[3H]thymidine Incorporation by PC-3 Cells .................................................................... 101
Cell Proliferation Based on ATP Content ........................................................................ 101
Cytotoxicity Assay............................................................................................................. 102
TUNEL Labeling ............................................................................................................... 102
Secretion of IL-8 ................................................................................................................ 103
Cell Cycle Analysis ........................................................................................................... 104
Statistical Analysis............................................................................................................. 105
Results and Discussion ............................................................................................................. 105
Proliferation of PC-3 cells ................................................................................................. 105
Lactate Rehydrogenase Release........................................................................................ 107
DNA Fragmentation (Apoptosis) ..................................................................................... 107
Interleukin-8 Secretion ...................................................................................................... 107
Cell Cycle Dynamics ......................................................................................................... 112
5 CHANGES IN EXPRESSION OF CELL-CYCLE RELATED GENES IN PC-3
PROSTATE CANCER CELLS CAUSED BY OVINE UTERINE SERPIN ...................... 118
Introduction ............................................................................................................................... 118
Materials and Methods .............................................................................................................. 119
Materials ............................................................................................................................. 119
Purification of rOvUS........................................................................................................ 120
PC-3 Cell Culture .............................................................................................................. 120
Proliferation Assay ............................................................................................................ 121
Cell Culture for RNA Extraction ...................................................................................... 121
RNA Extraction ................................................................................................................. 121
cDNA Synthesis and Real Time-PCR Array ................................................................... 122
Statistical Analysis............................................................................................................. 122
Results ........................................................................................................................................ 124
Inhibition of PC-3 Cell Proliferation by OvUS ............................................................... 124
Cell-cycle Related Gene Expression Profile at 12 h after Treatment with rOvUS ....... 124
Cell-cycle Related Gene Expression Profile at 24 h after Treatment with rOvUS ....... 127
Discussion .................................................................................................................................. 127
6 GENERAL DISCUSSION ....................................................................................................... 134
LIST OF REFERENCES ................................................................................................................. 141
BIOGRAPHICAL SKETCH ........................................................................................................... 156
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LIST OF TABLES
Table page
2-1 Primers used in RT-PCR procedure to obtain the full length coding sequence of the
equine uterine serpin gene. .................................................................................................... 55
2-2 Exon and intron sizes for the uterine serpin gene of the cow and dog. .............................. 63
2-3 Ratios (ω) of nonsynonymus (dN) to synonymous (dS) mutations, parameter
estimates and maximum log likelihood of models for positive selection within the
protein coding sequence of uterine serpins........................................................................... 74
2-4 Test of significance for models for positive selection within the protein coding
sequence of uterine serpins. ................................................................................................... 75
5-1 Cell cycle related genes screened using the RT2 Profiler
TM PCR Array. ......................... 123
5-2 Regulation of cell-cycle related genes of PC-3 cells after 12 h of treatment with 200
µg/ml recombinant ovine uterine serpin. ............................................................................ 126
5-3 Down-regulation of human cell cycle-related genes of PC-3 cells after 24 h of
treatment with 200 µg/ml recombinant ovine uterine serpin. ........................................... 128
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LIST OF FIGURES
Figure page
1-1 Structure of the inhibitory serpin α1-antitrypsin. A) Native-stressed conformation of
the protein. .............................................................................................................................. 18
1-2 Neighbor-joining tree of 110 serpin sequences. ................................................................... 34
2-1 Identification of an incorrectly annotated dog corticosteroid binding globulin (CBG)
as an uterine serpin (US)........................................................................................................ 61
2-2 Representative electrophoretogram of amplicons obtained by reverse transcriptase-
polymerase chain reaction (RT-PCR) of RNA fron canine tissue and canine uterine
serpin primers. ........................................................................................................................ 64
2-3 Expression and secretion of equine uterine serpin ............................................................... 65
2-4 Amino acid sequence alignment of the uterine serpins using the ClustalW algorithm. .... 68
2-5 Identification of the P1-P1’ site and hinge region (underlined) in uterine serpins. ........... 71
2-6 Phylogenetic tree of the uterine serpin proteins with the ovine uterine serpin (OvUS)
as out-group. ........................................................................................................................... 73
2-7 Selecton output generated for the uterine serpin group of proteins. ................................... 76
2-8 Amino acid sequence alignment of ovine uterine serpin (NP_001009304.1) and
human α1-antitrypsin (NP_000286.3) using the ClustalW algorithm ................................ 77
2-9 Phylogenetic tree of placentation in mammals (adapted from Vogel 2005) to
illustrate the existence of uterine serpin genes relative to type of placentation. ................ 79
3-1 Effect of OvUS on induction of apoptosis in D17 and PC-3 cells as determined by
TUNEL labeling. .................................................................................................................... 93
3-2 Inhibition of [3H]thymidine incorporation of P388D1 cells and PC-3 cells by native
(n) and recombinant (r) OvUS............................................................................................... 94
4-1 Inhibition of [3H]thymidine incorporation of PC-3 cells by recombinant ovine uterine
serpin (rOvUS) ..................................................................................................................... 106
4-2 Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin (rOvUS)
as determined by ATP content/well. ................................................................................... 108
4-3 Lack of cytotoxic effect of recombinant ovine uterine serpin (rOvUS) on PC-3 cells
was measured by the release of lactate dehydrogenase. .................................................... 109
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4-4 Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure
after 48 h of culture with either 100 (A) or 200 µg/ml (B) of rOvUS or 200 μg/ml of
the control protein ovalbumin. ............................................................................................ 110
4-5 Effect of recombinant ovine uterine serpin (rOvUS) on DNA fragmentation
(apoptosis) of PC-3 cells. ..................................................................................................... 111
4-6 Effect of recombinant ovine uterine serpin (rOvUS) on interleukin (IL) – 8
concentration in cell culture supernatants of PC-3 cells. ................................................... 113
4-7 Cell cycle dynamics of PC-3 cells as affected by recombinant ovine uterine serpin
(rOvUS). ............................................................................................................................... 114
4-8 Cell cycle dynamics of lymphocytes as affected by recombinant ovine uterine serpin
(rOvUS). ............................................................................................................................... 116
5-1 Inhibition of [3H]thymidine incorporation of PC-3 cells by 200 μg/ml of the
recombinant ovine uterine serpin (rOvUS) ........................................................................ 125
5-2 Points in the cell cycle where genes were differentially regulated by ovine uterine
serpin at 12 and 24 h are represented in panel A and B, respectively. ............................. 130
6-1 Possible pathways by which OvUS could block cell proliferation. .................................. 138
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LIST OF ABBREVIATIONS
Bo Bovine
Cap Caprine
CBG Corticosteroid Binding Globulin
CD Cluster of Differentiation
CDK Cyclin-Dependent Kinase
Con A Concanavalin A
DPBS Dulbecco’s Phosphate Buffered Saline
G-CSF Granulocyte Macrophage-Colony Stimulating Factor
HSP-47 Heat Shock Protein-47
IFN Interferon
IL Interleukin
LPS Lipopolysaccharide
LRP1 Low Density Lipoprotein Receptor Related Protein 1
Maspin Mammary Serine Proteinase Inhibitor
MENT Myeloid and Erythroid Nuclear Termination Stage-specific Protein
Mya Million Years Ago
NK Natural Killer
OVA Ovalbumin
Ov Ovine
PAI Plaminogen Activator Inhibitor
PC-3 Human Prostate Cancer Cells-3
PEDF Pigment Epithelium Derived Factor
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PHA Phytohemagglutinin
PK Protein Kinase
PolyI•PolyC Polyinosinic-Polycytidylic Acid
Po Porcine
PWM Pokeweed Mitogen
RAP Receptor Associated Protein
Rb Retinoblastoma protein
RCL Reactive Center Loop
RT-PCR Reverse Transcription-Polymerase Chain Reaction
rOvUS Recombinant Ovine Uterine Serpin
SERPIN Serine Proteinase Inhibitor
STZ Streptozotocin
TNF-α Tumor Necrosis Factor-α
TUNEL Terminal Deoxynucleotidyl Transferase (TdT) and fluorescein
isothiocyanate-conjugated dUTP nick end labeling
US Uterine Serpin
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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EVOLUTION AND CELLULAR BIOLOGY OF THE UTERINE SERPINS
By
Maria B. Padua
May 2009
Chair: Peter J. Hansen
Major: Animal Molecular and Cellular Biology
Uterine serpins (US) are a unique group of progesterone-induced glycoproteins in a
restricted group of mammals that belong to the serine proteinase inhibitor superfamily and which
are secreted in large quantities into the uterus during pregnancy. The US gene has been
identified in species with epitheliochorial placentation of the Ruminantia and Suidae orders of
the Laurasiatheria superorder of eutherian mammals.
One goal of this dissertation was to examine the evolution of the US gene in mammals. A
US gene was identified in horses and dogs and found expressed in the uterus during pregnancy.
The dog is a species with endotheliochorial placenta, suggesting that the US gene is not restricted
to species with epitheliochorial placentation. However, its absence in other mammals, and
apparent loss in the cat, suggests that the US gene evolved only within the Laurasiatheria
superorder.
The US do not appear to be functional proteinase inhibitors and to have species-specific
functions. The most studied member of the group, ovine uterine serpin (OvUS), inhibits
proliferation of several cell types including activated lymphocytes, bovine pre-implantation
embryos and some tumor cell lines. A second goal was to evaluate the mechanism by which
OvUS inhibits cell proliferation. Ovine US blocked cell cycle progression of human prostate
15
cancer (PC-3) cells, causing an accumulation of cells at G_2/M at 12 h and at G_0/G_1 at 24 h
after treatment addition. Additionally, OvUS blocked the cell cycle progression in
phytohemagglutinin-stimulated lymphocytes increasing the number of cells at the G_0/G_1 stage
at 96 h after treatment. Blocking of the cell-cycle progression by OvUS was caused specifically
by the up-regulation of cell cycle checkpoint and arrest genes such as CDKN1A (p21), CDKN2B
(p15) and CCNG2 (cyclin G2), and down-regulation of genes involved in DNA synthesis and
cell cycle regulation and progression.
The finding that the US gene is only retained in a limited group of mammals suggests its
importance for successful pregnancies in these species. It is also possible that the US gene
evolved a distinct and specie-dependent function from an ortholog serpin gene, rather than the
typical anti-proteolytic activity conserved in most members of the superfamily.
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CHAPTER 1
LITERATURE REVIEW
Introduction
Pregnancy in mammals has evolved in a process that included the appearance of new genes
for novel functions. Many of these new genes arose by gene duplication to generate paralogs
which underwent sequence divergence from the parental genes (Louis 2007). Recently, it was
determined specifically in the mouse placenta that most of the genes present during early events
of placentation, such as those involved in basic growth and metabolism, are evolutionary ancient
genes and their ortholog genes are also found within eukaryotes (Knox & Baker 2008). Similar
results were found in the human decidua where ortholog genes are also present in all vertebrates
(Knox & Baker 2008). In contrast, those genes expressed in the mature placenta are rodent
specific genes, which suggest that these genes are newly evolved since no orthologs were found
in other eukaryote or vertebrates (Knox & Baker 2008). Some examples of genes involved in
reproductive processes that were formed as a result of gene duplication are the primate chorionic
gonadotropins (Maston & Ruvolo 2002) and interferon-τ (Roberts et al. 1999). The uterine
serpins (US) present in the uterus of a limited group of mammals are another example. In sheep,
cattle, and pigs an ancestral gene or genes of the serine proteinase inhibitor (serpin) superfamily
underwent modification to exhibit high expression in the uterus and under the regulation of
progesterone. Unlike most of the members of the serpin superfamily, US probably have not
retained the anti-proteolytic activity that defines this superfamily (Irving et al. 2000). Instead,
evidence suggests they have divergent functions as for example, regulation of the immune
function in the sheep (Hansen 1998) and placental iron transport in the pig (Roberts & Bazer
1988). The purpose of this dissertation is to more closely examine the evolution of the US gene
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in mammals and to determine the mechanisms by which the US produced by the sheep inhibits
cell proliferation.
Serine Proteinase Inhibitor Superfamily
The serpins (serine proteinase inhibitors) are an important superfamily of proteins presents
in at least some members of all phyla. In vertebrates, serpins are involved in the regulation of
proteolytic pathways including blood coagulation and fibrinolysis (α1-antitrypsin, antithrombin),
complement cascade (complement C1-inhibitor) and tissue remodeling (plasminogen activator
inhibitor-1 and 2) (Irving et al. 2000). Although the inhibitory activity of serpins is mostly
towards serine proteinases, serpins can also function as inhibitors of proteinases that belong to
other families such as cysteine proteinases and papain-like cysteine proteinases (Silverman et al.
2001, Irving et al. 2002a, Hungtinton 2006).
Mechanism of Action
Inhibitory serpins inactivate their target proteinases through a unique suicide substrate-like
inhibitory mechanism. The structural conformation of serpins is typically formed by nine α-
helices (A-I) and 3 β-sheets (A-C) (Figure 1-1). The key region for inhibitory serpins is the
reactive center loop (RCL), which is a flexible structure localized on the top of the serpin and
contains a complementary sequence to the active site of the target proteinase. The RCL is
usually formed by 20-25 amino acids and the hinge region is enclosed in the P15-P9 portion of it
(Irving et al. 2000, Hungtinton 2006, Whisstock and Bottomley 2006). The hinge region
provides mobility to the RCL. There is a consensus pattern present in the sequence for inhibitory
serpins. Arginine is conserved at the P17 position, arginine, lysine or glutamic acid at P16,
glycine is usually present at P15 , threonine or serine is present at P14 position and acid residues
with short side chains such as alanine, glycine or serine are abundant in positions P12 to P9
(Irving et al. 2000).
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Figure 1-1. Structure of the inhibitory serpin α1-antitrypsin. A) Native-stressed conformation
of the protein. The helices are represented by blue cylinders and the A, B and C β-
sheets are shown in green, red and yellow respectively. The reactive center loop
(RCL) is shown in pink (1) with the hinge region (2). Also shown is the breach (3),
gate (4) and shutter (5). B) Cleaved-relaxed conformation of the protein. The
cleaved RCL is shown in 6 and the newly formed β-sheet A in 7. Reproduced from
Irving et al. (2000) with permission of Genome Research (© 2000) and after slight
modification.
3
1
4
2
5
6
7
A B
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The proteinase binds covalently to RCL and cleaves the scissile bond of the serpin at the
P1-P1’ site, releasing the P1’ residue and forming an ester bond between the P1 residue of the
serpin and the proteinase catalytic serine. Then, the amino-terminal portion of the RCL with the
attached proteinase moves as much as 70 Å to the opposite side to form the β-sheet A, and the
compression of the proteinase against the base of the serpin causes a distortion in the structure of
the proteinase causing its inactivation (Figure 1-1) (Irving et al. 2000, Silverman et al. 2001,
Hungtinton 2006). This conformational change in the structure, from the stressed (non-cleaved
RCL) to the relaxed (cleaved RCL) form, increases the overall stability of the serpin. This
characteristic makes serpins highly unusual. Most proteins are in a thermodynamically favorable
conformation in their native state. In contrast, serpins in their native state (i.e. the stress
conformation) require lower temperatures for denaturation (Silverman et al. 2001, Im et al. 2002,
Hungtinton 2006, Whisstock & Bottomley 2006).
There are some other important regions in the structure of the serpins besides the hinge
region of the RCL. One of them is the breach which is localized at the upper area of the β-sheet
A where the initial insertion of the RCL into the β-sheet A occurs (Figure 1-1) (Irving et al.
2000). There is also the shutter that is located close to the center of the β-sheet A. Together with
the breach, the shutter helps open the sheets to allow the insertion of the hinge region of the RCL
(Irving et al. 2000). Finally, the gate formed by strands s3C and s4C is region where the RCL
bypass around these strands to be completely inserted into the β-sheet A without cleavage (Irving
et al. 2000).
Some serpins are relatively inactive proteinase inhibitors, but they become fully activated
as inhibitors by the binding of cofactors such as heparin and vitronectin (Jin et al. 1997, Schvartz
et al. 1999, Zhou et al. 2003). This is the case of antithrombin, heparin cofactor II, plasminogen
20
activator inhibitor-1, protease nexin I and protein C inhibitor, where the RCL of these serpins is
partially inserted into the top of the β-sheet A, making the RCL and the P1 residue less
accessible for the proteinase. Once the cofactor interacts with the serpin, there is a
conformational change, where the RCL is expelled and the P1 residue becomes accessible for the
target proteinase (Law et al. 2006, Whisstock & Bottomley 2006). Likewise, heparin can
enhance the inhibitory function of already-active inhibitory serpins. A set of experiments
conducted by Gupta & Gowda (2008) showed that heparin can bind to α1-antitrypsin and thereby
increase the inhibitory potential of this serpin.
Non-Inhibitory Serpins
There is a group of serpins that do not inhibit proteinases. Examples of this growing group
includes ovalbumin (OVA), the chicken-egg storage protein (Benarafa & Remold-O’Donnell
2005), mammary serine protease inhibitor (maspin), which inhibits angiogenesis and tumor
growth and increases the sensitivity of cancer cells to undergo apoptosis (Zhang 2000, Sheng
2006), the hormone transport proteins corticosteroid and thyroxine binding globulin (Pemberton
et al. 1988), angiotensinogen, involved in blood pressure regulation, water and salt homeostasis
(Morgan et al. 1996, Stanley et al. 2006), the chaperone heat shock protein (HSP)-47 (Nagata
1998, Sauk et al. 2005) and the multifunctional pigment epithelium derived factor (PEDF),
which promotes differentiation of neurons and retinal photoreceptors, and neuronal degeneration
while inhibiting angiogenic processes in the retina, antagonizing the angiogenic effects of
vascular endothelial growth factor, platelet-derived growth factor and interleukin (IL)-8, and
promoting apoptosis in endothelial cells (Irving et al. 2000, Silverman et al. 2001, Tombran-
Tink & Barnstable 2003).
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Functions of Inhibitory Serpins Distinct from Proteinase Inhibition
Serpins are versatile proteins and can function in processes through mechanisms not
involving proteinase inhibition. A well studied example is the inhibitory serpin α1-antitrypsin,
which is produced by hepatocytes, neutrophils, macrophages, monocytes, intestinal epithelial
cells, astrocytes and some other cells to inhibit the enzymatic activity of different proteinases
such as neutrophil elastase, cathepsin G, proteinase 3, chymotrypsin, trypsin, thrombin (Travis &
Salvesen 1983, Congote 2007). The expression of α1-antitrypsin is increased by the pro-
inflammatory molecules IL-1, IL-6, tumor necrosis factor (TNF)-α, lipopolysaccharide (LPS)
(Congote 2007). Thus, it is assumed that the physiological role of this serpin is to prevent host
tissue from the proteolytic damage caused by the activity of these proteases.
In addition, α1-antitrypsin has functions that are unrelated to its anti-proteolytic activity.
Breit et al. (1983) demonstrated that purified α1-antitrypsin inhibited in a dose-dependent
manner the proliferative response of human peripheral blood lymphocytes induced by the
mitogens concanavalin A (Con A) and phytohemagglutin (PHA). However, α1-antitrypsin did
not completely inhibit these proliferative responses. Moreover, this serpin did not cause any
inhibitory activity against lymphocytes activated by the T and B cell mitogen pokeweed (PWM)
(Breit et al. 1983). Another set of experiments established that α1-antitrypsin competed with
diferric transferrin for the binding of the transferrin receptors inhibiting the proliferation of
human skin fibroblasts (Graziadei et al. 1998).
The effects of α1-antitrypsin on cytokines have been also pursued. Alpha1-antitrypsin, but
not the secretory leukocyte protease inhibitor, inhibited in a dose-dependent manner the release
of the pro-inflammatory cytokines TNF-α, monocyte chemoattractant protein-1, IL-8 and IL-1β
from LPS-stimulated human monocytes (Janciauskiene et al. 2004). Moreover, in the same
study, α1-antitrypsin increased IL-10 release from human monocytes stimulated with LPS.
22
However, the inhibitory effects of α1-antitrypsin on the pro-inflammatory cytokines were not
blocked when neutralizing antibodies against IL-10 were included in the experiments
(Janciauskiene et al. 2004).
Possible therapeutic roles for α1-antitrypsin in some human diseases such as type I diabetes
has been also studied. Alpha1-antitrypsin prevented the apoptotic effects of TNF-α and
streptozotocin (STZ, a diabetes inducer) on the MIN6 mouse insulinoma cell line through
caspase-3 activity inhibition (Zhang et al. 2007). In addition, administration of α1-antitrypsin to
the non-obese diabetic (NOD) mouse prevented the development of type 1 diabetes and reduced
insulin autoantibodies (Song et al. 2004). Moreover, Lewis et al. (2005) demonstrated that α1-
antitrypsin prolonged islet graft survival in C57BL/6 mice treated with STZ. Similar results
were obtained from experiments using C57BL/6 mice also treated with STZ, where the
administration of α1-antitrypsin reduced the levels of glucose in the blood, number of apoptotic
β-cells and rate of diabetes (Zhang et al. 2007).
Serpinopathies
The importance of the serine proteinase inhibitors is also highlighted by the loss of
function, deficiency and diseases (also known as serpinopathy) caused by mutations, leading to a
serpin existing in a non-functional state.
Serpin polymerization is a non-functional state which is achieved by the binding of the
RCL of one serpin to the β-sheet A, or in some cases to the β-sheet C of other serpin, forming a
long-inactive polymer. This phenomenon causes the accumulation of polymerized serpins as
misfolded protein, therefore decreasing the amount of functional serpins available (Kaiserman et
al. 2006, Law et al. 2006). In humans, α1-antitrypsin polymerization could lead to emphysema
and liver cirrhosis and the polymerization of neuroserpin could cause familial encephalopathy
(FENIB) (Kaiserman et al. 2006, Law et al. 2006). The other non-functional state is latency
23
where the RCL is inserted into the β-sheet A without being cleaved by the target proteinase
causing the inactivation of the serpin (Kaiserman et al. 2006).
Serpin mutations also alter the functionality and stability of the protein. Mutations can
induce polymerization (the neuroserpin pathology and α-1 antitrypsin Z mutant), cancer (loss of
function of maspin), change in serpin specificity (the α1- antitrypsin Pittsburg mutation, where
an amino acid substitution at the P1 site of this serpin generates a potent inhibitor of thrombin
and other coagulation enzymes, causing death by bleeding disorders), and also loss of cofactor
binding site to disrupt the interaction between heparin and antithrombin III (Law et al. 2006,
Kaiserman et al. 2006).
In addition, gain-of-function serpinopathy can occur when genetic mutations lead to
overexpression of a serpin. The excess in the synthesis of megsin, a serpin produced by human
mesangial cells, causes accumulation and polymerization of the protein in the endoplasmic
reticulum of the kidney glomerular epithelial cells (Inagi et al. 2005, Miyata et al. 2005). This
renal serpinopathy is characterized by mesangial matrix expansion, stress of the endoplasmic
reticulum, and augmented IgA deposition which leads to renal failure (Miyata et al. 2005,
Kaiserman et al. 2006). The overexpression of the mutant megsin, which lacks anti-proteolytic
activity and flexibility at the hinge region loop, does lead to polymerization, but not to the
generation of a serpinopathy, suggesting the importance of the overall structure and function of
megsin for the development of the disease (Inagi et al. 2005, Miyata et al. 2005).
Receptor Mediated Internalization of the Serpin-Proteinase Complex
Once the target proteinase is bound to the serpin, a stable complex is formed by the inactive
proteinase and the cleaved serpin. This serpin-proteinase complex is cleared rapidly from the
circulation by an internalization pathway mediated by hepatic receptors that leads to lysosomal
degradation. Perlmutter et al. (1990) showed a specific cell surface receptor in human
24
monocytes and hepatoma (HepG2) cells, which was referred as the serpin-enzyme complex
receptor (SEC receptor). The SEC receptor recognized and subsequently internalized the
following serpin-proteinase complexes α1-antitrypsin-elastase, α1-antichymotrypsin-cathepsin
G, antithrombin III-thrombin and also complement C1-inhibitor-C1s but with less affinity
(Perlmutter et al. 1990, Poller et al. 1995). Later, experimental data revealed that most of the
serpin-enzyme complexes are also internalized by the low-density lipoprotein (LDL) family of
endocytic receptors which includes the very-low-density lipoprotein (VLDLR), the low-density
lipoprotein-receptor-related protein 1 (LRP1), which was originally known as LRP or
occasionally as CD91, the glycoprotein 330 (gp330) also known as megalin or LRP2, and the
low-density lipoprotein (LDLR) receptors (Kounnas et al. 1996, Skeldal et al. 2006, Lillis et al.
2008).
Since the SEC receptor was never sequenced and the LRP1 is abundantly found in the
liver, this receptor is the most likely candidate for the internalization of the serpin-proteinase
complexes. Kounnas et al. (1996) demonstrated that addition of either LRP1 antibodies, the
antagonist chaperone receptor associated protein (RAP) or chloroquine (an inhibitor of
lysosomal degradation) to the HepG2 cells inhibited the internalization and degradation of α1-
antitrypsin-trypsin, antithrombin III-thrombin and heparin cofactor II-thrombin complexes.
Moreover, these serpin-proteinase complexes were not internalized by mouse fibroblasts lacking
LRP1 receptors (Kounnas et al. 1996). In addition, native or cleaved forms of the serpins alone
were not recognized as ligands and subsequently internalized by the LRP1 receptor, suggesting
the specificity of the receptor for serpin-proteinase complexes only (Kounnas et al. 1996).
Moreover, in vivo experiments using the rat as a model showed that the antagonist RAP delayed
the internalization of radiolabeled antithrombinIII-thrombin complexes from plasma circulation
25
(Kounnas et al. 1996). Similar experiments demonstrated that other serpin-proteinase complexes
such as complement C1-inhibitor-C1s, α1-antitrypsin-neutrophil elastase, protease nexin-1-
thrombin, protease nexin-1-factor XIa, neuroserpin-tissue-type plasminogen activator and
protease nexin-1-factor VII-activating protease are internalized by the endocytic LRP1 receptor
(Storm et al. 1997, Poller et al. 1995, Crisp et al. 2000, Knauer et al. 2000, Makarova et al.
2003, Muhl et al. 2007).
The role of heparin in the binding of the serpin-proteinase complexes to the LRP1
receptor and subsequent internalization has been also studied, in particular the binding of
protease nexin-1 to heparin after the serpin-proteinase complex has formed. Experiments
performed by Knauer et al. (1997) tested the role of heparin and LRP1 in the internalization of a
variant form of the protease nexin-1 (PN1-K7E) which does not bind heparin. They showed that
the complex PN1-K7E-thrombin could form but, internalization of the complex and further
degradation was greatly reduced in normal mouse embryonic fibroblasts and nearly abolished in
the LRP1-deficient mouse embryonic fibroblasts, when compared with the experimental
controls. Moreover, in the absence of endocytosis, the addition of soluble heparin inhibited the
binding of the serpin-proteinase complex to the surface of human and mouse embryonic
fibroblast cells (Knauer et al. 1997). The authors concluded that cell surface heparins mediated
the binding of protease nexin-1-thrombin complex to the LRP1 and further internalization.
Similar experiments were performed by Crisp et al. (2000) to study the role of heparins, in the
internalization of the protease nexin-1-urokinase plasminogen activator (uPA) complex through
the LRP1. These authors showed that the mechanisms of cell surface binding and internalization
of this serpin-proteinase complex are heparin-independent. These results indicate that the
26
interaction of the serpin-proteinase complex with cell surface receptors in the endocytic pathway
is determined by the specific serpin or proteinase involved.
There is also a specific receptor, the urokinase plasminogen activator receptor (uPAR)
known as well as CD87, that plays a role in the internalization of the uPA and tissue-type
plasminogen activator (tPA) complexes formed with either plasminogen activator inhibitor
(PAI)-1 or PAI-2 (Olson et al. 1992, Wind et al. 2002). Even though active uPA binds with high
affinity to uPAR, its internalization is much slower than the uPA-PAI-1 complex (Wind et al.
2002). Moreover, the internalization of uPA-PAI-1 bound to the uPAR receptor and the later
recirculation of the receptor requires the interaction of the α2-macroglobulin receptor/low
density lipoprotein receptor- related protein (α2 MR/LRP), a large cell-surface glycoprotein
(Binder et al. 2007). There is experimental evidence showing that the internalization of the uPA-
PAI-1 complex can be also mediated through the related receptor sorting protein-related receptor
(sorLA), LRP2, LRP1 and VLDLR, all members of the LDLR family of endocytosis receptors
(Wind et al. 2002, Skeldal et al. 2006). Likewise, it was recently demonstrated by Chroucher et
al. (2006) that the internalization of the uPA-PAI-2 complex is also achieved by the LRP1
receptor.
Evolution of Serpins and Their Functions
It was previously believed that serpin genes were restricted to higher eukaryotes. However,
recent studies indicate that serpins have an ancient origin, at least 1000 million years old, and
they are not only limited to higher eukaryotes organisms, but instead are present in at least some
species of all phyla on Earth.
Serpin genes have been found in some complete prokaryote genomes including Archaea
and Bacteria (Irving et al. 2002b). In the Bacteria genera, serpin genes are present in
cyanobacteria, actinobacteria and formicutes, but not in any proteobacteria family with complete
27
genomes (Irving et al. 2002b, Roberts et al. 2004, Ivanov et al. 2006). The conservation of
residues in functional key regions and secondary structural elements of these serpins suggests
their ability to inactivate proteinases (Irving et al. 2002b, Roberts et al. 2004). Ivanov et al.
(2006) demonstrated that the serpin found in the actinobateria Bifidobacterium longun inhibited
human neutrophil elastase and porcine pancreatic elastase. Kang et al. (2006) showed that there
are three serpin genes in the formicute Clostridium thermocellum (Clotm-serpin 1, 2 and 3). The
Clotm-serpins-1 and 2 are close related, with the hinge region of the RCL very conserved with
inhibitory serpins whereas Clotm-serpin-3 is a divergent serpin. In the same study, it was
demonstrated that serpin-1 did not inhibit trypsin, chymotrypsin or papain, but it had inhibitory
activity towards subtisilin, a bacterial serine proteinase. Multiple serpin genes were found in two
species of the Archaea and Bacteria domains, where the variability of the RCL suggested that
these genes were generated by duplication and later diversification (Roberts et al. 2004)
Despite the cases described above, serpin sequences are not broadly present in the
prokaryotic kingdom. No serpin sequences were identified in 13 Archaea and 56 Bacteria
genera with complete genome sequences, which suggested that in prokaryotes the serpin gene
was probably acquired by lateral transfer or lost when it was not vital (Irving et al. 2002b,
Roberts et al. 2004, Ivanov et al. 2006).
In viruses, serpin genes have been only identified in the poxviridae family and they can be
localized in extracellular and intracellular compartments (Brooks et al. 1995, Lucas & McFadden
2004). Three active serpin genes were found in the Orthopoxviruses genus. The viral serpin-1
(SPI-1) is a 40 kDa protein that blocked apoptosis in rabbitpox virus infected cells (Brooks et al.
1995). The viral serpin-2 (SPI-2) or CrmA inhibited efficiently caspase 1, preventing the
production of IL-1β and 18 (Brooks et al. 1995, Callus & Vaux 2007). Also, it was found that
28
SPI-2 inhibited caspases 3 and 8 (Brooks et al. 1995, Callus & Vaux, 2007). Finally, the viral
serpin-3 (SPI-3) is involved with regulation of virus-host cell fusion (Brooks et al. 1995).
Although the amino acid sequence of SPI-1 is almost 50% identical to SPI-2, the predicted RCL
of these two genes differs significantly (Brooks et al. 1995). Irving et al. (2000) suggested the
possibility of gene duplication from a single gene as the origin for SPI-1 and -2, but separately
from SPI-3. Another viral serpin gene, (SERP-I) was found in the myxoma virus of the
Leporipoxvirus genus. SERP-I is a 55 kDa glycosylated secreted protein which binds and
inhibits human host plasmin, uPA and tPA. The knockout SPI-1 myxoma virus demonstrated its
importance on reducing the immune response of host monocytes and macrophages (Lucas &
McFadden 2004).
Serpin genes are also present in several green algae and plants. In the plant kingdom,
serpin genes are present in moss and conifers (Roberts & Hejgaard 2008). Moreover, serpins
have been found in the phloem sap of the cucurbits pumpkin and cucumber and in very high
quantities in the seeds of barley, wheat, rye, oats and apple (Roberts & Hejgaard 2008). There is
very little information about the genomic organization of most of the members of the plant
kingdom. There is only one serpin gene present in the unicellular green algae Chlamydomonas
reinhardtii, 7 serpin genes in Arabidopsis and 14 in rice (Roberts & Hejgaard 2008). Although
the first plant serpin genes identified were inhibitory, recent findings in maize and rice have
shown the expression of close related non-inhibitory serpin genes (Roberts & Hejgaard 2008).
There are no studies on the evolution of serpin genes in green algae and plants, however, Roberts
& Hejgaard (2008) have postulated the possibility of a lateral gene transfer from bacteria to algal
ancestors and inherited from the most advanced green algae when they evolved to land plants.
29
The function of serpins in green algae and plants remains unclear. Most of the known
proteinases are found in plants. Typical animal caspases are absent, but cysteine proteinases are
involved in the programmed cell death (Roberts & Hejgaard 2008). Green algae and plants also
lack genes homologous to those encoding trypsin, elastase, thrombin, granzyme B. One
chymotrypsin-like proteinase has been discovered in plants, and in vitro studies have shown that
serpins from wheat, barley, rye, oat, pumpkin, apple and Arabidopsis are able to inhibit serine
proteinases of the chymotrypsin group (Roberts & Hejgaard 2008).
Silverman et al. (2001) postulated that phloem sap serpins could be involved in the host
mechanism of defense, since the phloem serpin-1 found in Cucurbita maxima decreased the
ability of the piercing-sucking aphids, Myzus persicae to survive and reproduce on pumpkins
(Yoo et al. 2000). Likewise, it has been proposed that the function of serpins found in high
concentrations in plant seeds protects storage proteins from degradation caused by proteolytic
enzymes of fungi or insects (Roberts & Hejgaard 2008).
Serpin sequences have been not found in complete genomes of Saccharomyces cerevisiae
(beaker’s yeast) and Schizosaccharomyces pombe (fission yeast) (Roberts et al. 2004).
However, a recent study conducted by Steenbakkers et al. (2008) revealed that the presence of
one serpin gene in the fungal kingdom. Analysis of the amino acid sequence of this so-called
‘celpin’ showed that it seems to have all the features of a prototypical functional serpin
(Steenbakkers et al. 2008). Moreover, the serpin gene is present in the unicellular eukaryote
Entamoeba histolytica which lacks of an N-terminus signal peptide even though it is secreted by
activated trophozoites (Riahi et al. 2004). The interaction between this serpin and trypsin is
typical of non-inhibitory serpins where the proteinase is still active while the serpin is cleaved in
30
its C-terminus (Riahi et al. 2004). In the same study, it was shown that the serpin had non-
inhibitory activity towards chymotrypsin, elastase and cathepsin G.
Additionally, serpins are present in early metazoans. Cole et al. (2004) cloned the full-
length cDNA of a serpin gene called ‘jellypin’ from the cnidarian jellyfish Cyanea capillata.
Analysis of the amino acid sequence showed that this serpin lacks of N-terminal signal sequence
as is characteristic of intracellular serpins. In the same study, it was determined that jellypin
contained a functional RCL and inhibited human chymotrypsin, elastase, and capthesin G,
however, under unphysiological conditions (Cole et al. 2004). Since the closest relative of
jellypin in the serpin superfamily are plant serpins, the authors suggest that this metazoan serpin
arose approximately 1000 million years ago (Mya), around the time of divergence between
plants and animals (Cole et al. 2004).
In nematodes, eight serpin genes have been found in Caenorhabditis elegans and the same
numbers of serpin genes are present in parasitic species such as Ascaris suum, Brugia malayi,
Onchocerca volvulus, Trichostrongylus vitrinus and Shistosoma mansoni (Zang & Maizels
2001). The role for these serpins has been related to the evasion of the host’s immune response
and limiting tissue damage in response to inflammation since BmSPN-2 inhibits cathepsin G and
elastase, both produced by neutrophils (Zang & Maizels 2001). In these species there are also
small serpins known as ‘smapins’ which have less than 100 amino acid residues, ten cysteine
residues forming five disulphide bonds where two of these are located at each side of the RCL,
and no structural relation with the well known members of the superfamily (Zang & Maizels
2001). In the dog hookworm A. caninum, smapins inhibits serine proteases that are involved in
the host’s blood coagulation pathway (Zang & Maizels 2001).
31
The percent of identity found between the C. elegans serpins CeSPN-3 and CeSPN-4 is
87% while the identity between CeSPN-7 and CeSPN-8 is 78% (Zang & Maizels 2001). The
authors concluded that these serpin genes in C. elegans arose as a local duplication event (Zang
& Maizels 2001). Moreover, it was also suggested that a possible intron gain-and-loss is the
model for the serpin gene evolution in nematodes (Zang & Maizels 2001).
Phylogeny of the Serpin Superfamily
In humans, serpins are classified in nine groups A-I where the largest group are the
extracellular clade A or SERPINA with 13 members found in chromosomes 1, 14 and X and the
intracellular clade B or SERPINB with 13 members on chromosomes 18 and 6 (van Gent et al.
2003, Law et al. 2006, Izuhara et al. 2008). The groups SERPINC and SERPIND are
represented by antithrombin and heparin cofactor II respectively (van Gent et al. 2003). The
serpins PAI-1 and glial-derived nexin are grouped together in the SERPINE group, α2-
antiplasmin and PEDF in SERPINF, and the complement C1 inhibitor in the SERPING group
(van Gent et al. 2003, Kaiserman et al. 2006). The SERPINH group is represented by the HSP-
47, the myoepithelium-derived serpin, also known as ‘pancpin’, and neuroserpin are in the
SERPINI group (van Gent et al. 2003, Kaiserman et al. 2006).
The phylogenetic analysis conducted by Irving et al. (2000) divided the serpin superfamily
into 16 clades or classes (A-P). The largest clades in the classification are Clade A and B and
they are formed by extracellular or antitrypsin-like serpins and intracellular or ov-like serpins
respectively. This last group of serpins is suggested to be ancestors to the majority of
extracellular serpins. The other clades are formed by serpins that have been found in insects,
nematodes, plants and virus; however, there are ten highly diverged orphan serpins which failed
to group within any of the clades; these proteins are also known as orphan serpins (Irving et al.
2000, Silverman et al. 2001). The viral serpins SPI-1 and -2 were clustered together in clade n,
32
but SPI-3 was classified alone in clade o (Irving et al. 2000). In addition, the myxoma serpin
SERP-I was placed in clade e clustered together with plasminogen activator inhibitor-1 and
protease nexin-1 (Irving et al. 2000).
A study performed only with the nematode serpins grouped all the C. elegans serpins
together in one cluster whereas serpins present in other species were separated in different
branches as an indication of divergence (Zang & Maizels 2001). These findings were similar to
those found by Irving et al. (2000) where the C. elegans serpins were clustered together in clade
l whereas the Schistosoma serpins are clustered in clade m and all the other nematode serpins are
consider orphans.
Cole et al. (2004) conducted also a phylogenetic study to determine the relationship of the
metazoan serpin jellypin with other members of the serpin family. The results classified jellypin
as an orphan, but its closest relatives were the plant serpins (Cole et al. 2004), which are
clustered in clade p (Irving et al. 2000).
More complex phylogenetic analyses were performed by Atchley et al. (2001) (Figure 1-2)
and Ragg et al. (2001) where they took into consideration amino acid sequences, exon-intron
structures and diagnostic of amino acid sites. The results from the study performed by Atchley
et al. (2001) were similar to those presented by Irving et al. (2000) where serpins were clustered
within ten clades or groups with two largest groups, the α1-antitrypsin and ovalbumin clades,
and several orphan serpins. Since the study by Ragg et al. (2001) was performed using only
vertebrate serpins, the number of clades or group was reduced to six with α1-antitrypsin and
ovalbumin groups being the largest. All three studies classified antithrombin III and HSP-47 in
separate clades or groups (Irving et al. 2000, Ragg et al. 2001, Atchley et al. 2001). However,
the intron-exon data from Atchley et al. (2001) and Ragg et al. (2001) showed that neuroserpins,
33
PAI-1 and glia derived nexin are grouped together within a clade suggesting a single
evolutionary lineage, whereas Irving et al. (2000) clustered neuroserpins into a separate group
from the other two serpins. Likewise, the intron-exon data clustered together complement C1-
inhibitor, PEDF and α2-antiplasmin (Atchley et al. 2001, Ragg 2001) whereas Irving et al.
(2000) placed the protease C1 inhibitor as a separate clade.
Positive Selection in Serpins
Positive or Darwinian selection refers to selective pressure acting on proteins that can
provide evolutionary advantage to an organism. The most used method to detect positively
selected sites in protein coding sequences is comparing the ratio (ω) between the non-
synonymous substitutions (dN), where a nucleotide substitution will lead to an amino acid
replacement of the encoded protein, with the synonymous substitutions (dS) where the
substitution of a nucleotide will not change the amino acid leaving the encoded protein
unchanged (Wong et al. 2004). When the ω ratio is greater than 1, the result is interpreted as
evidence of positive selection, but if the ω ratio is equal or less than 1, results are interpreted as
evidence of neutral or purifying selection respectively (Wong et al. 2004).
There is enough evidence suggesting that serpins, like a few other protein families, have
undergone positive or Darwinian selection. Positive selection has occurred at the RCL region of
the protein, in particular at the P1 site proximal to the scissile bond of the RCL which changes
the selectivity for the target proteinase (Brown 1987, Hill & Hastie 1987, Goodwin et al. 1996,
Zang & Maizels 2001, Barbour et al. 2002). Experimental data from Barbour et al. (2002)
showed that single α1-antitrypsin isoforms from two different species of mouse inhibited
different serine proteinases. Moreover, in the same study it was determined that this diversity in
the functionality of these isoforms was caused in particular by the variations in the RCL area
34
Figure 1-2. Neighbor-joining tree of 110 serpin sequences. The clusters of proteins show close
correspondence to the groups of proteins described by the analyses of exon-intron
structure. Atchely et al. (2001) by permission of the Oxford Journals, Oxford
University Press.
35
(Barbour et al. 2002). In contrast, van Gent et al. (2003) did not find any positive selection in a
study that compared some serpin groups between human and rodents, human and artiodactyla
and rat versus mouse.
The positive selection of the serpin genes has been reported in different species in which it
is suggested the advantage of this process during the evolution of the serpins in order to
neutralize a broader range of proteinases. For example, it has been postulated that caspase
inhibitor genes in viruses have undergone positive selection as a defense mechanism against the
host’s immune response (Brooks et al. 1995, Callus & Vaux 2007). Likewise, in nematodes, the
rapid diversification of the RCL has allowed novel changes in these serpins and smapins in order
to target mammalian host’s proteinases (Zang & Maizels 2001). There is also circumstantial
evidence suggesting that the evolution of the RCL of serpins present in plant seeds is because of
positive Darwinian selection to inhibit exogenous proteinases from insects and pathogens
(Roberts & Hejgaard 2008).
Novel Serpins and their Functions
As previously mentioned, the mechanisms of actions and functions of some serpins has
been extensively studied and well documented. However, recent discoveries have found new
serpins with novel characteristics and interesting biological functions. One example is the novel
nuclear serpin, myeloid and erythroid nuclear termination stage-specific protein (MENT) which
has an ‘M loop’, a large insertion between the C- and D-helices. The M loop is also known as
the CD loop and contains a nuclear localization signal and the AT-hook motif which is required
for chromatin and DNA binding (Silverman et al. 2001, Irving et al. 2002a). This intracellular
serpin is the major non-histone chromatin protein localized in the heterochromatin and it
functions by inducing higher order chromatin condensation by connecting separate nucleosomes
in vitro (Silverman et al. 2001, Irving et al. 2002a). In addition, MENT is one of those cross-
36
class serpins that inhibits cysteine proteinases and papain-like cysteine proteinases such as
cathepsins K, L and V (Irving et al. 2002a). Experimental data showed that MENT is involved
in cell cycle progression and therefore in cell proliferation by inhibiting the enzymatic activity of
the nuclear cysteine proteinase SPase, a cathepsin L-like proteinase, whose function has been
related to the degradation of the phosphorylated form of the retinoblastoma (Rb) protein, a
known regulator of the cell cycle (Irving et al. 2002a).
Plasminogen activator inhibitor type-2 is another interesting serpin with novel function.
The bi-topological PAI-2 is predominantly translated as an intracellular 47 kDa non-glycosylated
protein, but it can be also a 60 kDa glycosalyted secreted protein as a combination of an
inefficient nuclear localization signal and the lack of a conventional hydrophobic amino-terminal
sequence (Medcalf & Stasinopoulos 2005, Croucher et al. 2008). Like the serpin MENT, the CD
loop is present in PAI-2, which binds non-covalently to several proteins including the Rb protein
(Medcalf & Stasinopoulos 2005). Although the physiological role of the intracellular PAI-2
remains unknown, it has been suggested that PAI-2 could be involved in the cell cycle regulation
since this serpin protected Rb from degradation by a distinct anti-proteolytic mechanism
(Medcalf & Stasinopoulos 2005, Croucher et al. 2008).
A therapeutic role of PAI-2 in the reduction of tumor growth and metastasis of different
types of cancer has been extensively demonstrated using in vitro systems and rodent tumor
models where uPA was inhibited completely and a significant decrease in the extra cellular
matrix degradation achieved (Croucher et al. 2008). High levels of expression of PAI-2 in breast
cancer tumors are associated with reduced metastasis, tumor size and prolonged survival whereas
opposite effects are expected when this serpin is expressed at low levels (Croucher et al. 2008).
37
More recently, another novel serpin was discovered in the visceral adipose tissue of the
obese-type 2 diabetes rat model. This new serpin called visceral adipose tissue-derived serpin
(vaspin) was found to be up-regulated in the obese-type 2 diabetes rat model and down-regulated
in the non obese-diabetes resistant rat model (Hida et al. 2005). The vaspin transcript was up-
regulated in the subdermal white adipose tissue by the administrations of insulin and
thiazolidinediones (TZD), a compound that enhances insulin sensitivity (Hida et al. 2005). The
authors suggested from the experimental data that vaspin inactivates unknown target proteases
derived from fat or other tissue that inhibited the effects of insulin (Hida et al. 2005). However,
it was demonstrated in the same study that vaspin lacks anti-proteolytic activity towards common
serine proteinases such as trypsin, factor Xa, elastase, urokinase, collagenase and dipeptidyl
peptidase (Hida et al. 2005). The vaspin gene is also expressed in the white adipose tissue of
mouse and human (Hida et al. 2005).
The US are also novel with respect to serpins by virtue of their limited distribution among
mammals, since no human homologue of the US gene has been reported (van Gent et al. 2003).
The primary site of expression for the US is in the endometrium and regulation of their
expression by progesterone. A detailed description of their characteristics, properties and
plausible functions for this group of serpins are described below.
Uterine Serpins
Uterine serpins were first described as progesterone induced proteins in the uterine fluid of
the pregnant sheep, pig and cow (Moffatt et al. 1989, Baumbach et al. 1986, Leslie et al. 1990).
The protein in sheep, discovered first, was called uterine milk protein (UTMP) because it was the
major protein in uterine fluid (called uterine milk by Aristotle). The two US present in the pig
were originally known as uteroferrin-associated protein or uteroferrin-associated basic proteins
(UfAP/UABP) because they form non-covalently heterodimers with uteroferrin (Baumbach et al.
38
1986, Roberts & Bazer 1988). The connection between UTMP and UfAP was first made when
the genes were sequenced. Ing & Roberts (1989) found greatest homology between the cDNA
sequence of ovine UTMP with α1-antitrypsin and classified UTMP as a member of the serine
proteinase inhibitor (serpin) superfamily. Shortly thereafter, Malathy et al. (1990) found that
both pig proteins were serpins and also highly similar to ovine UTMP. Mathialagan & Hansen
(1996) identified the bovine UTMP as a serpin closely related to sheep UTMP and pig UfAPs
and proposed that old designations be eliminated and proteins termed as uterine serpins (US)
preceded by the name of the specie from where it was identified such as (Bo) for bovine, (Ov)
for ovine and (Po) for porcine. Since this paper, additional US were identified in the goat (Tekin
et al. 2005a) and a sequence for water buffalo (Bubalus bubalis) has been submitted to Genbank
(Accession number: DQ 661648.1)
Uterine serpins have been only characterized in species with epitheliochorial placenta. All
US proteins that have been described to date possess a 25-amino acid signal peptide sequence
and are secreted into the uterine lumen (Mathialagan & Hansen 1996, Tekin et al. 2005a).
Mathialagan & Hansen (1996) determined that most of the amino acids present in OvUS, BoUS
and PoUS-1 and -2, necessary for the serpin backbone structure, are conserved at the same
positions with those on α1-antitrypsin. However, analysis of the hinge regions of all US
indicates that these serpins are not conserved with inhibitory serpins and are probably not
functional proteinase inhibitors (Irving et al. 2000, Tekin et al. 2005a). In the hinge region of
US, the only amino acid that is conserved with the inhibitory serpins is alanine at the P12
position whereas the other positions are not (Tekin et al. 2005a). In addition, the amino acid at
P1-P1’ site for known uterine serpins is conserved among species where valine is usually found
with the exception of OvUS which has an alanine at this position (Tekin et al. 2005a).
39
The OvUS was screened for anti-proteinase activity against several proteinases without
identification of a target proteinase (Ing & Robets 1989). Both BoUS and OvUS are capable of
inhibiting pepsin at very high concentrations (Mathialagan & Hansen 1996, Peltier et al. 2000a),
but this activity occurs even when the protein is proteolytically cleaved and may be distinct from
the classical serpin inhibitory mechanism involving a functional RCL (Peltier et al. 2000a).
Uterine serpins have a conserved KVP sequence at the P4-P2 site of the RCL (Tekin et al.
2005a, Mathialagan & Hansen 1996). There is also a unique 39 amino acid insertion in the
BoUS, which incorporates two KAKEVPAVVKVPM repeats within the putative P1-P1’ site and
one KEVPVVVKVP sequence right after that P1-P1’ site (Mathialagan & Hansen 1996, Tekin et
al. 2005a). The KEVPVVVKVP motif is also found further upstream as a conserved sequence
among all known US (Mathialagan & Hansen 1996). The real function for all these repeats
remains unknown. Mathialagan & Hansen (1996) suggested that US may inhibit some members
of the aspartic proteinase family, because of the resemblance of the motif to the VVVK present
in pepsinogens. However, Peltier et al. (2000a) showed that the addition of a synthetic peptide
corresponding to the putative P7-P15’ region of OvUS did not inhibit pepsin activity.
Ovine Uterine Serpin
This is the most studied protein of the US group. Ovine US is a secreted protein which is
present in the uterine fluid as a pair of basic glycoproteins with molecular weights of 55,000 and
57,000, derived from a single 54,000 precursor (Moffatt et al. 1987, Hansen et al. 1987a). This
basic glycoprotein possess an isoelectric point of 9.2 and two N-linked glycosylation sites, which
suggests that the two major forms of the protein may differ in the number of carbohydrate chains
(Hansen et al. 1987a, Ing & Robets 1989). The amino acid composition was found to be rich in
lysine, leucine and threonine, but low in tyrosine and devoid of tryptophan (Hansen et al.
1987a). Analysis of the carbohydrate content of OvUS showed that it consists of 2.8% neutral
40
sugars, 2.5% amino sugars, and 0.3% sialic acid (Hansen et al. 1987a). Additionally, OvUS
possess a mannose 6-phoshate, the so-called lysosomal recognition marker (Hansen et al.
1987a).
Endometrial secretion. The major regulator of OvUS secretion is the hormone progesterone
(Moffatt et al. 1987, Ing et al. 1989, Leslie & Hansen 1991, Padua et al. 2005). Experiments
performed using the ovariectomized ewe as a model showed that after 6 days of treatment with
progesterone, OvUS can be detected in endometrial epithelial cells (Ing et al. 1989). Moreover,
progesterone therapy for 14-60 days induced a large increase in the secretion of the protein (Ing
et al. 1989, Leslie & Hansen 1991, Padua et al. 2005). Ovine US can be detected in uterine
secretions of ovariectomized ewes treated for 30 days with a combination of progesterone plus
estrone, however, the protein could not be detected when they were treated with estrone only
(Moffatt et al. 1987). In addition, levels of OvUS mRNA increased in the glandular epithelium
of ovariectomized ewes receiving uterine infusion of ovine placental lactogen and/or ovine
growth hormone combined with IFN-τ infusions and the administration of progesterone as
compared with the levels in ovariectomized ewes infused with control proteins (Spencer et al.
1999, Noel et al. 2003). It was also shown that the co-administration of estradiol with
progesterone, where estradiol up-regulates the expression of progesterone receptors in the
endometrium, decreased OvUS mRNA in the glandular epithelium (Spencer et al. 1999).
During the estrous cycle, OvUS mRNA can be detected in uterine endometrium around
days 13-16 (Stewart et al. 2000). In the pregnant sheep, the transcript for OvUS can be first
detected at days 13-15 (Ing et al. 1989, Stewart et al. 2000). Then, a 3-fold increase of steady-
state levels of OvUS mRNA in the glandular epithelium occurs between days 20 and 60, another
3-fold between days 60 and 80, and a decline at day 120 of pregnancy (Stewart et al. 2000).
41
Between days 20 and 50 of pregnancy, the expression of OvUS mRNA is lower in the deep
glandular epithelium than in the upper glandular epithelium of the uterine stratum spongiosum.
There was however, no difference in mRNA expression between the upper and lower glandular
epithelium of the stratum spongiosum between days 50 and 60 of gestation (Stewart et al. 2000).
High levels of expression of the transcript for OvUS can be detected in all glandular epithelium
in the stratum spongiosum between days 60 and 120 of pregnancy (Stewart et al. 2000). At day
1 of postpartum, OvUS mRNA was only detected in the stratum spongiosum of the glandular
epithelium, but during postpartum days 7 and 28, the transcript for OvUS was not longer
detected (Gray et al. 2003).
It appears that OvUS is initially produced only by the uterine glands and then its expression
spreads to the luminal epithelium. Although OvUS protein was found only in glandular
epithelium at day 60 of pregnancy, it was immunolocalized in the luminal and glandular
epithelium of the intercaruncular endometrium by days 120 to 140 of pregnancy (Moffatt et al.
1987, Stephenson et al. 1989a). Ovine US has been also detected in amniotic and allantoic
fluids (Newton et al. 1989, McFarlane et al. 1999), presumably because of transplacental
transport.
Biological function. The biological function of OvUS is still not known with certainty although
evidence suggests it functions primarily to inhibit cell proliferation. Experiments performed by
Ing & Roberts (1989) tested the anti-protease activity of OvUS against trypsin, chymotrypsin,
plasmin, thrombin, elastase and plasminogen activator without significant inhibition. Likewise,
OvUS had non-inhibitory activity towards recombinant human cathepsin D, porcine cathepsin D,
recombinant cathepsin E, cysteine proteinase cathepsin B (Mathialagan & Hansen 1996) or
dipeptidyl proteinase IV (DPPIV) (Liu & Hansen 1995).
42
Mathialagan & Hansen (1996) demonstrated that freshly purified OvUS inhibited pepsin A
and C (chymosin) activity at both pH 2.0 and 4.5, but an excess of 35- and 8-fold molar of OvUS
was required for a 50% inhibition of both aspartic proteinases, respectively. Moreover, the
complex formed by OvUS and pepsin could not be detected electrophoretically in the presence of
Sodium Dodecyl Sulfate (SDS) (Mathialagan & Hansen 1996) whereas, complexes formed by
inhibitory serpins and their partner proteinases (at 1:1 ratio) are so stable that they can be
resolved using the same approach (Potempa et al. 1994). Furthermore, the binding of OvUS to
pepsin seems to be electrostatic since it occurred at pH 8 and low salt concentration where OvUS
has positive charge and pepsin has a negative charge, but not at pH 10.25 where both proteins are
negatively charged (Peltier et al. 2000a).
Some studies have been conducted to determine the possible role of OvUS as a binding or
carrier protein. Ovine US can bind pregnancy-associated glycoproteins (Mathialagan & Hansen
1996), which are inactive aspartic proteinases secreted in large amounts by the ungulate placenta
(Xie et al. 1991, Green et al. 1998). Ovine US can also bind to activin, a member of the
transforming growth factor-β family present in the allantoic fluid (McFarlane et al. 1999).
However, the binding affinity of activin for OvUS is much lower than activin affinity towards
follistatin, suggesting that unlike follistatin, OvUS is not a candidate to neutralize the biological
activity of activin. In addition, OvUS can bind IgM and IgA (Hansen & Newton 1988).
However, the binding of OvUS to immunoglobulins was inhibited by the presence of high salt
concentrations, indicating the ionic nature of the binding (Hansen & Newton 1988).
A plausible function for OvUS is the inhibition of immune cell proliferation during
pregnancy to provide protection for the allogeneically-distinct conceptus (Hansen 1998).
Purified OvUS inhibited lymphocyte proliferation induced by mitogens such as the antigen
43
Candida albicans, Con A, PHA and in the mixed lymphocyte reactions (Segerson et al. 1984,
Hansen et al. 1987b, Stephenson et al. 1989b, Skopets & Hansen 1993, Skopets et al. 1995). In
addition, OvUS reduced the antibody titer in ewes immunized against the T-cell dependent
antigen OVA (Skopets et al. 1995). However, like α1-antitrypsin (Breit et al. 1983), OvUS did
not cause any inhibitory activity against lymphocytes activated by the T and B cell mitogen
PWM (Skopets & Hansen 1993).
Ovine US also inhibits NK cell activity. In vivo experiments using the pregnant mice as a
model, OvUS blocked abortion induced by poly(I)•poly(C), which is a 50-base-pair compound
consisting of double-stranded RNA that activates NK cells, and reduced basal splenocyte NK
cell activity (Liu & Hansen 1993). Additional experiments conducted by Liu & Hansen (1993)
demonstrated that OvUS inhibited NK-like activity in sheep lymphocytes and mouse splenocytes
against K562 and YAC-1 target cells. Moreover, the lytic activity of NK-like cells in sheep
peripheral blood lymphocytes and endometrial epithelium against D-17 cells infected with
bovine herpes virus-1 was inhibited by OvUS (Tekin & Hansen 2002).
Some experiments have been conducted to elucidate the mechanism by which OvUS
inhibits cell proliferation. The protein does not act by inducing cytotoxicity because OvUS did
not cause cytotoxic effects on lymphocytes (Stephenson et al. 1989b, Skopets & Hansen 1993).
Binding of OvUS to lymphocytes is specific, dose dependent and saturable (Liu et al. 1999).
However, it is not known if OvUS binds to a specific cell surface receptor or competes with
other molecules for a receptor binding site. Experiments with Rp-8-Cl-cAMPS, a selective
inhibitor of cAMP-dependent type-I protein kinase (PK) A, indicated that effects of OvUS on
proliferation of PHA-stimulated lymphocytes are not dependent on this kinase (Tekin et al.
2005b). A study performed by Peltier et al. (2000b) showed that OvUS reduced proliferation of
44
phorbol myristol acetate (PMA)-activated lymphocytes, suggesting that OvUS inhibits some
PKC mediated events. In the same study, the protein blocked IL-2 induced proliferation and
reduced expression of CD25 (IL-2Rα chain), but it did not affect the steady state amounts of IL-2
mRNA caused by Con A (Peltier et al. 2000b).
Skopets & Hansen (1993) demonstrated that the antiproliferative effect of OvUS on
lymphocytes was not blocked by addition of neutralizing antibody to transforming growth factor-
β. Thus, OvUS is not inhibiting lymphocyte proliferation by inducing secretion of this
immunosuppressive growth factor.
Porcine Uterine Serpin
The pig is the only species shown to contain two US genes (PoUS-1 and PoUS-2) with
predicted molecular weights of 45,123 and 46,009 respectively (Mathialagan & Hansen 1996).
The PoUS-1 has four potential N-glycosylation sites, whereas PoUS-2 presents only three,
lacking the one at (Asn107
) (Malathy et al. 1990, Mathialagan & Hansen 1996). Analysis of
purified PoUS showed three different peptides. There are two larger forms of the protein with
molecular weights of 50 and 46 kDa and identical NH2-amino terminus, but different in the
number or complexity of glycosylated chains (Baumbach et al. 1989, Murray et al. 1989). There
is also a small glycosylated polypeptide of 40 kDa, distinct from the other two in its NH2-amino
terminus (Baumbach et al. 1986, Murray et al. 1989) that may represent a proteolytic cleavage
product. These three polypeptides seemed to derive from a single 45 kDa precursor and like
OvUS, all of them posses the mannose 6-phosphate lysosome recognition signal (Murray et al.
1989).
Endometrial secretion. The porcine protein is present in uterine flushings of pregnant, pseudo-
pregnant and progesterone treated pigs (Baumbach et al. 1986). In addition, it can be detected in
allantoic fluid of conceptuses at mid pregnancy (Baumbach et al. 1986, Murray et al. 1989). In
45
endometrial tissue, the protein was immunolocalized only in the glandular epithelium of
pregnant pigs at days 45 and 60 (Murray et al. 1989).
During pregnancy, PoUS mRNA expression is low at days 13 and 30, but the expression of
the transcript increases until the last month of gestation, which in the pig lasts 115 days (Malathy
et al. 1990). Moreover, high level of expression of the PoUS transcript was also detected in
endometrial tissues from day 68 pseudo-pregnant gilts (Malathy et al. 1990). In contrast, PoUS
mRNA expression was no detected in either endometrium or liver of non-pregnant or
ovariectomized pigs (Malathy et al. 1990).
Biological function. There is little evidence that PoUS-1 or PoUS-2 are inhibitory serpins.
Purified PoUS did not inhibit proteinases such as trypsin and chymotrypsin (Malathy et al.
1990). Mathialagan & Hansen (1996) tested the ability of PoUS to inhibit porcine pepsin using
uterine flushes from long-term progesterone treated pigs. The inhibitory activity of the protein
was only present in some batches, had very low stability, and the activity was lost completely
when the batch was kept at 4oC for more than one day.
The main role of the PoUS has been related to the iron transport from the mother to the pig
conceptuses by its association with the uteroferrin protein. Porcine uteroferrin is a purple-
colored alkaline phosphatase whose secretion by the glandular epithelium is also stimulated by
progesterone (Chen et al. 1975, Buhi et al. 1982, Baumbach et al. 1986, Murray et al. 1989).
Uteroferrin is transported by the areolae of the placenta into the allantoic fluid (Chen et al. 1975,
Ducsay et al. 1982, Renegar et al. 1982). Uteroferrin carries two bound iron atoms per
polypeptide chain (Baumbach et al. 1986, Roberts & Bazer 1988, Nuttleman & Roberts 1990).
Data from experiments performed by Buhi et al. (1982) in which [59
Fe] labeled uteroferrin was
injected into selected allantoic sacs of days 58 and 67 pregnant gilts showed that the [59
Fe] was
46
released from uteroferrin and quickly associated with the protein transferrin, also present in the
allantoic fluid, and then from transferrin to the fetus. In addition, it was demonstrated that
uteroferrin was more susceptible to proteolysis after iron depletion (Buhi et al. 1982, Nuttleman
& Roberts 1990).
Porcine US binds non-covalently to uteroferrin. This pink color heterodimer has a
molecular weight of 80 kDa and is very stable for long periods of time in the presence of oxygen
whereas purple uteroferrin is not (Baumbach et al. 1989; Roberts & Bazer 1988). Also, the
association between PoUS and uteroferrin promotes the latter’s enzymatic activity in the
heterodimer conformation (Baumbach et al. 1989). Experimentally, the heterodimer can be
dissociated by low pH, antibodies raised against either uteroferrin or all three PoUS peptide
forms, or by dialysis at low salt concentration (Baumbach et al. 1989).
Vallet (1995) suggested that another possible role for PoUS during pregnancy is to prevent
embryonic losses caused by uteroferrin-ascorbic acid induced lipid peroxidation which is
harmful for smaller or less develop conceptuses. Experiments performed on microsomal
membranes from reproductive tissues showed that the uteroferrin-PoUS heterodimer in the
presence of ascorbic acid was less able to provoke lipid peroxidation damage than uteroferrin
itself (Vallet 1995).
Bovine Uterine Serpin
The bovine uterine serpin (BoUS) is also a basic protein and several molecular weight
forms can be detected in uterine secretions from pregnant and progesterone treated cows by
western blotting, with two major bands at 57,000 and 38,000 (Leslie et al. 1990, Leslie &
Hansen 1991). Unlike the porcine and ovine US, the BoUS has only one potential site for
glycosylation (Asn243
), which is conserved with OvUS and PoUS-1 and 2 (Mathialagan &
Hansen 1996).
47
Endometrial secretion. Secretion of BoUS into the uterus is dependent upon progesterone.
Using western blotting, Leslie & Hansen (1991) demonstrated induction of BoUS in uterine
secretions of ovariectomized cows treated with progesterone for 10 to 30 days. The bovine US is
secreted by the endometrial epithelium of the cow. The protein was immunolocalized in the
glandular epithelium of endometrial tissues from day 135 of pregnancy and ovariectomized cows
treated for 30 days with progesterone (Leslie et al. 1990, Leslie & Hansen 1991). However, no
immunostaining was detected in either the glandular or luminal epithelium of endometrial tissues
from cows on days 17, 19, 21 of pregnancy or days 17 and 19 of the estrous cycle (Leslie et al.
1990).
Unlike OvUS, the BoUS mRNA is expressed during estrus, with particular highly
expression in the cranial area of the uterine horns (Bauersachas et al. 2005). During estrous, the
mRNA for BoUS was highly expressed in the superficial uterine glands and more weakly
expressed in the deep glandular epithelium and some dispersed epithelial cells of the
endometrium (Bauersachas et al. 2005). In addition, the BoUS transcript can be detected in the
caruncule, ovary and cotyledon (Khatib et al. 2007).
Biological function. The biological function of BoUS remains unknown. Mathialagan &
Hansen (1996) showed that activity of porcine pepsin was inhibited by crude uterine flushing
obtained from progesterone treated cows. The importance of BoUS for physiologic function is
highlighted by recent experiments by Khatib et al. (2007) indicating that a single nucleotide
polymorphism (A/G) at position 1269 of the BoUS gene was associated with a significant
increase in productive life in cattle populations, where the G allele expression at that position has
been linked to the productive life. Productive life is in large part determined by culling rate and
48
culling is largely for problems with reproductive health or infectious diseases. Thus, BoUS is
likely to play a role in one or more processes controlling reproduction or immune function.
Caprine Uterine Serpin
This US was recently found discovered. Caprine (Cap) US was immunolocalized in the
endometrial glandular epithelium at day 25 of pregnancy, but it was not detected at day 5 of the
estrous cycle (Tekin et al. 2005a). Western blot of uterine fluid from a pregnant goat identified a
major band with a molecular weight of approximately 57,000 and some other low molecular
weight bands (Tekin et al. 2005a). Like the ovine and porcine, the CapUS had two predicted N-
glycosylation sites at positions 222 and 268, where the position 268 is conserved with the other
known US and the 222 position is also conserved in almost all the other US, with the exception
of the BoUS (Tekin et al. 2005a). No study has been performed to identify the possible
biological role of this US.
Evolution and Phylogeny of Uterine Serpins
Krem & Cera (2003) performed a study looking for possible evolutionary markers for
serpins, specifically serine residues at positions 53 and 56 (α1-antitrypsin numbering) of the
shutter region, where the serine 56 is related to the TCN-AGY codon usage dichotomy that is
associated to the protostome-deuterostome division. The authors determined that the OvUS,
BoUS, PoUS-1 and -2 posses a conserved serine (encoded by TCN codons) at position 53 which
is also preserved in all the sepins used for the study. At the position 56 they found that the
PoUS-1 and 2 also posses a conserved serine 56 (AGY codon) also found in almost all the
serpins present in chordates (Krem & Cera 2003). However, OvUS and BoUS were among
some other few serpins where distinct residue was found at position 56, where instead of serine,
alanine was present at this position (Krem & Cera 2003).
49
Uterine serpins have been classified as a highly-diverge group of the large α1-antitrypsin
clade (Irving et al. 2000). According to the results obtained from another phylogenetic study,
OvUS and PoUS-1 and 2 were grouped into a large clade containing serpins with different
functions including α1-antitrypsin, heparin cofactor II, α1-antichymotrypsin, angiotensinogen
where genes coding for these serpins contain three introns at homologous areas in the conserved
part of the coding region (Atchley et al. 2001). A similar study performed only on vertebrate
serpins by Ragg et al. (2001) inferred that OvUS and PoUS-1 and 2 could classify within the α1-
antitrypsin group. However, the authors stated the need for further verification for these results
due to the lack of genomic organization for these three US. In contrast, Irving et al. (2000)
created phylogenetic trees with preexisting alignments of the Pfam database. In this analysis, the
US and the angiotensin like-serpins were grouped as separate clades rather than being included
into the α1-antitrypsin group. A phylogenetic study by Peltier et al. (2000c) also resulted in US
being considered as a separate clade in the serpin superfamily.
The identity of the predicted amino acid sequence of OvUS with the CapUS is 96%, 82%
with the BoUS and 55 and 56% with the PoUS-1 and 2 respectively (Mathialagan & Hansen
1996, Tekin et al. 2005a). Peltier et al. (2000c) estimated that OvUS and BoUS diverged from
the PoUS-1 and 2 about 60 Mya and the PoUS-1 and 2 diverged from each other around 5 Mya.
Computational analysis of US motifs suggested that OvUS and BoUS contain similar amounts of
casein kinase-2 and cAMP phosphorylation sites. In contrast, neither PoUS-1 nor 2 contain
cAMP phosphorylation (Peltier et al. 2000c). The same study also showed that these three
species have similar amounts of phosphorylation sites for tyrosine kinases as well as for N-
myristoylation. However, the PoUS-1 and 2 contain more phosphorylation sites for casein
kinase-2 and PKC and N-linked glycosylation sites than OvUS and BoUS.
50
Synopsis and Objectives
Among the members of the serpin superfamily, the US group is a very intriguing group of
proteins. Based on this literature, there is evidence showing that US are hormonal-regulated
proteins and highly expressed into the uterus during pregnancy. The US group lacks apparently
of anti-proteinase activity and they plausibly diverge in biological function with respect to the
other members of the superfamily. Also, the limited group of species which posses the US gene
suggests the possibility of the evolution of the US gene within mammals for specific functions.
The specific objectives that addressed in this dissertation are
1) To determine the presence of the US gene in species with non-epitheliochorial placenta and
also to examine the evolution of the US gene in mammals.
2) To establish whether the US produced by the sheep (OvUS) inhibits cell proliferation by a)
causing cell death such as apoptosis or necrosis b) altering cell cycle dynamics to cause cell
cycle arrest.
3) To determine which cell cycle related genes are regulated by OvUS.
51
CHAPTER 2
MOLECULAR PHYLOGENY OF UTERINE SERPINS AND ITS RELATIONSHIP TO
EVOLUTION OF PLACENTATION
Introduction
Evolution of placentation in mammals has been dependent upon new uses of existing genes
as well as appearance of new genes arising from gene duplication and selection for sequence
divergence. Knox & Baker (2008) have shown that genes expressed preferentially by the
placenta and decidua early in development tend to be ancient genes while the genes expressed
preferentially by the mature placenta have been formed recently. Examples of such trophoblast
genes are the interferon-τ genes found in ruminants (Roberts et al. 1999) and the primate
chorionic gonadotropins (Maston & Ruvolo 2002).
Another group of uterine genes formed by gene duplication are the uterine serpins (US)
(also called uterine milk proteins). These proteins are members of the serine proteinase inhibitor
(serpin) superfamily and have been identified as secretory products of the endometrial epithelium
of the pregnant sheep, goat, cow and pig (Moffatt et al. 1987, Leslie et al. 1990, Malathy et al.
1990, Tekin et al. 2005a). Uterine serpins have been classified as either a separate clade of the
serpin superfamily (Peltier et al. 2000c) or as a highly-diverge group of the α1-antitrypsin clade
(Irving et al. 2000). Recently, they have been designated as SERPINA14.
The prototypical serpins are competitive inhibitors of serine and cysteine proteinases. The
proteinase binds covalently to reactive center loop (RCL), which is localized on the top of the
serpin and contains a complementary sequence to the active site of the target proteinase. The
scissile bond at the P1-P1’ of the RCL is cleaved and then incorporated as the central strand of
the serpin also known as β-sheet A (Irving et al. 2000, Hungtinton 2006, Whisstock &
Bottomley, 2006). The inactivation of the proteinase is accompanied by an irreversible
52
conformational change of the serpin structure that increases its overall stability (Silverman et al.
2001, Hungtinton 2006, Whisstock & Bottomley 2006).
A few serpins such as the US have evolved biological functions that do not involve
proteinase inhibitory activity. Other examples are the intracellular serpin mammary serine
protease inhibitor (maspin) (Sheng 2006), corticosteroid and thyroxine binding globulins
(Pemberton et al. 1998), pigment epithelium derived factor (PEDF) (Tombran-Tink &
Barnstable 2003) and the heat shock protein 47 (Sauk et al. 2005). Inhibitory serpins are usually
recognized by a consensus sequence in the hinge region which is localized within the RCL of the
serpin (Irving et al. 2000) but the putative hinge region of US is not conserved with inhibitory
serpins (Irving et al. 2000, Tekin et al. 2005a). The ovine (Ov) and bovine (Bo) US exhibit
either no or very weak inhibitory activity towards a range of serine and aspartic proteinases (Ing
& Roberts 1989, Liu & Hansen 1995, Mathialagan & Hansen 1996, Peltier et al. 2000a).
All of the species shown to possess US genes are in the Ruminantia and Suidae orders of
the Laurasiatheria superorder of eutherian mammals and all have epitheliochorial type of
placentation characterized by limited invasiveness and maintenance of epithelial layers in
endometrium and trophoblast. The epitheliochorial placenta has evolved from a more primitive
hemotropic placenta three separate times in evolution – in a subgroup of Laurasiatheria
consisting of cetartiodactyls, pigs, horses, and spiny anteaters, in a species of mole, and in lemurs
(Vogel 2005). The evolutionary pressure for development of an epitheliochorial placenta has
been attributed to increased efficiency of nutrient transport and gas exchange (Leiser et al. 1997),
greater maternal control of nutrient distribution to the conceptus (Mess & Carter 2007) and an
altered immunological relationship between mother and conceptus (Moffett & Loke 2006).
53
The objectives of this study were to identify novel US genes in other species within and
outside of the Laurasiatheria superorder to determine whether the presence of the US gene is
restricted to species with epitheliochorial placentation and to evaluate whether US gene has been
subject to positive selection.
Materials and Methods
Data Base Queries to Identify Uterine Serpin Genes
The nucleotide and protein sequences for OvUS were used as query sequences to perform a
blastn search in the nucleotide collection (nr/nt) database of the National Center for
Biotechnology Information (NCBI) website to identify known and novel uterine serpin genes.
The Genbank accession number for the OvUS nucleotide sequence was NM_001009304.1 and
the accession number for the protein sequence was NP_001009304.1. Subsequently, a genomic
blast (blastn) of completed genomic database sequences in the NCBI was performed to identify
sequences that have similarities with the OvUS query sequence. Species examined were human
(Homo sapiens), rhesus macaque (Macaca mulata), chimpanzee (Pan troglodytes), dog (Canis
lupus familiaris), cow (Bos taurus) mouse (Mus musculus) rat (Rattus norvegicus), opossum
(Monodelphis domestica) and duck-billed platypus (Ornithorhynchus anatinus), as well as other
vertebrates like chicken (Gallus gallus), zebra fish (Danio rerio) and puffer fish (Tetraodon
nigroviridis). Additionally, the unfinished whole shotgun genomic sequences for the horse
(Equus cabalus - previously unfinished) and cat (Felis catus) were included in this search.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Uterine endometrial tissues were collected from pregnant mares at days 21 and 59 of
gestation, a pregnant bitch at day 60 of gestation, a pregnant queen at day 60 of gestation, as well
as from the intercaruncular area from ovariectomized ewes treated with 100 mg/ml of
progesterone for 60 days (Padua et al. 2005). Dog liver tissue and ovaries were also tested.
54
Total RNA was isolated after homogenization with the TRI reagent (Sigma-Aldrich, St Louis,
MO) according to the manufacturer’s instructions. RNA purity and concentration were
determined spectrophotometrically.
The RT-PCR was performed using the SuperScript One Step RT-PCR kit with Platinum
Taq (Invitrogen, Carlsbad, CA). cDNA for OvUS, equine (Eq), Canine (Can) and Feline (Fe)
US were amplified from total RNA using primer sets for sheep (forward ACA GAT GCT TTA
CAG CCG GTC AGA; reverse TGA ACT TAA CAA CCA CCG GGA CCT), horse (forward
GCT GCA GAA ATG TCC CAC AGG AAA; reverse AGA GGA AAT CCC TGT GCT TCA
GGT) dog (forward ACC CAG TCT CGT CAT GGG AAG TTT; reverse TCA CGT CAT ACA
TCG CCT GTG TGT) and cat (forward TAC GAG ATC CAC AAC GCG CAC TAA; reverse
AAG TCA GTC ATC TGG GCC TTC ACA). To verify that PCR products were amplified from
RNA only, the SuperScript reverse transcriptase/Platinum Taq mix was omitted from control
reactions and an equivalent concentration of Taq DNA polymerase (Invitrogen) was added.
cDNA synthesis and pre-denaturation reactions were 1 cycle of 50oC for 30 min and 94
oC for 2
min respectively. PCR amplification reactions were performed in 40 cycles of 94oC for 15 sec
for denaturation, 50oC for 30 sec for annealing and 72
oC for 1 min extension. A final extension
cycle was performed at 72oC for 5 min.
Identification of cDNA for Equine Uterine Serpin Gene
The complete sequence of the EqUS gene was obtained by primer walking and the rapid
amplification of cDNAs ends (RACE) to obtain the 5’ and 3’ ends. The RACE reactions were
performed using RACE core system kits (Takara Bio Inc., Japan), following the manufacturer’s
recommendations. The set of primers used to obtain the full length of the 5’ end as well as the
oligo dT-3sites adaptor primer used to obtain the 3’end were gene specific primers and are
55
Table 2-1. Primers used in RT-PCR procedure to obtain the full length coding sequence of the
equine uterine serpin gene. Amplicons are designated based on the Genbank
Accession Number of the sequences obtained from the RT-PCR. The full length was
obtained by overlapping seven sequences as shown in the diagram.
Amplicon Primer Sequence (5’ - 3’)
EU 810388 Fw GCTGCAATGTCCCACAGGAAA; Rv AGAGGAAATCCCTGTGCTTCAGGT
EU 810389 Fw CTCTGGATTGCTGCAGAAATG; Rv CTACTCAGCTATGGGGTTGAA
EU 810390 Fw CTCTGGATTGCTGCAGAAATG; Rv CTACTCAGCTATGGGGTTGAA
EU 810391 Fw TCGACCTCCAAAGAATCAGAGCGT; Rv AGGACACGTTTCCAGTGTAAGGCA
EU 810392 Fw AAACGTGTCCTTAGTCCTCGTGCT; Rv TTGAAGACTTGGCCCACAAAGAGC
EU 810393
(5’ RACE)
Rv /Phos/TGTCCCTAAAGGAGA
Fw TGTTCGAGGCTCTGTCAGTTGAGT; Rv TGTGCTTCAGGTGCCTCTGTCTAT
Fw GCTGCAGAAATGTCCCACAGGAAA; Rv ACTCAACTGACAGAGCCTCGAACA
EU 810394 (3’RACE)
Fw AAACGTGTCCTTAGTCCTCGTGCT
EU810388
EU810389
EU810390EU810391
EU810392
EU810393
EU810394
EqUS gene (1380 bp)5’ 3’
850 1380
819 1262
1 215
33322
40033
325 829 1245954
EU810388
EU810389
EU810390EU810391
EU810392
EU810393
EU810394
EqUS gene (1380 bp)5’ 3’
850 1380
819 1262
1 215
33322
40033
325 829 1245954
56
shown in Table 2-1. Amplicons were electrophoresed on 3% (w/v) agarose gel containing 1
µg/ml ethidium bromide in Tris acetate-EDTA buffer (40 mM Tris-acetate, 2 mM EDTA pH
8.5), visualized on a ultraviolet transluminator and photographed with a Canon G-7 (Canon Inc,
Japan) power shot digital camera using the Digi-Doc-ITTM
imaging system (UVP, LLC, Upland,
CA). Amplicons were purified from the corresponding agarose gel bands using the QIAquick®
gel extraction kit (Qiagen Inc, Valencia, CA) according to the manufacturer’s instructions. DNA
concentration was determined spectrophotometrically and purity was assessed by electrophoresis
as previously described.
Sequencing of Amplicons
DNA amplicons were sequenced in both directions at the University of Florida DNA
Sequencing Core Laboratory using the ABI Prism® 3130 genetic analyzer (Applied Biosciences,
Foster City, CA). Briefly, ABI prism BigDye Terminators v.1.1 cycle sequencing reactions were
assembled in a total volume of 20 µl containing 500 ng DNA, 10 pmols primer, 4µl of BigDyer
terminator, and 5% (v/v) dimethyl sulfoxide. Sequencing reactions were performed on an ABI
GeneAmp 9700 thermal cycler (Applied Biosystems) with an initial denaturation at 95oC for 10
min and followed by 45 cycles of denaturation at 95oC for 30 sec, annealing at 55
oC for 20 sec
and extension at 60oC for 4 min. The excess of dye-labeled terminators were removed by using
MultoScreen® 96-well filtration system (Millipore, Bedford, MA). The purified extension
products were dried in SpeedVac® (ThermoSavant, Holbrook, NY) and then suspended in Hi-di
formamide. Sequencing reactions were performed using POP-7 sieving matrix on 50-cm
capillaries in the ABI Prism® 3130 Genetic Analyzer and were analyzed by ABI Sequencing
Analysis software v. 5.2 and KB Basecaller.
Analyses of the deduced amino acid sequence were conducted using the program Scan
Prosite at the ExPASy Molecular Biology Server (http://ca.expasy.org). The predicted signal
57
peptide cleavage sites were obtained from SignalP 3.0 (Bendtsen et al. 2004) and predicted
glycosylation sites were obtained from NetNGlyc 1.0 both from the ExPASy Molecular Biology
Server.
Detection of Equine Uterine Serpin by Western Blotting
Ovariectomized pony mares were treated daily with progesterone (150 mg/ml), estradiol
benzoate (10 mg/ml), progesterone plus estradiol benzoate or vehicle as a control. After 28 days,
uterine fluids were collected by flushing the uterus with 0.9% (w/v) NaCl (Hansen et al. 1985).
Samples were concentrated using Centricon plus-20 devices (Millipore Corporation, Billerica,
MA). Aliquots of 20 µl of concentrated mare samples were separated under reducing conditions
using one-dimensional discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis
at 4-15% (w/v) polyacrylamide, Tris-HCl gels (Bio-Rad, Hercules, CA). Aliquants of 0.5 µg of
protein from uterine fluid of an ovariectomized ewe treated with 100 mg/ml of progesterone for
60 days and from the control serpin ovalbumin (OVA) were used as positive and negative
control, respectively. Western blotting was performed as described elsewhere (Padua et al.
2005) with minor modifications. Briefly, the monoclonal anti-OvUS (HL-218; Leslie et al.
1990) and IgG1 (MOPC21) (Sigma-Aldrich) (1:32,000 dilution) were used as a primary and
control antibodies, respectively. The second antibody used was ECLTM
anti-mouse IgG
peroxidase-linked; 1:25,000 dilution (Amersham-GE Healthcare Bio-Sciences Corp, Piscataway,
NJ) and the ECL plus western blotting detection kit (Amersham) was used as a detection system
according to the vendor’s instructions.
Amino Acid Sequence Alignments and Analysis of Phylogenetic Tree
ClustalW (Chenna et al. 2003) was used to obtain the amino acid alignments which were
then prepared for publication using the Boxshade multiple alignments designer program version
3.21 (http://www.ch.embnet.org).
58
The phylogenetic tree that includes corticosteroid binding globulin (CBG) was constructed
using the Neighbor Joining (NJ) method (Saitou & Nei 1987) of the Molecular Evolutionary
Genetics Analysis (MEGA4) software, version 4.0 (Tamura et al. 2007). The NJ tree was based
on the distance calculation under the Jones-Taylor-Thornton (JTT) matrix substitution model
(Jones et al. 1992) after removing position containing gaps (complete deletion option).
The NJ method (Saitou & Nei 1987) was also used to construct the phylogenetic tree for
the US group of proteins based on a gamma shape parameter (α) corrected JTT distance matrix
(Jones et al. 1992). The plotted tree was obtained by using the drawgram software of PHYLIP
(Phylogeny Inference Package) version 3.5c (Felsenstein 1989, 1993). The gamma shape
parameter and amino acid frequencies were estimated from the data using the Tree-Puzzle
software by maximum likelihood analysis for amino acids (Strimmer & von Haeseler 1996,
Schmidt et al. 2002) and the reliability of the trees was estimated by the bootstrap test with 1,000
repetitions (Felsenstein 1985).
Analysis of Ratio of Non-Synonymous and Synonymous Substitutions
The ratio of non-synonymous and synonymous substitutions (dN/dS; termed ω) of codons
was used to determine whether the pressure of selection induces purifying or positive selection at
specific areas of the sequence (Yang & Nielsen 2002). Sites with ω values significantly higher
or lower than 1 are an indication of positive (Darwinian) or purifying selection, respectively.
The codon-based substitution model in the CODEML program of the phylogenetic analysis by
maximum likelihood (PAML, version 4.0b) software package (Yang 2007) was used to identify
the effect of selective pressures on the uterine serpin genes. Aligned sequences were tested using
different models of variable dN/dS ratios among sites which includes M0 (null model), M1
(nearly neutral, where ω>1 is not allowed), M2 (positive selection, ω>1 is allowed), M7 (ω>1 is
59
not allowed, beta distribution) and M8 (ω>1 is allowed, beta distribution) (Yang et al. 2000,
Bielawski & Yang 2003, Wong et al. 2004). The likelihood ratio test (LRT) was obtained by
twice the difference in log likelihood (2L) from two different models and the significance of
the test was estimated by using the 2 distribution with degrees of freedom calculated from the
estimated parameters (Anisimova et al. 2001, Yang & Nielsen 2002). The models M1 and M7
were tested against models M2 and M8, respectively. Additionally, the Selecton server (Server
for the identification of site-specific positive and purifying selection, version 2.4) (Stern et al.
2007) was used to identify ω at each codon site based on an empirical Bayesian method.
Aligned coding sequences in FASTA format for uterine serpins were tested with the M8 model,
(extra category ωs ≥ 1, beta distribution and positive selection is allowed). The significance of ω
scores were also tested by using LRT test which compares the positive selection model (M8) and
the null M8a model (extra category ωs set to 1); allowing only for purifying and neutral selection,
respectively.
Results
Identification of Coding Sequences of Known and New Uterine Serpins Using Blastn
Uterine serpin sequences have been previously described for goat (Capra hircus), cow (Bos
taurus) and pig (Sus scrofa). Results from the blastn search in the nucleotide collection database
of the NCBI identified an additional uterine serpin coding sequence that has been submitted to
Genbank for water buffalo (Bubalus bubalis, Genbank: DQ 661648.1). This is a species with
epitheliochorial placenta that is closely related to the cow. The blast search also identified a
gene in the dog (Canis lupus familiaris, Genbank: XM_850115.1) with similarity to the query
sequence, OvUS. The dog is a member of Laurasiatheria but, unlike other species with uterine
serpin genes, the dog has an endotheriochorial placenta. Currently, XM_850115.1 is annotated
as one of the two CBG genes in the dog. Phylogenetic analysis shows, however, that while the
60
other predicted dog CBG (Genbank: XP_547960.2) is clustered together with CBG proteins
present in other species such as human, chimpanzee, squirrel and rhesus monkey, pig, sheep,
mouse and hamster, XP_855208.1 clusters with the US (Figure 2-1).
A genomic blastn search of complete genomic sequences identified uterine serpin genes for
the cow and dog. No US genes were identified in the order Anthropoidea, represented by
human, rhesus monkey and chimpanzee, or in Rodentia, represented by the mouse and rat.
Likewise, the US gene was no identified either in the opossum, a species with a primitive
epitheliochorial placentation within the Marsupialia order, in the duck-billed platypus, an egg-
laying mammal that belongs to the Monotremata order, or in any of the other vertebrates with
complete genome sequences (chicken, puffer and zebra fish).
A genomic blast was also performed using OvUS as query for the whole shotgun genomic
sequence of the horse, which was incomplete at the time of analysis. There were four stretches
of nucleotides of 664, 232, 72 and 86 nucleotides in length (Genbank: gbAAWR02019930) that
matched with the coding sequence of OvUS (68,75, 86 and 79% identity, respectively). These
sequences were all localized on chromosome 24 and are flanked at the 5’ side by SERPINA11.
A genomic blast search of the whole shotgun genomic sequence of the cat using OvUS as
the query identified one stretch of 198 nucleotides in length (Genbank: gbACBE01215383.1)
that matched with the coding sequence of OvUS with 77% of identity. However, using CanUS
as the query sequence identified four stretches of nucleotides of 525, 273, 146 and 199
nucleotides in length (same accession number) that matched the coding sequence of CanUS with
82, 82, 80 and 89% identity, respectively.
61
Figure 2-1. Identification of an incorrectly annotated dog corticosteroid binding globulin (CBG)
as an uterine serpin (US). The sequence incorrectly annotated as CBG [Genbank:
XP_855208.1] clusters with the US while another dog CBG [Genbank:
XP_547960.2] clusters with CBG in other species. The Neighbor Joining tree was
constructed using the Molecular Evolutionary Genetics Analysis (MEGA4)
software. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is shown next to the branches. The
tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary
distances were computed using the Jones-Taylor-Thornton (JTT) matrix-based
method and are in the units of the number of amino acid substitutions per site. All
positions containing gaps and missing data were eliminated from the dataset
(Complete deletion option).
CBG-Human (AAB59523.1)
CBG-Chimpanzee (XP 510143.2)
CBG-Rhezus monkey (XP 001098128.1)
CBG-Squirrel monkey (AAC60614.1)
CBG-Dog (XP 547960.2)
CBG-Pig (AAG45431.1)
CBG-Sheep (CAA52000.1)
CBG-Mouse (NP 031644.1)
CBG-Hamster (AAA37065.1)
CanUS (XP 855208.1)
PoUS-1 (AAA31137.1)
PoUS-2 (NP 999010.1)
OvUS (NP 001009304.1)
CapUS (DAA05634.1)
BoUS (NP 777222.1)
BuUS (ABG56442.1)
100
100
99
99
100
99
89
100
100
99
95
100
100
0.1
62
Uterine Serpin Gene Organization in the Bovine and Canine
The BoUS gene is localized in chromosome 21 for the cow and is organized in 6 exons,
with two small untranslated regions (UTR) contained in exons 1 and 6 respectively. The CanUS
is localized in chromosome 8 and also contains 6 exons but with no UTR present. The BoUS
gene clusters with SERPINA1, SERPINA11 and SERPINA12. The CanUS gene clusters with
SERPINA1, SERPINA9, SERPINA11, and SERPINA12. There are variations in the length of
exons and introns between BoUS and CanUS gene as indicated in Table 2-2. However, the size
of exon 3 is identical in both species.
Characteristics of CanUS and Expression in Tissues
The gene for CanUS (Genbank: XM_850115) is 1416 bp in length that encodes a
polypeptide of 471 amino acids with a theoretical isoelectric point of 9.43 and predicted
molecular mass of 54.25 kDa. The CanUS protein sequence lacks a signal peptide suggesting
the intracellular localization of the protein. Using XM_850115 primers were designed for RT-
PCR. Amplicons were obtained from endometrium of a bitch at 60 days of gestation, but there
was no expression in the liver (Figure 2-2). In addition, transcripts for CanUS were identified in
the ovary by RT-PCR (data not shown).
Uterine Expression and Amino Acid Sequence of EqUS
Using primers designed from the EqUS sequences identified in the blast search, expression
of EqUS in endometruim from a pregnant mare at Day 21 (data not shown) and Day 59 of
gestation was identified (Figure 2-3A). The complete sequence of EqUS was obtained by
performing RT-PCR, 5’RACE and 3’ RACE using endometrial cDNA of a mare at Day 59 of
gestation. In this manner, seven overlapping sequences were obtained (Genbank: EU810388,
EU810389, EU810390, EU810391, EU810392, EU810393, EU810394) with a total length of
1380 bp that includes 9 and 108 nucleotides for the 5’ and 3’ UTR of the gene, respectively. The
63
Table 2-2. Exon and intron sizes for the uterine serpin gene of the cow and dog.
Cow exon1
(UTR) exon2 intron2 exon3 intron3 exon4 intron4 exon5
exon6
(UTR) Total bp
16 637 3204 268 1808 148 2205 288 65 8639
Dog exon1 intron1 exon2 intron2 exon3 intron3 exon4 intron4 exon5 Total bp
210 1863 148 1580 268 876 666 5034 124 10768
64
Figure 2-2. Representative electrophoretogram of amplicons obtained by reverse transcriptase-
polymerase chain reaction (RT-PCR) of RNA fron canine tissue and canine uterine
serpin primers. RNA was isolated from dog liver or endometruim from a pregnant
bitch at Day 60 of gestation. Control reactions excluded reverse transcriptase (w/o
RT). Arrows on the left represent size of standards while the arrow on the right
shows the expected amplicon size.
Pre
gn
an
t b
itc
h w
/o R
T
Pre
gn
an
t b
itc
h w
/o R
T
DE
PC
-wa
ter
Do
g l
ive
r –w
/o R
T
DN
A l
ad
de
r
(1 K
b)
Pre
gn
an
t b
itc
h
Do
g l
ive
r
Do
g l
ive
r –w
/o R
T
Do
g l
ive
r
Pre
gn
an
t b
itc
h
387 bp396 bp
344 bp
Pre
gn
an
t b
itc
h w
/o R
T
Pre
gn
an
t b
itc
h w
/o R
T
DE
PC
-wa
ter
Do
g l
ive
r –w
/o R
T
DN
A l
ad
de
r
(1 K
b)
Pre
gn
an
t b
itc
h
Do
g l
ive
r
Do
g l
ive
r –w
/o R
T
Do
g l
ive
r
Pre
gn
an
t b
itc
h
387 bp396 bp
344 bp
65
Figure 2-3. Expression and secretion of equine uterine serpin. Panel A represents an
electrophoretogram of amplicons obtained by reverse transcriptase-polymearse
chain reaction (RT-PCR) of either RNA from endometruim of an ovariectomized
ewe treated with progesterone for 60 days (P4-treated ewe) or RNA from
endometruim of a pregnant mare at Day 59 of gestation. Control reactions excluded
reverse transcriptase (w/o RT). Arrows show a size of the standard and expected
size of the amplicons for the sheep and mare respectively. Panel B represents
results of a western blotting experiment using antibody to OvUS and either uterine
fluid from an ovariectomized ewe treated with progesterone for 60 days, uterine
flushings from ovariectomized mares treated for xx days with either vehicle,
progesterone (P4), estradiol benzoate (E), or the combination (P4+E), or
endometrial tissue from a pregnant mare at 59 days of gestation. As negative
control, the serpin ovalbumin (OVA) was also subjected to western blotting.
500 bp500 bp467 bp467 bp436 bp436 bp
AD
EP
C-w
ate
r
Ew
e (
P4)
DN
A lad
der
(1 K
b)
Ew
e (
P4)
Pre
gn
an
t m
are
Pre
gn
an
t m
are
Ew
e (
P4)
w/o
RT
Pre
gn
an
t m
are
w/o
RT
500 bp500 bp467 bp467 bp436 bp436 bp
AD
EP
C-w
ate
r
Ew
e (
P4)
DN
A lad
der
(1 K
b)
Ew
e (
P4)
Pre
gn
an
t m
are
Pre
gn
an
t m
are
Ew
e (
P4)
w/o
RT
Pre
gn
an
t m
are
w/o
RT
Pre
gn
an
t m
are
ST
D
OV
A
Ew
e (
P4)
Mare
(veh
icle
)
Mare
(E
)
Mare
(P
4+
E)
Mare
1 (
P4)
Mare
2 (
P4)
Uterine fluid Uterine flushings Endometruim
250 kD
150 kD
20 kD
10 kD
37 kD
50 kD
75 kD
100 kD
B
Pre
gn
an
t m
are
ST
D
OV
A
Ew
e (
P4)
Mare
(veh
icle
)
Mare
(E
)
Mare
(P
4+
E)
Mare
1 (
P4)
Mare
2 (
P4)
Uterine fluid Uterine flushings Endometruim
250 kD
150 kD
20 kD
10 kD
37 kD
50 kD
75 kD
100 kD
B
66
sequence was submitted to NCBI GenBank as TPA (Third Party Annotation, GenBank accession
number: BK 006618). The complete coding sequence of the EqUS was 1263 bp which is 100%
identical to genomic sequence.
The deduced amino acid sequence is shown in Figure 2-4. The gene encodes a polypeptide
of 421 amino acids with a theoretical isoelectric point of 9.49 and predicted molecular mass of
48.79 kDa. Analysis of the EqUS protein sequence showed a predicted signal peptide, most
likely between positions (Cys25
) and (Glu26
) and two potential glycosylated sites at (Asn212
) and
(Asn246
).
Endometrial Secretion of EqUS
Uterine flushings from ovariectomized mares treated with steroids were examined by
western blotting using an antibody to OvUS to determine if EqUS was secreted into uterine
lumen and, if so, whether as for other uterine serpins secretion was under control of
progesterone. The western blot is shown in Figure 2-3B. Immunoreactive protein bands were
obtained from uterine flushes from ovariectomized mares treated with estradiol benzoate,
progesterone or progesterone and estrogen. There was an absence of immunoreactive protein in
flushes of ovariectomized mares treated with vehicle. The molecular weight of the
immunoreactive EqUS was similar to OvUS identified by western blotting of uterine fluid from a
progesterone treated ewe (55,000-57,000). The immunoreactive EqUS is larger than the
predicted molecular mass of 48.79 kDa, suggesting the protein is glycosylated. Multiple
immunoreactive bands were also seen for supernatant from lysed endometrial tissue of a mare at
Day 59 of gestation. The variety of lower molecular weight bands probably represent proteolytic
products as has been described for OvUS (Moffatt et al. 1987, Leslie et al. 1990, Peltier et al.
2000a, Leslie & Hansen 1991). No immunoreactive bands were identified for the control serpin
OVA (Figure 3B) or when IgG replaced the primary antibody (data not shown).
67
Lack of Expression of the Uterine Serpin Gene in the Pregnant Cat
RT-PCR experiments with primers designed for a putative FeUS sequence identify by
blastn against CanUS (525 bp in length) failed to yield an amplicon from cDNA obtained from
pregnant endometruim of a pregnant queen at Day 60 of gestation (data not shown). Moreover,
stop codons were identified after translation of the deduced amino acid sequences for at least two
of the four stretches of nucleotides identified by blastn. Depending upon the frame used for
codon identification, the 525 bp sequence had 1-10 stop codons, the 272 bp sequence had 1-6
stop codons, the 199 bp sequence had from 0-3 stop codons, and the 146 bp sequence had from
0-4 stop codons. Similarly, the 198 bp sequence identified in cat by genomic blast search using
OvUS had 1-4 stop codons.
Amino Acid Sequence Conservation
The ClustalW amino acid sequence alignments of uterine serpins are shown in Figure 2-4.
There is a 9-amino acid insert present in OvUS and CapUS (KINLKHLLP) that is not present in
uterine serpins from other species. Likewise, the water buffalo US (BuUS) contains a 13-amino
acid insert (MNAKEVPVVVKVP) that is highly conserved with 39-amino acid insert for BoUS.
There is also a 3-amino acid insert in the same relative position for the PoUS-1, PoUS-2, CanUS
and EqUS. This insert is conserved between the PoUS-1 and -2, but the insert for CanUS and
EqUS is not conserved with any other species.
The CanUS sequence is unique among uterine serpins in that there are two start codons
upstream from the start codons for other uterine serpins (Figure 2-4) so that the CanUS
polypeptide could be as much as 48-amino acid longer than other serpin sequences and not
contain a signal peptide.
There is a KEVPVVVK motif located near the putative P1-P1’ site originally described for
the BoUS that has been postulated to be a pepstatin-like domain (Mathialagan & Hansen 1996).
68
Figure 2-4. Amino acid sequence alignment of the uterine serpins using the ClustalW
algorithm. Sites of conservation are shown in shaded columns using the Boxshade
software. Black-shaded columns show identical amino acids and grey-shaded
columns represent similar amino acids. Distinctive amino acid motifs limited to a
subset of uterine serpins are marked with red rectangles. Start codons are shown by
red arrows.
|AAA31137.1-PoUS-1| 1 ------------------------------------------------MSHGKMPLVLSLVLILCGLFNS
|NP_999010.1-PoUS-2| 1 ------------------------------------------------MSHGKMPLVLSLVLILCGLFNS
|NP_001009304.1 OvUS| 1 ------------------------------------------------MSHRRMQLALSLVFILCGLFNS
|DAA05634.1-CapUS| 1 ------------------------------------------------MSHRRMQLALSLVFILCGLFNS
|NP_777222.1-BoUS| 1 ------------------------------------------------MSHGRMNLALSLVFILCGLFNS
|ABG56442.1-BuUS| 1 ------------------------------------------------MSHGRMNLALSLVLILCGLLNS
|XP_855208.1-CanUS| 1 MEKGWERLSKQGPGKEADDISHKASSSDMRVRWEARTKEEETPWIAAEMSHRKMHLALSLALILCGLFNS
|BK006618-EqUS| 1 ------------------------------------------------MSHRKMHLALSLVLTLCGLLNS
CONSENSUS 1 MSH kMqLaLSLvliLCGLfNS |AAA31137.1-PoUS-1| 23 ISCEKQQTSPKTITPVSFKRIAALSQKMEANYKAFAQELFKTLLIEDPRKNMIFSPVSISISLATLSLGL
|NP_999010.1-PoUS-2| 23 ISCEKQQTSPKTITPVSFKRIAALSQKMEANYKAFAQELFKTLLIEDPRKNMIFSPVSISISLATLSLGL
|NP_001009304.1-OvUS| 23 IFCEKQQHSQQHANLVLLKKISAFSQKMEAHPKAFAQELFKALIAENPKKNIIFSPAAMTITLATLSLGI
|DAA05634.1-CapUS| 23 IFSEKQQHSQQHANLVLLKKISAFSQKMEAHPKAFAQELFKALIAEDPKKNIIFSPVAMTITLATLSLGI
|NP_777222.1-BoUS| 23 IFCEKQQHSQKHMNLVLLKKISALSQKMEAHPKDFAQELFKALIIEDPRKNIIFSPMAMTTTLATLSLGI
|ABG56442.1-BuUS| 23 IFCEKQQHSQKHVNLVLLKKISALSQKMEAHPKDFAQELFKALIIEAPRKNIIFSPMAMTTTLATLSLGI
|XP_855208.1-CanUS| 71 IFCQVKQRSKWSIYPVSSWEVSPVHHKIEAGNKAFAHKLFKTLLMEHPRKNIIFSPLSISASFAMLSLGT
|BK006618-EqUS| 23 IFCETQPNPKRDPHPVSSRKTSPPSHETDTDHEAFAYKLFEALSVEYHRKNLIFSPESISTALAMLSLGT
CONSENSUS 71 Ifcekqqhsqkhin V lkkisalsqkmeahpkaFAqeLFkaLiiEdprKNiIFSPv isitlAtLSLGi
|AAA31137.1-PoUS-1| 160 KMDIQMIDFKDKEKTKKAINQFVADKIDKKAKNLITHLDPQTLLCLVNYIFFKGILERAFQTNLTKKEDF
|NP_999010.1-PoUS-2| 163 KVDIQMIDFKDKEKTKKAINQFVADKIDKKAKNLITHLDPQTLLCLVNYVFFKGILERAFQTNLTKKEDF
|NP_001009304.1-OvUS| 160 KMDIQMIDFSDTEKAKKAISHHVAEKTHTKIRDLITDLNPETILCLVNHIFFKGILKRAFQPNLTQKEDF
|DAA05634.1-CapUS| 160 KMDIQMIDFSDTEKAKKAISHHVAEKTHTKITDLITDLNPETILCLVNHIFFKGILKRAFQPNLTQKEDF
|NP_777222.1-BoUS| 160 GMDIQMIDFTDIEKAKKTISHHVAEKTHTKITNLITDLNPETILCLVNHIFFKGILKRAFQPKLTQKEVF
|ABG56442.1-BuUS| 160 GMDIQMIDFTDTEKARKTISHHVAEKTHTKISDLITDLNPETILCLVNHVSLKGILKRAFQPELTQKEDF
|XP_855208.1-CanUS| 211 DVRARMIDFRDVVKTKKQINHFVAEKTHKRTKELITSLNPHTFLFLVDYVFFKGTWEMAFHSNLMHKEDF
|BK006618-EqUS| 163 EVEAQVADFRHRGIAKEQINQFVAQRLAHRIEEVVTSLHPHTFLFLLNYIFFKGVWEVAFQTRFTQKENF
CONSENSUS 211 kmdiqmiDF d ekakkaI h VAekthtkikdliTdLnPeTiLcLvn iffKGil rAFqpnltqKEdF
|AAA31137.1-PoUS-1| 93 RSATRTNAIDVLDVALKNLAVMLMAQAPTALLEIVHELVN-RTAKHQDIL-IDR-TEMNQMFLKEIDRYI
|NP_999010.1-PoUS-2| 93 RSATRTNAIDVLERDLRNLRVWDKHQALQHLVEMLHELEKKKQLKHKDIFFIDRNKKMNQMFLKEIDRVY
|NP_001009304.1-OvUS| 93 KSTMSTNHPEDLELELK---LLDAHKCLHHLVHLGRELVKQKQLRHQDILFLNSKMMANQMLLHQIRKLQ
|DAA05634.1-CapUS| 93 KSTMSTNHPEDLELELK---LLDAHKCLHHLLHLGRELVKQKQLKHQDILFLNSKMMANQMLLHQISKLQ
|NP_777222.1-BoUS| 93 KSTMRTHHPEDLKLEPK---LLDVHKYLQPLVHVGRELVKQKVLKHQHILFINRKMMVNQMLLQQISKLQ
|ABG56442.1-BuUS| 93 KSTMNAHHPEDLKLEPK---LLDVHKYLQTLVHVEHELVKQKLLKHQHILFINSKMVVNQMLLQQIGKLQ
|XP_855208.1-CanUS| 141 RSTTLTNLLEGLGFDLKVIKVWDVHHGFQSVIQMLKQLNRAGHLMHRDMLFIDSNRKINSPFLWDTQAMY
|BK006618-EqUS| 93 RSTTLSNLVAGLGFDLQRIRAWEVHTSFQRLVQTLNELDRQRHLKHRDFLFIDINRRIKPKFLQETERLY
CONSENSUS 141 rSt tnhpedLdlelk l lldvhk lqhlvhivheLvkqk lkHqdilfi skmmmnqm Lkqi klq
|AAA31137.1-PoUS-1| 1 ------------------------------------------------MSHGKMPLVLSLVLILCGLFNS
|NP_999010.1-PoUS-2| 1 ------------------------------------------------MSHGKMPLVLSLVLILCGLFNS
|NP_001009304.1 OvUS| 1 ------------------------------------------------MSHRRMQLALSLVFILCGLFNS
|DAA05634.1-CapUS| 1 ------------------------------------------------MSHRRMQLALSLVFILCGLFNS
|NP_777222.1-BoUS| 1 ------------------------------------------------MSHGRMNLALSLVFILCGLFNS
|ABG56442.1-BuUS| 1 ------------------------------------------------MSHGRMNLALSLVLILCGLLNS
|XP_855208.1-CanUS| 1 MEKGWERLSKQGPGKEADDISHKASSSDMRVRWEARTKEEETPWIAAEMSHRKMHLALSLALILCGLFNS
|BK006618-EqUS| 1 ------------------------------------------------MSHRKMHLALSLVLTLCGLLNS
CONSENSUS 1 MSH kMqLaLSLvliLCGLfNS |AAA31137.1-PoUS-1| 23 ISCEKQQTSPKTITPVSFKRIAALSQKMEANYKAFAQELFKTLLIEDPRKNMIFSPVSISISLATLSLGL
|NP_999010.1-PoUS-2| 23 ISCEKQQTSPKTITPVSFKRIAALSQKMEANYKAFAQELFKTLLIEDPRKNMIFSPVSISISLATLSLGL
|NP_001009304.1-OvUS| 23 IFCEKQQHSQQHANLVLLKKISAFSQKMEAHPKAFAQELFKALIAENPKKNIIFSPAAMTITLATLSLGI
|DAA05634.1-CapUS| 23 IFSEKQQHSQQHANLVLLKKISAFSQKMEAHPKAFAQELFKALIAEDPKKNIIFSPVAMTITLATLSLGI
|NP_777222.1-BoUS| 23 IFCEKQQHSQKHMNLVLLKKISALSQKMEAHPKDFAQELFKALIIEDPRKNIIFSPMAMTTTLATLSLGI
|ABG56442.1-BuUS| 23 IFCEKQQHSQKHVNLVLLKKISALSQKMEAHPKDFAQELFKALIIEAPRKNIIFSPMAMTTTLATLSLGI
|XP_855208.1-CanUS| 71 IFCQVKQRSKWSIYPVSSWEVSPVHHKIEAGNKAFAHKLFKTLLMEHPRKNIIFSPLSISASFAMLSLGT
|BK006618-EqUS| 23 IFCETQPNPKRDPHPVSSRKTSPPSHETDTDHEAFAYKLFEALSVEYHRKNLIFSPESISTALAMLSLGT
CONSENSUS 71 Ifcekqqhsqkhin V lkkisalsqkmeahpkaFAqeLFkaLiiEdprKNiIFSPv isitlAtLSLGi
|AAA31137.1-PoUS-1| 160 KMDIQMIDFKDKEKTKKAINQFVADKIDKKAKNLITHLDPQTLLCLVNYIFFKGILERAFQTNLTKKEDF
|NP_999010.1-PoUS-2| 163 KVDIQMIDFKDKEKTKKAINQFVADKIDKKAKNLITHLDPQTLLCLVNYVFFKGILERAFQTNLTKKEDF
|NP_001009304.1-OvUS| 160 KMDIQMIDFSDTEKAKKAISHHVAEKTHTKIRDLITDLNPETILCLVNHIFFKGILKRAFQPNLTQKEDF
|DAA05634.1-CapUS| 160 KMDIQMIDFSDTEKAKKAISHHVAEKTHTKITDLITDLNPETILCLVNHIFFKGILKRAFQPNLTQKEDF
|NP_777222.1-BoUS| 160 GMDIQMIDFTDIEKAKKTISHHVAEKTHTKITNLITDLNPETILCLVNHIFFKGILKRAFQPKLTQKEVF
|ABG56442.1-BuUS| 160 GMDIQMIDFTDTEKARKTISHHVAEKTHTKISDLITDLNPETILCLVNHVSLKGILKRAFQPELTQKEDF
|XP_855208.1-CanUS| 211 DVRARMIDFRDVVKTKKQINHFVAEKTHKRTKELITSLNPHTFLFLVDYVFFKGTWEMAFHSNLMHKEDF
|BK006618-EqUS| 163 EVEAQVADFRHRGIAKEQINQFVAQRLAHRIEEVVTSLHPHTFLFLLNYIFFKGVWEVAFQTRFTQKENF
CONSENSUS 211 kmdiqmiDF d ekakkaI h VAekthtkikdliTdLnPeTiLcLvn iffKGil rAFqpnltqKEdF
|AAA31137.1-PoUS-1| 93 RSATRTNAIDVLDVALKNLAVMLMAQAPTALLEIVHELVN-RTAKHQDIL-IDR-TEMNQMFLKEIDRYI
|NP_999010.1-PoUS-2| 93 RSATRTNAIDVLERDLRNLRVWDKHQALQHLVEMLHELEKKKQLKHKDIFFIDRNKKMNQMFLKEIDRVY
|NP_001009304.1-OvUS| 93 KSTMSTNHPEDLELELK---LLDAHKCLHHLVHLGRELVKQKQLRHQDILFLNSKMMANQMLLHQIRKLQ
|DAA05634.1-CapUS| 93 KSTMSTNHPEDLELELK---LLDAHKCLHHLLHLGRELVKQKQLKHQDILFLNSKMMANQMLLHQISKLQ
|NP_777222.1-BoUS| 93 KSTMRTHHPEDLKLEPK---LLDVHKYLQPLVHVGRELVKQKVLKHQHILFINRKMMVNQMLLQQISKLQ
|ABG56442.1-BuUS| 93 KSTMNAHHPEDLKLEPK---LLDVHKYLQTLVHVEHELVKQKLLKHQHILFINSKMVVNQMLLQQIGKLQ
|XP_855208.1-CanUS| 141 RSTTLTNLLEGLGFDLKVIKVWDVHHGFQSVIQMLKQLNRAGHLMHRDMLFIDSNRKINSPFLWDTQAMY
|BK006618-EqUS| 93 RSTTLSNLVAGLGFDLQRIRAWEVHTSFQRLVQTLNELDRQRHLKHRDFLFIDINRRIKPKFLQETERLY
CONSENSUS 141 rSt tnhpedLdlelk l lldvhk lqhlvhivheLvkqk lkHqdilfi skmmmnqm Lkqi klq
69
Figure 2-4. Continued
|AAA31137.1-PoUS-1| 230 FVNEKTIVQVDMMRKTERMIYSRSEELLATMVKIPCKENASIILVLPDTGKFNFALKEMAAKRARLQKTN
|NP_999010.1-PoUS-2| 233 FVNEKTIVQVDMMRKTERMIYSRSEELLATMVKMPCKENASIILVLPDTGKFDFALKEMAAKRARLQKTN
|NP_001009304.1-OvUS| 230 FLNDKTKVQVDMMRKTEQMLYSRSEELFATMVKMPFKGNVSLILMLPDAGHFDNALKKLTAKRAKLQKIS
|DAA05634.1-CapUS| 230 FLNDKTKVQVDMMRKTEQMLYSRSEELFATMVKMPFKGNVSLILMLPDAGHFDNALKKLTAKRAKLQKTS
|NP_777222.1-BoUS| 230 FVNDQTKVQVDMMRKTERMLYSRSEELHATMVKMPCKGNVSLTLMLPDAGQFDTDLKKMTAKRAKLQKIS
|ABG56442.1-BuUS| 230 LLDDKTKVQVDMMRKTERMLYSRSEELHTTMVKMPCKGNVSLILMLPDAGQFDTALKKVTAKRAKLQKIS
|XP_855208.1-CanUS| 281 FVDKHTTVPVDMIWKTGQMIYSRSEELFATMVKIPFVGNMSIVLVLPDVGQPDSAVKEIVVQRATLLQSS
|BK006618-EqUS| 233 FLEDNTSVQVHMMRKTERMIYSRAEHLFATIVKLPYTGNVSLVLVLPDAGRFDFVAKELAARRARPLQSR
CONSENSUS 281 fvndkTkVqVdMmrKTerMiYSRsEeLfaTmVKmPckgNvSliLvLPDaGkfd alK mtakRAklqkts
|AAA31137.1-PoUS-1| 300 DFRLVHLVVPKIKDNLQDRFKHLLP---------KIGINDIFTTKAVTWNTTGTS--TILEAVHHAVIEV
|NP_999010.1-PoUS-2| 303 ELQIGALSCAQDQDHLQDRFKHLLP---------KIGINDIFTTKAVTWNTTRTS--TILEAVHHAVIEV
|NP_001009304.1-OvUS| 300 NFRLVHLTLPKFKITFDINFKHLLPKINLKHLLPKIDPKHTLTTTASSQHVTLKAPLPNLEALHQVEIEL
|DAA05634.1-CapUS| 300 NFRLVHLTLPKFKITFEINFKHLLPKINLKHLLPKIDPKHTLTTTASSQDVTLKAPLPNLEALHQVEIEL
|NP_777222.1-BoUS| 300 DFRLVRLILPKLKISFKINFKHLLP---------KIDPKHILTATAISQAITSKAPLPNLEALHQAEIEL
|ABG56442.1-BuUS| 300 DFRLVRLTLPKLKISFKINFKHLLP---------KIGPKHTLTTTAISQAITLKAPLPNLEALHQAEIEL
|XP_855208.1-CanS 351 NMRWVHIIMPKFKISSKIDLKKILP---------KMGISNVFTTGANFSGITKEDFPTIFEAMHEATMEV
|BK006618-EqUS| 303 DTRLVHLILPKFKISSRIDLNRLLP---------KVGIEDIFSRRANFSGITDETFPTIFEAIHEARLEV
CONSENSUS 351 dfrlvhl lpkfkisfkinfkhlLP Kig khi tttAvsqaiT kapl lEAlHqaeiEv
|AAA31137.1-PoUS-1| 359 KEDGLTKNAAKDKD-FWKVP---------- -----------------------------VDKKEVPVVV
|NP_999010.1-PoUS-2| 362 KEDGLTKNAAKDKD-FWKVP---------- -----------------------------VDKKEVPVVV
|NP_001009304.1-OvUS| 370 SEHALTTDTAIHTDNLLKVP---------- -----------------------------ANTKEVPVVV
|DAA05634.1-CapUS| 370 SEHALTTDTAIHTDNLLKVP---------- -----------------------------VNAKEVPVVV
|NP_777222.1-BoUS| 361 SEHALTVDTAIHTDNLLKVPVKAKEVPAVV KVPMKAKEVPAVVKVPMNTKEVPVVVKVPMNTKEVPVVV
|ABG56442.1-BuUS| 361 NEHALTVDTAIHTDNLLKVP---------- ----------------MNAKEVPVVVKVPTNAKEVPVDV
|XP_855208.1-CanUS| 412 SKEGKMDTSKDMDSRNTIAC---------- -----------------------------HTYTATPLVV
|BK006618-EqUS| 364 NEKG-VIKAAAEDVHAKRAH---------- -----------------------------HAPHADATVV
CONSENSUS 421 sehglt dtaihtdnllkvp vn kevpvvV
|AAA31137.1-PoUS-1| 388 KFDRPFFLFVEDEITRRDLFVAKVFNPKTE
|NP_999010.1-PoUS-2| 391 KFDRPFFLFVEDEITRRDLFVAKVFNPKTE
|NP_001009304.1-OvUS| 400 KFNRPFLLFVEDEITQTDLFVGQVLNPQVE
|DAA05634.1-CapUS| 400 KFNRPFLLFVEDEMTQTDLFVGQVLNPQVE
|NP_777222.1-BoUS| 430 KVNRPFLLFVEDEKTQRDLFVGKVLNPQVE
|ABG56442.1-BuUS| 404 KVNRPFLLFVEDERTQRDLFVGQVLNPQVE
|XP_855208.1-CanUS| 442 KFNRPFFLFVEDWMTQRAILMGKVFNPIAE
|BK006618-EqUS| 393 KFNRPFLLFVEDELNQRQLFVGQVFNPLQ-
CONSENSUS 491 KfnRPFlLFVEDeitqrdlfvg V NPqve
|AAA31137.1-PoUS-1| 230 FVNEKTIVQVDMMRKTERMIYSRSEELLATMVKIPCKENASIILVLPDTGKFNFALKEMAAKRARLQKTN
|NP_999010.1-PoUS-2| 233 FVNEKTIVQVDMMRKTERMIYSRSEELLATMVKMPCKENASIILVLPDTGKFDFALKEMAAKRARLQKTN
|NP_001009304.1-OvUS| 230 FLNDKTKVQVDMMRKTEQMLYSRSEELFATMVKMPFKGNVSLILMLPDAGHFDNALKKLTAKRAKLQKIS
|DAA05634.1-CapUS| 230 FLNDKTKVQVDMMRKTEQMLYSRSEELFATMVKMPFKGNVSLILMLPDAGHFDNALKKLTAKRAKLQKTS
|NP_777222.1-BoUS| 230 FVNDQTKVQVDMMRKTERMLYSRSEELHATMVKMPCKGNVSLTLMLPDAGQFDTDLKKMTAKRAKLQKIS
|ABG56442.1-BuUS| 230 LLDDKTKVQVDMMRKTERMLYSRSEELHTTMVKMPCKGNVSLILMLPDAGQFDTALKKVTAKRAKLQKIS
|XP_855208.1-CanUS| 281 FVDKHTTVPVDMIWKTGQMIYSRSEELFATMVKIPFVGNMSIVLVLPDVGQPDSAVKEIVVQRATLLQSS
|BK006618-EqUS| 233 FLEDNTSVQVHMMRKTERMIYSRAEHLFATIVKLPYTGNVSLVLVLPDAGRFDFVAKELAARRARPLQSR
CONSENSUS 281 fvndkTkVqVdMmrKTerMiYSRsEeLfaTmVKmPckgNvSliLvLPDaGkfd alK mtakRAklqkts
|AAA31137.1-PoUS-1| 300 DFRLVHLVVPKIKDNLQDRFKHLLP---------KIGINDIFTTKAVTWNTTGTS--TILEAVHHAVIEV
|NP_999010.1-PoUS-2| 303 ELQIGALSCAQDQDHLQDRFKHLLP---------KIGINDIFTTKAVTWNTTRTS--TILEAVHHAVIEV
|NP_001009304.1-OvUS| 300 NFRLVHLTLPKFKITFDINFKHLLPKINLKHLLPKIDPKHTLTTTASSQHVTLKAPLPNLEALHQVEIEL
|DAA05634.1-CapUS| 300 NFRLVHLTLPKFKITFEINFKHLLPKINLKHLLPKIDPKHTLTTTASSQDVTLKAPLPNLEALHQVEIEL
|NP_777222.1-BoUS| 300 DFRLVRLILPKLKISFKINFKHLLP---------KIDPKHILTATAISQAITSKAPLPNLEALHQAEIEL
|ABG56442.1-BuUS| 300 DFRLVRLTLPKLKISFKINFKHLLP---------KIGPKHTLTTTAISQAITLKAPLPNLEALHQAEIEL
|XP_855208.1-CanS 351 NMRWVHIIMPKFKISSKIDLKKILP---------KMGISNVFTTGANFSGITKEDFPTIFEAMHEATMEV
|BK006618-EqUS| 303 DTRLVHLILPKFKISSRIDLNRLLP---------KVGIEDIFSRRANFSGITDETFPTIFEAIHEARLEV
CONSENSUS 351 dfrlvhl lpkfkisfkinfkhlLP Kig khi tttAvsqaiT kapl lEAlHqaeiEv
|AAA31137.1-PoUS-1| 359 KEDGLTKNAAKDKD-FWKVP---------- -----------------------------VDKKEVPVVV
|NP_999010.1-PoUS-2| 362 KEDGLTKNAAKDKD-FWKVP---------- -----------------------------VDKKEVPVVV
|NP_001009304.1-OvUS| 370 SEHALTTDTAIHTDNLLKVP---------- -----------------------------ANTKEVPVVV
|DAA05634.1-CapUS| 370 SEHALTTDTAIHTDNLLKVP---------- -----------------------------VNAKEVPVVV
|NP_777222.1-BoUS| 361 SEHALTVDTAIHTDNLLKVPVKAKEVPAVV KVPMKAKEVPAVVKVPMNTKEVPVVVKVPMNTKEVPVVV
|ABG56442.1-BuUS| 361 NEHALTVDTAIHTDNLLKVP---------- ----------------MNAKEVPVVVKVPTNAKEVPVDV
|XP_855208.1-CanUS| 412 SKEGKMDTSKDMDSRNTIAC---------- -----------------------------HTYTATPLVV
|BK006618-EqUS| 364 NEKG-VIKAAAEDVHAKRAH---------- -----------------------------HAPHADATVV
CONSENSUS 421 sehglt dtaihtdnllkvp vn kevpvvV
|AAA31137.1-PoUS-1| 388 KFDRPFFLFVEDEITRRDLFVAKVFNPKTE
|NP_999010.1-PoUS-2| 391 KFDRPFFLFVEDEITRRDLFVAKVFNPKTE
|NP_001009304.1-OvUS| 400 KFNRPFLLFVEDEITQTDLFVGQVLNPQVE
|DAA05634.1-CapUS| 400 KFNRPFLLFVEDEMTQTDLFVGQVLNPQVE
|NP_777222.1-BoUS| 430 KVNRPFLLFVEDEKTQRDLFVGKVLNPQVE
|ABG56442.1-BuUS| 404 KVNRPFLLFVEDERTQRDLFVGQVLNPQVE
|XP_855208.1-CanUS| 442 KFNRPFFLFVEDWMTQRAILMGKVFNPIAE
|BK006618-EqUS| 393 KFNRPFLLFVEDELNQRQLFVGQVFNPLQ-
CONSENSUS 491 KfnRPFlLFVEDeitqrdlfvg V NPqve
70
This domain is completely conserved in OvUS, CapUS, PoUS-1 and PoUS-2, is nearly
completely conserved in BuUS (only one amino acid substitution), but is not conserved in
CanUS and EqUS.
Identification of the Putative Hinge Region and P1-P1’ Site of the RCL
ClustalW alignment was used to align all US with inhibitory serpins including human α1-
antitrypsin (Genbank: NP_000286), human and porcine α1-antichymotrypsin (Genbank:
NP_001076 and NP_998952.1), human plasminogen activator inhibitor (Genbank: NP_000593),
bovine α2-plasmin inhibitor (Genbank: NP_777095), human leukocyte elastase inhibitor
(Genbank: P30740), human placental thrombin inhibitor (Genbank: P35237) and human
squamous cell carcinoma antigen 1 (Genbank: NP_536722) (Figure 2-5). Note that while
inhibitory serpins usually have a serine or cysteine at the P1’ site, most of the US have a valine at
the same position. The exception is for EqUS and CanUS which have an isoleucine and arginine
at this position, respectively.
The hinge region of the RCL (underlined in Figure 2-5) is highly conserved among
inhibitory serpins. The consensus sequence of RCL involves an arginine at P17, arginine, lysine
or glutamic acid at P16, glycine at P15, serine or threonine at P14 and for the hinge region an
alanine, glycine or serine at P12-P9 (Irving et al. 2000). For the US, the conserved arginine at
P17 is present in almost all US with the exception of CanUS which has a lysine at this position.
For the P16, US possess an aspartic acid or histidine with the exception of the CanUS (glutamic
acid) and EqUS (lysine). Moreover, the conserved glycine at P15 is present in only PoUS-1,
PoUS-2, EqUS and CanUS. Uterine serpins do not contain threonine or serine at P14 nor
alanine, glycine or serine at positions P12 to P10 (with the exception of the PoUS-1 and -2 which
have an alanine at P10). However, US have a conserved serine or alanine at position P9. Taken
71
Figure 2-5. Identification of the P1-P1’ site and hinge region (underlined) in uterine serpins.
Represented alignment of a portion of the amino acid sequences of some inhibitory
members of the serpin superfamily and the uterine serpin (blue square). Inferred
amino acid sequences of uterine serpins were aligned with inferred amino acid
sequences of selected inhibitory serpins using the ClustalW software. Shown is a
portion of the polypeptides near the P1-P1’ site. Sites of conservation are shown in
shaded columns using the Boxshade software. Black-shaded columns show
identical amino acids and grey-shaded columns represent similar amino acids.
NP_001076.2| 330 -QLGIEEAFT-SKADLSGITGARNLAVSQVVHKAVLDVFEEGTEASAATAVKITLLSAL-
NP_998952.1| 328 -QLGIEEIFG-DNANLSGITNTKPLKVSQVVHSAVLDVNEEGTEAAAATGIDINVRS---
NP_000286.3| 329 -QLGITKVFS-NGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMS---
P30740.1| 290 -RLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFCM---
P35237.3| 288 -NLGMTDAFELGKADFSGMS-QTDLSLSKVVHKSFVEVNEEGTEAAAATAAIMMMRC---
NP_536722.1| 316 -DMGITDIFDETRADLTGISPSPNLYLSKIIHKTFVEVDENGTQAAAATGAVVSERS---
NP_001009304.1 333 PKIDPKHTLT-TTASSQHVTLKAPLPNLEALHQVEIELSEHALTTDTAIHTDNLLKVP--
DAA05634.1| 333 PKIDPKHTLT-TTASSQDVTLKAPLPNLEALHQVEIELSEHALTTDTAIHTDNLLKVP--
NP_777222.1| 325 -KIDPKHILT-ATAISQAITSKAPLPNLEALHQAEIELSEHALTVDTAIHTDNLLKVPVK
ABG56442.1| 325 -KIGPKHTLT-TTAISQAITLKAPLPNLEALHQAEIELNEHALTVDTAIHTDNLLKVP--
AAA31137.1| 325 -KIGINDIFT-TKAVTWNTTGTS--TILEAVHHAVIEVKEDGLTKNAAKDKD-FWKVP--
NP_999010.1| 328 -KIGINDIFT-TKAVTWNTTRTS--TILEAVHHAVIEVKEDGLTKNAAKDKD-FWKVP--
XP_855208.1| 376 -KMGISNVFT-TGANFSGITKEDFPTIFEAMHEATMEVSKEGK-MDTSKDMDSRNTIAC-
BK 006618| 328 -KVGIEDIFS-RRANFSGITDETFPTIFEAIHEARLEVNEKG--VIKAAAEDVHAKRAH-
NP_000593.1| 316 --LGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNESGTVASSSTAVIVSARMAP-
NP_777095.1| 354 -QLGLQELFQ--APDLRGISDER-LVVSSVQHQSALELSEAGVQAAAATSTAMSRMSLS-
NP_001076.2| 387 ----------------------------------------VETRTIVRFNRPFLMIIVPT
NP_998952.1| 383 -----------------------------------------LERIALHFNRPFLFVIISK
NP_000286.3| 384 ------------------------------------------IPPEVKFNKPFVFLMIEQ
P30740.1| 346 ----------------------------------------LMPEENFTADHPFLFFIRHN
P35237.3| 343 ----------------------------------------ARFVPRFCADHPFLFFIQHS
NP_536722.1| 372 ----------------------------------------LRSWVEFNANHPFLFFIRHN
NP_001009304.1 390 -------------------------------------ANTKEVPVVVKFNRPFLLFVEDE
DAA05634.1| 390 -------------------------------------VNAKEVPVVVKFNRPFLLFVEDE
NP_777222.1| 383 AKEVPAVVKVPMKAKEVPAVVKVPMNTKEVPVVVKVPMNTKEVPVVVKVNRPFLLFVEDE
ABG56442.1| 381 ------------------------MNAKEVPVVVKVPTNAKEVPVDVKVNRPFLLFVEDE
AAA31137.1| 378 -------------------------------------VDKKEVPVVVKFDRPFFLFVEDE
NP_999010.1| 381 -------------------------------------VDKKEVPVVVKFDRPFFLFVEDE
XP_855208.1| 432 -------------------------------------HTYTATPLVVKFNRPFFLFVEDW
BK 006618| 383 -------------------------------------HAPHADATVVKFNRPFLLFVEDE
NP_000593.1| 373 --------------------------------------------EEIIMDRPFLFVVRHN
NP_777095.1| 409 ---------------------------------------------SFIVNRPFLFFILED
1P17 15 13 11 9 7 35 1’
NP_001076.2| 330 -QLGIEEAFT-SKADLSGITGARNLAVSQVVHKAVLDVFEEGTEASAATAVKITLLSAL-
NP_998952.1| 328 -QLGIEEIFG-DNANLSGITNTKPLKVSQVVHSAVLDVNEEGTEAAAATGIDINVRS---
NP_000286.3| 329 -QLGITKVFS-NGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMS---
P30740.1| 290 -RLGVQDLFNSSKADLSGMSGARDIFISKIVHKSFVEVNEEGTEAAAATAGIATFCM---
P35237.3| 288 -NLGMTDAFELGKADFSGMS-QTDLSLSKVVHKSFVEVNEEGTEAAAATAAIMMMRC---
NP_536722.1| 316 -DMGITDIFDETRADLTGISPSPNLYLSKIIHKTFVEVDENGTQAAAATGAVVSERS---
NP_001009304.1 333 PKIDPKHTLT-TTASSQHVTLKAPLPNLEALHQVEIELSEHALTTDTAIHTDNLLKVP--
DAA05634.1| 333 PKIDPKHTLT-TTASSQDVTLKAPLPNLEALHQVEIELSEHALTTDTAIHTDNLLKVP--
NP_777222.1| 325 -KIDPKHILT-ATAISQAITSKAPLPNLEALHQAEIELSEHALTVDTAIHTDNLLKVPVK
ABG56442.1| 325 -KIGPKHTLT-TTAISQAITLKAPLPNLEALHQAEIELNEHALTVDTAIHTDNLLKVP--
AAA31137.1| 325 -KIGINDIFT-TKAVTWNTTGTS--TILEAVHHAVIEVKEDGLTKNAAKDKD-FWKVP--
NP_999010.1| 328 -KIGINDIFT-TKAVTWNTTRTS--TILEAVHHAVIEVKEDGLTKNAAKDKD-FWKVP--
XP_855208.1| 376 -KMGISNVFT-TGANFSGITKEDFPTIFEAMHEATMEVSKEGK-MDTSKDMDSRNTIAC-
BK 006618| 328 -KVGIEDIFS-RRANFSGITDETFPTIFEAIHEARLEVNEKG--VIKAAAEDVHAKRAH-
NP_000593.1| 316 --LGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNESGTVASSSTAVIVSARMAP-
NP_777095.1| 354 -QLGLQELFQ--APDLRGISDER-LVVSSVQHQSALELSEAGVQAAAATSTAMSRMSLS-
NP_001076.2| 387 ----------------------------------------VETRTIVRFNRPFLMIIVPT
NP_998952.1| 383 -----------------------------------------LERIALHFNRPFLFVIISK
NP_000286.3| 384 ------------------------------------------IPPEVKFNKPFVFLMIEQ
P30740.1| 346 ----------------------------------------LMPEENFTADHPFLFFIRHN
P35237.3| 343 ----------------------------------------ARFVPRFCADHPFLFFIQHS
NP_536722.1| 372 ----------------------------------------LRSWVEFNANHPFLFFIRHN
NP_001009304.1 390 -------------------------------------ANTKEVPVVVKFNRPFLLFVEDE
DAA05634.1| 390 -------------------------------------VNAKEVPVVVKFNRPFLLFVEDE
NP_777222.1| 383 AKEVPAVVKVPMKAKEVPAVVKVPMNTKEVPVVVKVPMNTKEVPVVVKVNRPFLLFVEDE
ABG56442.1| 381 ------------------------MNAKEVPVVVKVPTNAKEVPVDVKVNRPFLLFVEDE
AAA31137.1| 378 -------------------------------------VDKKEVPVVVKFDRPFFLFVEDE
NP_999010.1| 381 -------------------------------------VDKKEVPVVVKFDRPFFLFVEDE
XP_855208.1| 432 -------------------------------------HTYTATPLVVKFNRPFFLFVEDW
BK 006618| 383 -------------------------------------HAPHADATVVKFNRPFLLFVEDE
NP_000593.1| 373 --------------------------------------------EEIIMDRPFLFVVRHN
NP_777095.1| 409 ---------------------------------------------SFIVNRPFLFFILED
1P17 15 13 11 9 7 35 1’
72
together, results indicate that the RCL and the hinge region required for inhibitory activity has
not been conserved in uterine serpins.
Phylogenetic Analysis of Uterine Serpins
The phylogenetic tree constructed using the NJ method is presented in Figure 2-6. The
sheep and goat proteins as wells as the water buffalo and cow proteins were clustered together,
and the horse and dog were clustered together in a sister group and the pig proteins were closer
to the horse and dog than to the ruminants.
Positive Selection of the Uterine Serpin Gene
Aligned sequences of uterine serpins were tested for evidence of positive selection using
different models of variable dN/dS ratios among sites (Table 2-3). The LRT for positive
selection was significant when comparing M2 and M8 against their null model (Table 2-4). The
Bayes empirical Bayes method used in PAML to calculate the posterior probabilities at each
codon site (Yang et al. 2005) indicate A121, G343 and K380 as positive selected sites for M2
and I35, A121, G343, K365, V378, F373 and K380 as positive selected sites for M8. These
results partially agreed with those obtained with the Selecton program where six amino acid
residues (Figure 2-7) were identified as sites with potential positive selection (I35, A121, D156,
G343, K365 and K380) and thirty nine amino acid residues were likely to be under purifying
selection (M1, H3, M6, S22, I23, V38, A58, F62, E68, K72, I75, F76, A86, D167, I178, V182,
T195, T201, K226, E227, F229, T235, V237, V239, M241, K244, M248, S251, E254, N268,
D277, K324, E349, E357, K388, F393, E398, V411 and N413).
To identify whether codons undergoing positive selection were associated with regions
critical for antiproteinase activity, the amino acid sequence of OvUS was aligned to human α1 -
antitrypsin to identify helices and β-sheets of the protein (Figure 2-8). A total of six amino acid
residues were identified as being subject to positive selection by at least two of the three
73
Figure 2-6. Phylogenetic tree of the uterine serpin proteins with the ovine uterine serpin
(OvUS) as out-group. The Jones-Taylor-Thornton matrix was used for distance
calculation with gamma corrected distances and tree was inferred by the Neighbor-
joining method. The tree reliability was tested with 1000 bootstrap replicates (not
shown).
74
Table 2-3. Ratios (ω) of nonsynonymus (dN) to synonymous (dS) mutations, parameter
estimates and maximum log likelihood of models for positive selection within the
protein coding sequence of uterine serpins.
a The numbers of free parameters are represented by (p) assigned to each class of ω
Model dN/dS Parameter estimate(s) a L
M0 (one ratio) 0.8815 ω = 0.8815 -6270.25
M1 (Nearly Neutral) 0.7506 p0= 0.29151, p1= 0.70849, ω0= 0.14433, ω1= 1 -6233.37
M2 (Positive selection) 1.0717 p0= 0.25346, p1= 0.61790, p2= 0.12864, ω0= 0.16249, ω1= 1, ω2= 3.20732 -6219.15
M7 (Beta) 0.7799 p= 0.02945 q= 0.00709 -6235.54
M8 (Beta & ω) 1.0860 p= 0.49271, q= 0.19375, p0= 0.83214, p1= 0.16786, ωs= 2.91144 -6219.83
75
Table 2-4. Test of significance for models for positive selection within the protein coding
sequence of uterine serpins.a
Models 2L 2 value df P values
Positive selected sites
(BEB)b
M1 versus M2 2(-6219.15+6233.37) 28.44 2 <0.001 A121**, G343*, K380*
M7 versus M8 2(-6219.83+6235.54) 31.42 2 <0.001 I35*, A121**, G343*, K365*
V378*, F373*, K380**
a The likelihood ratio test statistics were calculated as two times the difference in the log
likelihood between models (2L). Significance was tested by using the 2 test with degrees of
freedom calculated from the parameter estimates.
b
Sites of positive selection were estimated by calculating the posterior probability that each
codon was from the site class of positive selection under models M2 and M8 by the Bayes
empirical Bayes method (* P >0.95; ** P >0.99).
76
Figure 2-7. Selecton output generated for the uterine serpin group of proteins. Shown is the
sequence for PoUS-1. Color intensity indicates likelihood of purifying (dark purple)
and positive selection (dark yellow) at each amino acid residues. Six amino acid
residues were estimated to undergo positive selection (I35, A121, D156, G343,
K365 and K380) while thirty nine were identified to be under purifying selection.
Amino acid residues identified as under positive selection by the Bayes empirical
Bayes method for models M2 and M8 of the PAML program are shown by an
asterisk (*) and diamond (), respectively. The putative hinge region of the reactive
center loop is underlined and the putative scissile bond at P1-P1’ site is indicated by
an arrow.
*
*
*
P1-P1’
*
*
*
P1-P1’
77
Figure 2-8. Amino acid sequence alignment of ovine uterine serpin (NP_001009304.1) and
human α1-antitrypsin (NP_000286.3) using the ClustalW algorithm. Helices are
shown as green cylinders and β-pleated sheets are shown as yellow arrows. The
location and designation of these regions are based on Silverman et al. (2004).
Amino acid residues identified as being subject to positive selection by at least two
of three statistical methods are indicated by arrowheads.
s1C s4B s5B
s5A
HA HBs6B
s3A s4C s3C
HG HHs1C s2B s3B
HC HD s2A
HE HFs1A
s6As2C HI
s1C s4B s5Bs1C s4B s5B
s5A
HA HBs6B
s3A s4C s3C
HG HHs1C s2B s3B
HC HD s2A
HE HFs1A
s6As2C HI
s5As5A
HA HBs6BHA HBs6B
s3A s4C s3Cs3A s4C s3C
HG HHs1C s2B s3B HG HHs1C s2B s3B
HC HD s2AHC HD s2A
HE HFs1AHE HFs1A
s6As2C HIs6As2C HI
78
statistical methods. Of the six positive selected sites, two are in colied regions, one is in the
putative Helix D, one is in the putative Helix E, one is in the reactive center loop and one is the
5’ site relative to the putative scissile bond (Figure 2-8).
Discussion
Serpin genes are of ancient origin and are found in bacteria, fungus, nematodes, archaea
and virus (Irving et al. 2000, 2002b, Steenbakkers et al. 2008). The fact that uterine serpins
(SERPINA14) are apparently limited to species in the Laurasiatheria superorder means that they
either arose recently, after Laurasiatheria diverged from Euarchontoglires (~87 Mya) (Murphy et
al. 2007), or they have been retained during evolution in this superorder only. Their uterine
expression and the loss of amino acid motifs required for proteinase inhibitory activity in the P1-
P1’ site and hinge region indicates that the genes have been selected for a new function required
for maintenance of pregnancy. Based on experiments with sheep and pigs, uterine serpins may
function to inhibit cell proliferation (Padua & Hansen 2008) or interact with other uterine
proteins (Baumbach et al. 1986, Hansen & Newton 1988, McFarlane et al. 1999). The uterine
serpins are thus an example of a new gene arising from gene duplication and selection for
sequence divergence (Louis 2007) that plays a particular role in pregnancy.
While it was not possible to examine complete genomic sequences in each order of
mammals, the available evidence points strongly to restriction of the uterine serpin to
Laurasiatheria only. The distribution of known uterine serpins as well as orders where blast
search of complete genomic sequences fail to identify a uterine serpin is shown in Figure 2-9.
All the identified uterine serpins are in Ruminantia, Suidae, Perissodactyla or Carnivora orders
of Laurasiatheria. No gene that significantly matched OvUS was found in within the orders
79
Figure 2-9. Phylogenetic tree of placentation in mammals (adapted from Vogel 2005) to
illustrate the existence of uterine serpin genes relative to type of placentation.
Shown are the 4 major superorders of eutherian mammals (Laurasiatheria,
Euarchontoglires, Xenarthra and Afrotheria) with the order Marsupiala as the out-
group of the tree. Blue branches represent orders with epitheliochorial placenta and
orange branches represent orders with either endotheliochorial or hemochorial type
of placentation. The yellow branches represent unresolved situations in the
phylogeny. The symbol represents those orders within the Laurasiatheria
superorder where the uterine serpin gene has been identified. The symbol
represents those orders where the uterine serpin gene was not identified after blast
search of complete genomic sequences.
Proboscidea
Sirenia
Xenarthra
Tenrecomorpha
Marsupialia
Laurasiatheria
Afrotheria
Xenarthra
Euarchontoglires
Cetacea
Hippopotamidae
Carnivora
Suidae
Pholidota
Perissodactyla
Ruminantia
Talpa
Rodentia
Lemuriformes
Scandentia
Tarsiphormes
Scalopus
Anthropoidea
Lagomorpha
Chiroptera
Erinaceomorpha
Proboscidea
Sirenia
Xenarthra
Tenrecomorpha
Marsupialia
Laurasiatheria
Afrotheria
Xenarthra
Euarchontoglires
Cetacea
Hippopotamidae
Carnivora
Suidae
Pholidota
Perissodactyla
Ruminantia
Talpa
Rodentia
Lemuriformes
Scandentia
Tarsiphormes
Scalopus
Anthropoidea
Lagomorpha
Chiroptera
Erinaceomorpha
80
Anthropoidea and Rodentia of the superorder Euarchontoglires, Marsupialia, or in non-
mammalian species examined.
Given the fact that all uterine serpins identified before this study were species exhibiting
epitheliochorial placentation (Figure 2-9), it was hypothesized that uterine serpin genes serve
sole role uniquely required for species with this type of placentation. While this hypothesis may
be correct it is not true that the gene is only present in species with epitheliochorial placentation.
Of the two new uterine serpins identified in the present study, one is for species with
epitheliochorial placentation (horse), but one is for species with endotheliochorial placentation
(dog). Phylogenetic analysis showed that the genes in the dog and horse are closely related to
each other. In fact, CanUS and EqUS are closer to each other than to PoUS-1 and PoUS-2. For
example, the KEVPVVVK in other uterine serpins including pig is lost in both horse and dog. In
some mammalian phylogenies, the horse is more closely related to the pig than carnivores
(Vogel 2005) whereas in others, the horse is closer to carnivores than to pigs (Nishihara et al.
2006) or the horse is equidistant from carnivores and pigs (Murphy et al. 2007).
Even though the dog has a uterine serpin gene, there was no evidence for functional uterine
serpin gene in the closely-related cat. Results must be tentative because a complete genomic
sequence of cat is not yet available. However, the fact that nucleotide sequences identified in cat
as being similar to uterine serpins contained stop codons suggests that a functional uterine serpin
gene has been lost in this species . Additionally, the dog uterine serpin has undergone significant
change from other serpins in that it has acquired two putative start codons upstream from the
start codons in other species. If in fact this start codon is correct, the CanUS gene encodes for an
intracellular protein without a signal peptide while all other species with uterine serpin encode
for secretory proteins (Moffatt et al. 1987, Leslie et al. 1990, Malathy et al. 1990, Tekin et al.
81
2005a). The newly discovered EqUS was also shown to be secreted into the uterine lumen as
revealed by western blotting of uterine flushings.
Taken together, it is possible that the uterine serpin gene in species with epitheliochorial
placentation has been required as a secretory protein while the gene in species with
endotheliochorial placentation has either being changed to become an intracellular protein (dog)
or a pseudogene (cat). If this idea is correct, uterine serpins play some important role in the
uterine lumen of species with epitheliochorial placentation that is not required for species with
either endotheliochorial placentae. This hypothesis is also supported by the absence of a
homologous uterine serpin gene in human and rodents, species with hemochorial placentation.
The function of uterine serpins is probably not to inhibit serine or cysteine proteinases.
Ovine and BoUS exhibit no inhibitory activity towards a range of proteinases (Ing & Roberts
1989, Liu & Hansen 1995, Mathialagan & Hansen 1996, Peltier et al. 2000a). In addition, there
is evidence, as shown here, that the P1-P1’ site and hinge region of the uterine serpins differ
from inhibitory serpins. The RCL contains a complementary sequence to the active site of the
target proteinase and the hinge region of RCL is highly conserved among inhibitory serpins
(Irving et al. 2000). In uterine serpins, however, the amino acids of the consensus sequence is
absent at P15 (except for PoUS-1, PoUS-2, EqUS and CanUS), P14 and P12 to P10 (except for
PoUS-1 and -2 which have an alanine at P10). Furthermore, an amino acid in the RCL and at the
5’ position were identified as positions subject to positive selection and the P1 site is not
conserved with inhibitory serpins. Among the serpin superfamily, the RCL is important for
selectivity of the serpin for the target proteinase and has undergone an accelerated rate of
evolution (Brown 1987, Hill & Hastie 1987, Goodwin et al. 1996, Zang & Maizels 2001,
Barbour et al. 2002).
82
The identification of the P1-P1’ site in this study is different by two amino acids (K-V
instead of P-V) that was identified in the original paper by Ing & Robets (1989) and used as the
basis for assigning its location in other papers (Mathialagan & Hansen 1996, Peltier et al. 2000c,
Tekin et al. 2005a). The accessibility to more complete sequences from inhibitory serpins whose
P1-P1’ sites have already been determined and also more uterine serpin sequences in addition to
improvement in alignment programs results in a more reliable estimate of the P1-P1’site for the
uterine serpins.
Unique functions of uterine serpins in species with epitheliochorial placentae must be
linked to the unique characteristics of placentation in this species. Unlike other type of placentae
in eutherian mammals, where maternal blood either spills directly onto trophoblast (hemochorial
placenta) or where trophoblast invasion and erosion of endometrial tissue leaves the trophoblast
in contact with maternal endothelium, the epitheliochorial placenta is characterized by limited
invasiveness of trophoblast and an intact endometrial epithelium that sometimes forms a
synctium with trophoblast cells. The efficiency of placental transport is improved in species
with epitheliochorial placentae because of countercurrent exchange of nutrients and gases
between fetal and maternal blood vessels (Leiser et al. 1997). It has also been speculated that the
presence of an intact maternal endometrial epithelium may reduce the recognition of trophoblast
antigens by maternal immune system (Moffet & Loke 2006) although available evidence
indicates that the maternal immune response to the trophoblast is similar in species with
epitheliochorial placentation to that of other species (Oliveira & Hansen 2008).
Ovine uterine serpin has been shown to inhibit proliferation of lymphocytes and a variety
of cancer cells (Padua & Hansen 2008) and it is possible that one or more uterine serpins
function to inhibit cell proliferation during pregnancy. Inhibition of lymphocyte proliferation
83
has been interpreted as signifying role for uterine serpins in prevention of rejection of conceptus
tissue by the maternal immune system (Hansen 1998). In addition, the limited invasiveness of
trophoblast tissue in species with epitheliochorial placentation could be achieved, at least in part,
by inhibition of trophoblast proliferation. Through inhibition of the cell cycle (Padua & Hansen
2008), it may be also that uterine serpins participate in the process of trophoblast binucleate cell
formation, which occurs in sheep, goat, cattle and horse (Hoffman & Wooding 1993).
Another characteristic of uterine serpins is the propensity to bind other proteins. Ovine
uterine serpin has been found to bind activin (McFarlane et al. 1999), IgA and IgM (Hansen &
Newton 1988), and the so-called pregnancy associated glycoproteins (Mathialagan & Hansen
1996) that are inactive members of the aspartic proteinase family (Xie et al. 1991, Green et al.
1998). The pig uterine serpins bind to an iron-binding protein secreted by the uterus called
uteroferrin and stabilize the iron bound to uteroferrin (Baumbach et al. 1989). In some cases,
this binding can be ascribed to the highly basic isoelectric point for uterine serpins. Binding to
IgA and pepsin can be reduced by high salt concentrations (Hansen & Newton 1988, Peltier et al.
2000a). However, a physiological role for protein binding remains a possibility. Binding to
uteroferrin may enhance iron transport to the fetus since this protein delivers its iron to fetal
fluids (Buhi et al. 1982). The function of pregnancy associated glycoproteins is not known but
the conservation of the KEVPVVVK motif located near the P1-P1’ site described as pepstatin-
like domain (Mathialagan & Hansen 1996) may mean that binding to pregnancy associated
glycoproteins is an important function.
Progesterone is the hormone that induces the synthesis and secretion of uterine serpins into
the uterine lumen of sheep, pig, cow and goat (Ing et al. 1989, Leslie et al. 1990, Malathy et al.
1990, Tekin et al. 2005a). It is likely that the equine protein is also stimulated by progesterone.
84
In addition, in the cow (Khatib et al. 2007) as well as in dog, the uterine serpin gene is expressed
in the ovary. The presence of uterine serpin in other reproductive tissues distinct to the uterus
raises the possibility that uterine serpins may have different functions according to the cell type
and stage of differentiation. It is possible that uterine serpin functions in the ovary to regulate
follicular growth by binding to activin. Activin improves follicular growth and granulosa cell
proliferation (Knight & Glister 2006). OvUS binds to this member of the TGF-β family
(McFarlane et al. 1999).
In summary, the uterine serpin gene is present only in a restricted group of species within
the Laurasiatheria superorder of eutherian mammals and likely evolved under positive selection
which suggests diversifying functionality of these proteins from the proteinase inhibitory activity
of most members of the serpin superfamily. This evidence also suggests the uterine serpins gene
is an example of gene duplication followed by selection. The finding that the uterine serpin has
been retained as a secretory protein in species with epitheliochorial placentation within the
Laurasiatheria superorder also suggests that the protein has an important role during pregnancy
in these species while in species with endotheliochorial placenta the gene has undergone changes
suggesting that it is playing either a new role in those species (dog) or no required (cat) during
pregnancy.
85
CHAPTER 3
COMPARISON OF THE NATIVE AND RECOMBINANT FORMS OF OVINE UTERINE
SERPIN FOR INHIBITION OF CELL PROLIFERATION
Introduction
The uterine serpins (US) are a group of glycosylated proteins secreted into the uterus of the
sheep, cow, goat and pig during mid and late pregnancy (Moffatt et al. 1987, Leslie et al. 1990,
Baumbach et al. 1986, Tekin et al. 2005a). These proteins are members of the superfamily of
serine proteinase inhibitors (serpins) (Ing & Robets 1989, Mathialagan & Hansen 1996). Most
of the members of the serpin superfamily are inhibitors of serine or cysteine proteinases, which
are inactivated by an irreversible suicide substrate-like mechanism (Silverman et al. 2001).
However, there are some members of this superfamily that have roles distinct from the inhibition
of proteinases. Some examples of non-inhibitory serpins include the hormone transport proteins
corticosteroid and thyroxine binding globulin (Pemberton et al. 1988), the chaperone heat shock
protein 47 (Nagata 1998) and angiotensinogen which is involved in the regulation of blood
pressure (Morgan et al. 1996).
Another serpin that seems to have a divergent function is ovine uterine serpin (OvUS).
This US has been linked to the protection of the allogeneic conceptus through inhibition of
immune cell proliferation during pregnancy (Hansen 1998). Several experiments have
demonstrated that OvUS inhibited lymphocyte proliferation induced by mitogens such as
concanavalin A (Con A), the antigen Candida albicans, phytohemagglutinin and in the mixed
lymphocyte reactions (Segerson et al. 1984, Hansen et al. 1987b, Stephenson et al. 1989b,
Skopets & Hansen 1993, Skopets et al. 1995). Ovine US also inhibits natural killer (NK) cell
activity (Liu & Hansen 1993). Moreover, OvUS inhibited the lytic activity of NK-like cells in
sheep peripheral blood lymphocytes and endometrial epithelium against D17 cells infected with
bovine herpes virus-1 (Tekin & Hansen 2002).
86
However, OvUS did not cause any inhibitory activity against lymphocytes activated by the
T and B cell mitogen PWM (Skopets & Hansen 1993). Ovine US had no effect on the reduction
of skin-fold thickness caused by Mycobacterium tuberculosis in sheep (Skopets et al. 1995) and
failed to inhibit expression of CD25 on γδ-T + cells induced by Con A (Peltier et al. 2000b),
suggesting the apparent selectivity of the protein to inhibit some immune cell types.
Little is known about the antiproliferative actions of OvUS in other non-immune cell types.
The objectives pursued on this study are to test whether OvUS inhibited proliferation of non-
immune cells, specifically tumor cells, and to determine whether OvUS inhibits cell proliferation
by apoptosis. Finally, the antiproliferative potency of a recombinant form of OvUS (rOvUS)
was compared with the native form of the protein.
Materials and Methods
Materials
The Eagle’s Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium
Nutrient Mixture F-12 Ham (DMEM-F12), Dulbecco’s Modified Eagle’s Medium (DMEM),
Dulbecco’s phosphate buffered saline (DPBS) and penicillin–streptomycin were purchased from
Sigma-Aldrich (St Louis, MO). The fetal bovine serum was from Intergen (Purchase, NY) and
the heat-inactivated horse serum was from Hyclone (Logan, UT). The D-17 (canine primary
osteogenic sarcoma), PC-3 (human prostate cancer) and P388D1 (mouse lymphoma) cell lines
were purchased from ATCC (Rockville, MD). The centricon ultrafiltration devices were from
Amicon (Beverly, MA) or Millipore (Bedford, MA). Carboxymethyl Sepharose, Sephacryl S-
200 and His-Trap columns were obtained from Amersham Biosciences (Piscataway, NJ) and
[3H]thymidine (6.7 Ci/mmol) was from ICN (Irvine, CA). The RQ1 RNase-free DNase was
from Promega (Madison, WI), in situ cell death detection kit [terminal deoxynucleotidyl
transferasemediated dUTP nick end labeling (TUNEL)] was from Roche (Indianapolis, IN). The
87
ProLong Antifade kit was purchased from Molecular Probes (Eugene, OR), RNase A was from
Qiagen (Valencia, CA) and the Precast Tris-HCl gradient gels® were obtained from Bio-Rad
(Richmond, CA). Other reagents were from either Sigma-Aldrich or Fisher.
Collection of Uterine Fluid and Purification of Native OvUS
Ewes of Rambouillet genotype (n=4) were made unilaterally pregnant as described
elsewhere (Bazer et al. 1979) by ligating one uterine horn. Ewes were slaughtered at day 140 of
pregnancy by captive bolt stunning and exsanguination. Crude uterine fluid was collected from
the ligated uterine horn after slaughter by aspiration. Uterine fluid was clarified by
centrifugation at 3600 x g and stored at –20oC until purification of native ovine uterine serpin
(nOvUS) by a combination of cation-exchange chromatography with carboxymethyl Sepharose
and gel filtration chromatography with Sephacryl S-200 as previously described (Liu & Hansen
1993). The purity of the protein was assessed by sodium dodecyl sulfate, polyacrylamide gel
electrophoresis (SDS-PAGE) using 4-15% polyacrylamide Precast Tris-HCl gradient gels and
staining by Coomassie Blue G-250. After purification, nOvUS was buffer-exchanged with
DPBS and concentrated using Centricon PL-20 ultrafiltration devices. Protein concentration was
determined using the Bradford assay with bovine serum albumin as standard (Bradford 1976).
[3H]thymidine Incorporation by D17 and PC-3 Cells
The D17 cells were cultured continuously in complete medium [MEM supplemented with
10% (v/v) heat-inactivated fetal bovine serum, 200 U/ml penicillin and 2 mg/ml streptomycin].
The PC-3 cells were grown in DMEM-F12 medium supplemented with 10% (v/v) heat-
inactivated fetal bovine serum, 200 U/ml penicillin and 2 mg/ml streptomycin. Both cell lines
were cultured continuously at 37oC in a humidified 5% (v/v) CO2. At confluence, cells were
trypsinized, centrifuged for 5 min at 110 x g and resuspended in fresh complete medium. Cell
88
viability was assessed by trypan blue exclusion and cell concentration adjusted to 1 x 105
cells/ml.
For the [3H]thymidine incorporation assay, aliquots of 100 μl containing 1 x 10
4 cells were
cultured in 96-well plates at 37oC and 5% (v/v) CO2 with all treatments in triplicate. After 24 h
in culture, treatments consisting of 1 mg/ml of nOvUS or ovallbumin (OVA, as negative control)
were added. For control wells, an equivalent volume of DPBS was added instead of proteins.
The final volume was adjusted to 200 μl with complete culture medium. After 48 h of culture,
0.1 μCi of [3H]thymidine in 10 μl of complete culture medium were added and cells were
harvested onto glass-fiber filters using a cell harvester device at 24 h after thymidine addition.
Filters were counted for radioactivity using scintillation spectrometry.
Induction of Apoptosis in D17 and PC-3 Cells
To determine apoptosis, 1 x 104 cells in 100 μl were cultured with vehicle, 1 mg/ml nOvUS
or OVA. For the vehicle treatment, an equivalent volume of DPBS was added instead of OvUS
or OVA. The final volume in all wells was adjusted with culture medium to 200 μl. Cells were
cultured at 37oC and 5% (v/v) CO2 for 24 h and then harvested for the detection of apoptotic
cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). All
treatments were performed in triplicate and the experiment was performed on three (D17) or one
(PC-3) separate occasions.
Cells were processed for TUNEL staining as follows. Cells were washed by centrifugation
with 0.1 M sodium phosphate, pH 7.4 with 0.9% (w/v) NaCl (PBS) and containing 1 mg/ml
polyvinyl pyrrolidone (PBS/PVP), and then resuspended in 200 μl 4% (w/v) paraformaldehyde
in 0.2 M sodium phosphate, pH 7.4 with 0.9% (w/v) NaCl for 1 h at room temperature. Cells
were washed again with PBS/PVP, resuspended in 200 μl PBS/PVP, and 100 μl cell suspension
was transferred to a poly-l-lysine coated slide and allowed to dry for at least 24 h at room
89
temperature. For TUNEL staining, slides were washed twice in PBS/PVP (2 min each) and then
incubated with permeabilization solution [0.1% (v/v) Triton X-100 containing 0.1 % (w/v)
sodium citrate in PBS] for 1 h at room temperature. Positive controls were incubated with RQ1
RNase-free DNase (50 U/ml) at 37oC for 1 h. Slides were washed in PBS/PVP and then
incubated with 50 μl TUNEL reaction mixture (containing fluorescein isothiocyanate-conjugated
dUTP and the enzyme terminal deoxynucleotidyl transferase as prepared by the manufacturer)
for 1 h at 37oC in the dark. Negative controls were incubated in the absence of the enzyme.
After washing, slides were incubated with RNase A (50 μg/ml) for 1 h at room temperature and
then with 12.5 μg/ml propidium iodide for 30 min at room temperature. Cells were washed 4
times in PBS/PVP to remove excess propidium iodide and coverslips mounted with mounting
medium containing Prolong Antifade®
as recommended by the manufacturer. Slides were
observed using a Zeiss Axioplan 2 fluorescence microscope with dual filter (Carl Zeiss, Inc.,
Göttingen, Germany). Images were acquired using AxioVision software and a high-resolution
black and white AxioCam MRm digital camera (Carl Zeiss, Inc., Thorwood, NY). Percent
apoptotic cells were determined by counting the total number of TUNEL-labeled nuclei in at 10
different sites on the slide.
Purification of His-Tagged rOvUS from Conditioned Medium
The human embryonic kidney (HEK)-293F (Gibco-Invitrogen, Carlsbad, CA) cells
transfected with a plasmid construct containing the gene for OvUS (Tekin et al. 2006) was
cultured continuously in selective medium [FreeStyleTM
293 expression medium containing 700
μg/ml of Geneticin] at 37oC in a humidified 8% (v/v) CO2 incubator according to the
manufacturer’s recommendations. Conditioned medium from 293F-rOvUS cells was clarified
by centrifugation at 4000 x g for 15 min and the supernatant was removed and concentrated
using Centricon PL-80 or 20 concentration devices (molecular weight exclusion limit = 30000).
90
Purification of the His-tagged rOvUS was achieved using HisTrap columns. The binding buffer
was 20 mM sodium phosphate buffer, pH 8.0, 15 mM imidazole, 0.3 M NaCl, and the elution
buffer was 20 mM sodium phosphate buffer, pH 8.0, 0.3 M NaCl and with 300 or 500 mM
imidazole. The concentrated medium was diluted with at least an equal volume of binding buffer
and loaded onto the His Trap columns. The protein recovered after elution was buffer-
exchanged with DPBS and concentrated using Centricon PL-20 filters. Purity of rOvUs was
assessed by SDS-PAGE using 4-15% polyacrylamide Precast Tris-HCl gradient gels and
concentration was determined using the Bradford assay (Bradford 1976).
Expression and Purification of β-Galactosidase
The pcDNA3.1/V5-His-TOPO/lacZ®
plasmid containing the gene for β-galactosidase
(Gibco-Invitrogen, Grand Island, NY) was used to express a His-tagged β-galactosidase as a
control protein. FreestyleTM
293-F cells were transfected as described somewhere else (Tekin et
al. 2006). Transfected cells were then selected as described for the rOvUS. After harvesting, the
cell pellet was collected by centrifugation at 100 x g for 10 min at room temperature. The pellet
was resuspended in 20 mM sodium phosphate, pH 8.0 containing 0.3 M NaCl and 35 mM
imidazole and a proteinase inhibitor cocktail diluted as per manufacturer’s recommendations
(Sigma-Aldrich). Cells were lysed by two freeze-thaw cycles. The supernatant was collected by
centrifugation and the recombinant His-tagged protein purified as previously described for
rOvUS.
Proliferation of P388D1 and PC-3 Cells
The P388D1 cells were cultured continuously in DMEM supplemented with 10% (v/v)
heat-inactivated horse serum, 200 U/ml penicillin and 200 μg/mL streptomycin. The PC-3 cells
91
were grown in DMEM-F12 medium (Gibco-Invitrogen) supplemented with 10% (v/v) heat-
inactivated fetal bovine serum, 200 U/ml penicillin and 200 μg/ml streptomycin.
The proliferation assay was performed in 96-well culture plates at 37oC and 5% (v/v) CO2
with all treatments in triplicate. For P388D1 cells, 2 x 103 cells in 100 μl of culture medium
were cultured for 6 h before the addition of various concentrations (0, 125, 250, 500 and 1000
μg/ml) of nOvUS, rOvUS and OVA (negative control). For PC-3 cells, 1 x 104
cells in 100 μl
were cultured for 24 h before addition of 0, 62.5, 125, 250, 500 and 1000 μg/ml of nOvUS,
rOvUS or OVA. For control wells, an equivalent volume of DPBS was added instead of
proteins. The final volume was adjusted to 200 μl with culture medium. After 24 h of culture,
0.1 μCi of [3H]thymidine in 10 μl culture medium were added and cells were harvested onto
glass-fiber filters using a cell harvester device at 24 h after thymidine addition. Filters were
counted for radioactivity using scintillation spectrometry. Experiments were performed on 3 and
5 separate occasions for P388D1 and PC-3 cells respectively. Another experiment with PC-3
cells was performed as described above except with recombinant β-galatcosidase at
concentrations of 50, 100 and 200 μg/ml in nine replicates in one assay.
Statistical Analysis
Data on [3H]thymidine incorporation were analyzed by least squares analysis of variance
using the General Linear Models Procedure of SAS (SAS for Windows, Release 8.02; SAS Inst.,
Inc., Cary, NC). Replicate was considered as a random effect and other main effects were
considered fixed. Error terms were determined based on calculation of expected mean squares.
In some analyses, the pdiff mean separation test of SAS was performed to determine treatments
that differed from untreated cells.
92
Results
Inhibition of Proliferation and Induction of Apoptosis in D17 and PC-3 Cells
Ovine uterine serpin inhibited proliferation (P<0.05) of both D17 and PC-3 cells (Figure 3-
1A). The proportion of cells that were TUNEL-positive was very low (1.1% for control D17
cells and 0.5% for control PC-3 cells) and was slightly increased by OvUS (2.2% for D17 cells
treated with OvUS and 3.3% for PC-3 cells treated with OvUS; Figure 3-1B). Nonetheless, the
number of apoptotic cells was very low for all treatments (see Figures 3-1C for examples of
TUNEL staining).
Antiproliferative Actions on P388D1 and PC-3 Cell Lines: Comparison of the Native and
Recombinant Forms of OvUS
Both nOvUS and rOvUS inhibited proliferation of P388D1 cells as measured by
incorporation of [3H]thymidine into DNA (Fig. 3-2). All concentrations of rOvUS tested were
inhibitory, with the lowest concentration being 125 μg/ml. For nOvUS, in contrast, there was
inhibition only at 500 μg/ml (P<0.10) and 1000 μg/ml (P<0.001) and the magnitude of the
reduction in proliferation at any concentration was greater for rOvUS than for OvUS. The
control protein, ovalbumin, did not inhibit proliferation.
Similar results were obtained for PC-3 cells (Figure 3-2) except that, in this case, the only
significant inhibition was for rOvUS. All concentrations tested, including a concentration as low
as 62.5 μg/ml, reduced [3H]thymidine incorporation (P<0.001). While not significant, the
highest concentration of nOvUS (1000 μg/ml) tended to reduce [3H]thymidine incorporation. In
another experiment, there was no inhibitory effect of recombinant β-galactosidase on
proliferation of PC-3 cells. Incorporation of [3H]thymidine in cells treated with 50, 100 and 200
μg/ml β-galactosidase was 114, 110, and 112% of values for cells cultured without β-
galactosidase.
93
Figure 3-1. Effect of OvUS on induction of apoptosis in D17 and PC-3 cells as determined by
TUNEL labeling. Data in panel A illustrate that addition of 1 mg/ml OvUS reduced
incorporation of [3H]thymidine into D17 cells and PC-3 cells. Results for D17 cells
are least square means + SEM and are based on three separate assays and with two
separate batches of OvUS. Results for PC-3 cells are least square means + SEM
and are based on one assay with two separate batches of OvUS. Asterisks represent
means that differ from the control value at P<0.05. Data in panel B represent the
percent of cells that were TUNEL-positive after 24 h culture. Data are from a
representative assay. Panel C shows representative patterns of TUNEL labeling for
D17 cells (top row) and PC-3 cells (bottom row). Note that yellow cells are
considered apoptotic.
94
Figure 3-2. Inhibition of [3H]thymidine incorporation of P388D1 cells and PC-3 cells by native
(n) and recombinant (r) OvUS. Cells were cultured with various concentrations of
nOvUS (open circles), rOvUS (filled circles and solid line) or ovalbumin (OVA)
(filled circles and dashed line). Data represents least square means ± SEM. Means
that differ from untreated cells are indicated by symbols (†P<0.06; ***P<0.001).
0 200 400 600 800 1000
0
2000
4000
6000
8000
10000
12000 PC-3
[3H
]th
ym
idin
e i
nco
rpo
rati
on
(d
pm
)
Col 1 vs Col 2
Col 1 vs Col 4
Col 1 vs Col 6
0
5000
10000
15000
20000
25000
30000
35000
P388D1
Protein concentration (μg/ml)
****** ***
***
******
***
******
***
†
95
Discussion
Results from the present study showed that the antiproliferative effect of OvUS is not
restricted to immune cells, specifically the mitogen-stimulated lymphocytes. Ovine US can
inhibit the proliferation of three different tumor cell lines (D17, PC-3 and P388D1). It is
important to mention that there are differences between the signaling pathways of these tumor
cells and the mitogen-activated lymphocytes. Usually, tumor cells present mutations on genes
that are related to cell growth control. In contrast, lymphocytes are in a resting state and once
activated by mitogens, the expression and production the IL-2 receptor is crucial for proliferation
and activation of these immune cells (Ellery & Nicholls 2002). Thus, it is possible that the
signaling pathways affected by OvUS to inhibit cell proliferation of tumor cells and lymphocytes
are different. It was previously shown that OvUS blocked IL-2 induced proliferation and
reduced expression of CD25 (IL-2Rα chain), but it did not affect the steady state amounts of IL-2
mRNA caused by the mitogen Con A (Peltier et al. 2000c).
There are few serpins that inhibit cell proliferation. The mammary serine proteinase
inhibitor (maspin) is one example where the function of this serpin has been related to the
suppression of tumor growth by the induction of apoptosis of cancer cells and the reduction of
angiogenesis (Sheng 2006). Another example is the pigment epithelium derived factor (PEDF)
which promotes the expression of FasL, activating signal transduction pathways, in particular
caspases 8 and 3 leading to endothelial cell death (Tombran-Tink & Barnstable 2003). Unlike
maspin and PEDF, which inhibit proliferation by inducing apoptosis, OvUS had only a slight
effect on DNA fragmentation (apoptosis) of D17 and PC-3 cells.
There are two other serpins that inhibit cell proliferation, but by a caspase- independent
mechanisms. One is the intracellular serpin, myeloid and erythroid nuclear termination stage-
specific protein (MENT) which is involved in cell cycle progression and therefore in cell
96
proliferation. This serpin inhibits the activity of the nuclear cysteine proteinase SPase, whose
function has been related to the degradation of the phosphorylated form of the retinoblastoma
(Rb) protein, a known regulator of the cell cycle (Irving et al. 2002a). The other serpin is the
intracellular plasminogen activator inhibitor type-2 (PAI-2). This protein could be involved in
the regulation of the cell cycle since it protected Rb from degradation by an independent anti-
proteolytic mechanism (Medcalf & Stasinopoulos 2005, Croucher et al. 2008). However, the
effects of OvUS on cell cycle progression as a mechanism to inhibit cell proliferation remain to
be determined.
97
CHAPTER 4
REGULATION OF DNA SYNTHESIS AND THE CELL CYCLE IN HUMAN PROSTATE
CANCER CELLS AND LYMPHOCYTES BY OVINE UTERINE SERPIN
Introduction
Serine proteinase inhibitors (serpins) inactivate their target proteinases through a suicide
substrate-like inhibitory mechanism. The proteinase binds covalently to the reactive center loop
(RCL) of the serpin and cleaves the scissile bond at the P1-P1’ site. The RCL then moves to the
opposite side to form the β-sheet A and a distortion in the structure of the proteinase that results
in its inactivation (Irving et al. 2000, Van Gent et al. 2003, Law et al. 2006). Not all serpins,
however, exert proteinase inhibitory activity. Some examples are corticosteroid and thyroxine
binding globulins, which function as hormone transport proteins (Pemberton et al. 1988), the
chaperone heat shock protein 47 (Sauk et al. 2005), mammary serine protease inhibitor (Maspin),
which increases the sensitivity of cancer cells to undergo apoptosis (Sheng 2006), and pigment
epithelium derived factor (PEDF), which has neurotrophic, neuroprotective, antiangiogenic, and
proapoptoticactions (Fernandez-Garcia et al. 2007).
Another class of serpins without apparent proteinase activity is the uterine serpins. These
proteins, which are produced by the endometrial epithelium of the pregnant cow, sow, sheep, and
goat (Moffatt et al. 1987, Ing & Roberts 1989, Malathy et al. 1990, Leslie et al. 1990,
Mathialagan & Hansen 1996, Tekin et al. 2005a), have been classified as either a separate clade
of the serpin superfamily (Peltier et al. 2000c) or as a highly-diverge group of the α1-antitrypsin
clade (Irving et al. 2000). The best characterized protein of this unique group of serpins is ovine
uterine serpin (OvUS). This basic glycoprotein is a weak inhibitor of aspartic proteinases
(pepsin A and C) (Mathialagan & Hansen 1996, Peltier et al. 2000a), but it does not inhibit a
broad range of serine proteinases (Ing & Roberts 1989, Liu & Hansen 1995). Additionally,
amino acids in the hinge region of inhibitory serpins are not conserved in uterine serpins and
98
OvUS behaves different in the presence of guanidine HCl than for inhibitory serpins (Peltier et
al. 2000a).
The biological function of OvUS during pregnancy may be to inhibit immune cell
proliferation during pregnancy and provide protection for the allogeneically-distinct conceptus
(Hansen 1998). Ovine US decreases proliferation of lymphocytes stimulated with concanavalin
A, phytohemagglutinin (PHA), Candida albicans, and the mixed lymphocyte reaction (Segerson
et al. 1984, Hansen et al. 1987b, Skopets & Hansen 1993, Skopets et al. 1995, Peltier et al.
2000b). In addition, OvUS decreases natural killer cell cytotoxic activity, abortion induced by
poly(I)•poly(C) in mice (Liu & Hansen 1993) and the production of antibody in sheep
immunized with ovalbumin (Skopets et al. 1995). The antiproliferative actions of OvUS are not
limited to lymphocytes. Ovine US decreases development of the bovine embryos and
proliferation of mouse lymphoma, canine primary osteogenic sarcoma and human prostate
cancer cell lines (Tekin et al. 2005b, 2006).
The mechanism by which OvUS inhibits proliferation of cells is unknown. The protein
could block activation of cell proliferation, inhibit the cell cycle at other points or induce
apoptosis or other forms of cell death. For the PC-3 prostate cancer line, inhibition of cell
proliferation by OvUS might involve reduction in interleukin-8 (IL-8) secretion because of the
importance of autosecretion of this cytokine for cell androgen-independent proliferation (Araki
et al. 2007). The goal of the present study was to evaluate the mechanism by which OvUS
inhibits cell proliferation. Using PC-3 cells as a model system, it was tested whether inhibition
of DNA synthesis involves cytotoxic action of OvUS, induction of apoptosis or disruption of the
IL-8 autocrine loop. It was also tested whether OvUS blocks specific steps in the cell cycle for
PC-3 cells and lymphocytes.
99
Materials and Methods
Materials
The human prostate cancer (PC-3) cell line was purchased from ATCC (Rockville, MD),
the FreeStyleTM 293 expression medium, Dulbecco’s Modified Eagle Medium Nutrient Mixture
F-12 (DMEM-F12) and 0.25% Trypsin-EDTA were obtained from Gibco-Invitrogen (Carlsbad,
CA), the RQ1 RNase-free DNase and the CellTiter-Glo® Luminescent Cell Viability Assay kit
were obtained from Promega, (Madison, WI), the DHLTM Cell Cytotoxicity Assay kit was from
Anaspec (San Jose, CA) and the ELISA MAXTM Set Deluxe kit for human IL-8 was obtained
from BioLegend (San Diego, CA). The in situ cell death detection kit [terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)] was purchased from
Roche (Indianapolis, IN), the DNase-free RNase A was obtained from Qiagen (Valencia, CA),
Precast Tris-HCl gradient Ready gels® were from BioRad (Richmond, CA) and [3H]thymidine
(6.7 Ci/mmol) was from ICN (Irvine, CA). The Prolong Antifade® kit was purchased from
Molecular Probes (Eugene, OR), Geneticin was from Research products international (Mount
Prospect, IL), Centricon filter devices were from Millipore Corporation (Bedford, TX), niquel
Sepharose chromatography medium (high performance) was from Amersham Biosciences
(Piscataway, NJ), fetal bovine and horse serum from Atlanta Biologicals (Norcross, GA). Other
reagents were obtained from either Fisher (Pittsburg, PA) or Sigma-Aldrich (St. Louis, MO).
Purification of rOvUS
The His-tagged rOvUS was purified from conditioned medium of FreeStyleTM human
embryonic kidney (HEK)-293F cells (Gibco-Invitrogen, Carlsbad, CA) transfected with a
plasmid construct containing the gene for OvUS. Details of the cell line are provided elsewhere
(Tekin et al. 2006). Cells were cultured continuously in selective medium [FreeStyleTM 293
expression medium containing 700 μg/ml of Geneticin®] at 37oC in a humidified 8% (v/v) CO2
100
incubator according to the manufacturer’s recommendations. Conditioned medium containing
rOvUS was diluted 1:1 (v/v) in binding buffer [20 mM sodium phosphate buffer, 35 mM
imidazole, 0.3 M NaCl, pH 8.0] and loaded into a nickel Sepharose column that was pre-
equilibrated with binding buffer. The His-tagged rOvUS was eluted with 20 mM phosphate
buffer, 500 mM imidazole, 0.3 M NaCl, pH 8.0, concentrated and buffer-exchanged into
Dulbecco’s phosphate buffered saline (DPBS) using Centricon plus-20 concentration devices.
Purity of the rOvUS was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
using precast 4-15% polyacrylamide Tris-HCl gradient gels. The protein concentration was
determined by Bradford assay (Bradford 1976) using bovine serum albumin as standard.
For each experiment, rOvUS and the control protein, OVA, were added to culture wells of
PC-cells or lymphocytes dissolved in DPBS. The vehicle control included addition of DPBS at
the same volume as for rOvUS and OVA. The actual volume of protein or vehicle added varied
between experiments but was generally 14-25 μl and never more than 50 μl. Cultures were set up
so that the volume of DPBS was the same in all wells.
PC-3 Cell Culture
The PC-3 cell line was cultured continuously in Dulbecco’s Modified Eagle Medium
Nutrient Mixture F-12 (DMEM-F12) supplemented with 10 % (v/v) heat-inactivated fetal bovine
serum, 200 U/ml penicillin and 2 mg/ml streptomycin at 37oC in a humidified 5% (v/v) CO2
incubator. For the IL-8 experiment only, the medium was modified to reduce the fetal bovine
serum concentration to 4% (v/v). For all the experiments, cells were cultured in 75 cm2
flasks
until they reached 50-70% of confluence. Cells were then trypsinized, centrifuged at 110 x g for
5 min and resuspended in fresh complete medium. Cell viability was assessed by trypan blue
exclusion and cell concentration was adjusted according to the requirements of each experiment.
101
[3H]thymidine Incorporation by PC-3 Cells
PC-3 cells (100 μl) were plated overnight at a final concentration of 1 x 105 cell/ml in a 96-
well plate. Afterwards, various concentrations of rOvUS (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 125 and
250 μg/ml) or vehicle were added to each well in a total volume (including additional culture
medium) of 200 μl. After 48 h of culture, 0.1 μCi [3H]thymidine in 10 μl of culture medium
were added. Cells were harvested 24 h after [3H]thymidine addition onto fiber-glass filters using
a cell harvester (Brandel, Gaithersburg, MD). Filters were counted for radioactivity using
scintillation spectrometry (Beckman Coulter Inc., Fullerton, CA). Each concentration of protein
was tested in triplicate and the experiment was performed in six different replicates using a
different batch of rOvUS for each replicate.
Cell Proliferation Based on ATP Content
Aliquots of 50 μl of PC-3 cells (1 x 105 cells/ml) were cultured for 24 h in a dark wall-clear
bottom 96 well plate. Then, treatments consisting of vehicle (DPBS) or three different
concentrations (50, 100 and 200 μg/ml) of rOvUS or a control protein (ovalbumin; OVA) and
culture medium added to bring the final volume to 100 μl. Additional control wells without cells
were prepared to determine background. At 48 h after addition of treatments, ATP content per
well was determined using the CellTiter-Glo® Luminescent Cell Viability Assay kit according to
the manufacturer’s instructions. Briefly, 100 μl of the CellTiter-Glo® reagent were added to
each well, contents of the plate were mixed on a shaker for 2 min and then incubated at room
temperature for 10 min. Chemiluminescence was quantified using a multi-detection microplate
reader (FLX-800, BioTek, Winooski, VT). All treatments were performed in triplicates and the
assay was performed on three different occasions using a different batch of rOvUS for each
replicate.
102
Cytotoxicity Assay
The assay was based on the release of lactate dehydrogenase into culture medium following
loss of cell membrane integrity accompanying cell death (Decker and Lohmann-Matthes, 1988).
Procedures for cell culture and treatments were similar to those described for the ATP assay. At
48 h after addition of the treatments, release of lactate dehydrogenase into the medium was
determined using the DHLTM Cell Cytotoxicity Assay kit following the vendor’s instructions.
Briefly, the plate was equilibrated at room temperature for 20 min before adding 10 μl of lysis
solution or DPBS. To facilitate cell lysing, the plate was placed on a shaker for 2 min. A total of
50 μl lactate dehydrogenase assay solution was then added to each well. After 10 min at room
temperature, the reaction was stopped using 20 μl of the stop solution and the fluorescence
intensity was measured using a multi-detection microplate reader (FLX-800) with excitation and
emission wavelengths of 530-560 nm and 590 nm, respectively. Percent cytotoxicity was
calculated by dividing 100 x fluorescence from the unlysed cells by fluorescence of the lysed
cells. For each assay, each treatment was performed in six wells. The assay was replicated five
different times using a different batch of rOvUS for each replicate.
TUNEL Labeling
An aliquot of 100 μl of PC-3 cells were cultured overnight in chamber slides at a final
concentration of 1 x 104 cells/ml. Then, treatments consisting of vehicle (DPBS), 50, 100 or 200
μg/ml rOvUS, or 200 μg/ml OVA were added and additional culture medium added to produce a
final volume of 300 μl. After 24 and 48 h in culture with treatments, cells were washed with
PBS/PVP [100 mM sodium phosphate pH 7.4, 0.9% (w/v) NaCl, 1 mg/ml polyvinyl pyrrolidone]
and fixed with 4% (w/v) paraformaldehyde for 1 h at room temperature. Cells then were washed
in PBS/PVP and stored at 4oC for the TUNEL (terminal deoxynucleotidyl transferase and
fluorescein isothiocyanate-conjugated dUTP nick end labeling) procedure.
103
For TUNEL labeling, fixed cells were incubated for 1 h at room temperature with
permeabilization solution [PBS, pH 7.4, 0.1 (v/v) Triton X-100, 0.1% (w/v) sodium citrate).
After washing with PBS/PVP, slides were incubated with 50 μl of TUNEL reaction mixture
containing terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-conjugated
dUTP, for 1 hour at 37oC. Positive controls were preincubated with RQ1 RNase-free DNase (50
U/ml) and negative controls were incubated without transferase. Slides were washed with
PBS/PVP, incubated for 1 h with 50 μg/ml of RNase A and then for 30 min with propidium
iodide (2.5 μg/ml) at room temperature. Slides were washed with PBS/PVP and Prolong
Antifade® was used to mount coverslips. Samples were observed using a Zeiss Axioplan 2
fluorescence microscope with dual filter (Carl Zeiss, Inc., Göttingen, Germany). Percent of cells
with DNA fragmentation was determined by counting the total number of nuclei and total
number of TUNEL-labeled nuclei at 10 different sites on the slide. The experiment was
performed using three different batches of rOvUS.
Secretion of IL-8
PC-3 cells (100 μl) were cultured in wells of a 96-well plate overnight at a final
concentration of 1 x 105 cells/ml. Treatments were then added including vehicle (DPBS, similar
volume as for rOvUS and OVA treatments), and three different concentrations of rOvUS and
OVA (50, 100 and 200 μg/ml). The volume of each well was brought to 200 μl with culture
medium. At 48 h after addition of treatments, cell culture supernatants were collected,
centrifuged and stored at -20oC until ELISA for IL-8. Treatments were performed in triplicate
for each assay; the experiment was repeated on three different occasions using three different
batches of the recombinant protein. For the measurement of IL-8, the ELISA MAXTM Set Deluxe
kit for human IL-8 was used according to the manufacturer’s instructions using 100 μl of
conditioned medium.
104
Cell Cycle Analysis
PC-3 cells (100 μl) were cultured in 4 well plates at a final concentration of 4 x 105
cells/ml. After 24 h, treatments consisting of vehicle, 100 and 200 μg/ml rOvUS, and 200 μg/ml
OVA were added with additional culture medium for a total volume of 400 μl. At 12 and 24 h
after addition of treatments, cells were collected by trypsinization and washed with DPBS. Cells
were fixed overnight in 70% (v/v) ethanol at 4oC, washed with DPBS and resuspended with 500
μl of staining solution [DPBS pH 7.4, 0.1% (v/v) Triton X-100, 0.05 mg/ml DNase-free RNase
A, 50 μg/ml propidium iodide]. Cells were then analyzed by flow cytometry using a FACSort
flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and the red fluorescence of single events
was recorded at wavelengths of 488 nm (excitation) and 600 nm (emission). Data were gated
using pulse width and pulse area to exclude doublets, and the percent of cells present in each
phase of the cell cycle was calculated using ModFITLT V3.1 software (Verity Software,
Topsham, ME). The experiment was performed on three occasions with five different batches of
rOvUS.
For the sheep lymphocyte experiment, mononuclear cells were purified by density gradient
centrifugation from the buffy coat of heparinized peripheral blood collected by jugular
venipuncture from non pregnant Rambouillet ewes (Tekin & Hansen 2002). After removing red
blood cells by incubation with red cell lysis buffer (0.01 M Tris-HCl pH 7.5 containing 8.3 g/L
of ammonium chloride), cell viability was assessed by trypan blue exclusion, and concentration
adjusted to 4 x 106 cells/ml. Cells were then suspended in a culture medium consisting of Tissue
Culture Medium-199 containing 5% (v/v) horse serum, 200 U/ml penicillin, 0.2 mg/ml
streptomycin, 2 mM glutamine and 10-5
M β-mercaptoethanol and aliquots of 100 μl cells
cultured in 4 well plates in the presence or absence of 4 μg/ml PHA and with treatments of
DPBS vehicle, 200 μg/ml rOvUS, and 200 μg/ml OVA. Total culture volume was 400 μl. After
105
72 and 96 h in culture at 37oC in a humidified 5% (v/v) CO2 incubator, lymphocytes were
collected and washed with DPBS. Thereafter, lymphocytes were fixed and treated as described
above. The experiment was performed separately for lymphocytes from four different sheep.
Three different batches of rOvUS were tested for each sheep.
Statistical Analysis
Data were analyzed by least-squares means analysis of variance using the General Linear
Models Procedures of SAS (SAS System for Windows, Version 9.0; SAS Institute, Cary, NC,
USA). Error terms were determined based on calculation of expected mean squares with
replicate considered random and other main effects considered fixed. For the cytotoxicity and
IL-8 data, orthogonal polynomial contrasts were used to determine the linear and quadratic
effects of rOvUS and OVA. In other analysis, the pdiff mean separation test of SAS was used to
distinguish the difference of various levels of a treatment.
Results and Discussion
Proliferation of PC-3 cells
The antiproliferative effects of rOvUS on proliferation of PC-3 cells were evaluated by two
different assays. In the first experiment, it was shown that rOvUS caused a concentration-
dependent decrease in incorporation of [3H]thymidine into DNA (P<0.001) with the minimum
effective concentration being 8 μg/ml (Figure 4-1). The antiproliferative actions of OvUS using
[3H]thymidine uptake as the measure of proliferation has been demonstrated previously for PC-3
cells and other cell types (Segerson et al. 1984, Hansen et al. 1987b, Skopets & Hansen 1993,
Skopets et al. 1995, Peltier et al. 2000b, Tekin et al. 2005b, 2006). To confirm this effect of
rOvUS reflected an inhibition in cell proliferation and not a disruption in [3H]thymidine uptake
by the cells, antiproliferative effects were also evaluated by an assay in which the relative total
106
Figure 4-1. Inhibition of [3H]thymidine incorporation of PC-3 cells by recombinant ovine
uterine serpin (rOvUS). The inset graph is provided to clarify the effects of rOvUS
at lower concentrations (≤ 8 μg/ml). Data represent least-squares means ± SEM.
Values that differ from untreated cells are indicated by asterisks (***P<0.001).
rOvUS concentration (μg/ml)
0 50 100 150 200 250 300
[3H
]th
ym
idin
e in
co
rpo
rati
on
(d
pm
)
1500
2000
2500
3000
3500
4000
4500
Protein concentration (μg/ml)
0 2 4 6 8 10
[3H
]thym
idin
e incorp
ora
tion (
dpm
)
2000
2500
3000
3500
4000
4500
***
***
***
***
***
***
rOvUS concentration (μg/ml)
0 50 100 150 200 250 300
[3H
]th
ym
idin
e in
co
rpo
rati
on
(d
pm
)
1500
2000
2500
3000
3500
4000
4500
Protein concentration (μg/ml)
0 2 4 6 8 10
[3H
]thym
idin
e incorp
ora
tion (
dpm
)
2000
2500
3000
3500
4000
4500
***
***
***
***
***
***
107
number of cells per well was estimated by the ATP content per well. Treatment with rOvUS
reduced ATP content per well at all concentrations tested (50, 100 and 200 μg/ml) (Figure 4 -2).
In contrast, the control serpin, ovalbumin, did not cause effect in the ATP content per well. The
finding that rOvUS reduced ATP content per well confirms that the effects of OvUS to reduce
[3H]thymidine incorporation reflect a reduction in cell proliferation rather than interference with
[3H]thymidine transport into the cell.
Lactate Rehydrogenase Release
Possible cytotoxic effects of rOvUS on PC-3 cells were evaluated by measurements of
lactate dehydrogenase release into culture medium (Figure 4-3). None of the concentrations of
rOvUS or OVA tested caused an increase in the percent of lysed cells during culture. Thus,
rOvUS does not inhibit proliferation through induction of cell death.
DNA Fragmentation (Apoptosis)
The TUNEL procedure was used to test whether rOvUS decreased cell proliferation by
induction of DNA fragmentation characteristic of apoptosis and other forms of cell death.
Representative images of TUNEL labeled cells are shown in Figure 4-4 and the average percent
of cells that were TUNEL positive is shown in Figure 4-5. Treatment of PC-3 with either rOvUS
or the control protein OVA did not increase the percent of cells that were TUNEL positive at
either 24 or 48 h after treatment; the percentage of cells that were TUNEL positive was low for
all groups (< 5.7 %). The fact that rOvUS did not induce apoptosis makes the action of this
serpin distinct from that of two other serpins that inhibit cell proliferation. Both maspin (Sheng
2006) and PEDF (Fernandez-Garcia et al. 2007) are proapoptotic serpins.
Interleukin-8 Secretion
Interleukin-8 accumulation in the medium was measured because of the autocrine effect of
this chemokine on prostate cell proliferation (Araki et al. 2007). In addition, at least one class of
108
Figure 4-2. Inhibition of proliferation of PC-3 cells by recombinant ovine uterine serpin
(rOvUS) as determined by ATP content/well. Ovalbumin (OVA) was used as a
negative control. Data represent least-squares means ± SEM. Means that differ
from untreated cells are indicated by symbols (†P<0.1; **P<0.01;***P<0.001).
AT
P C
on
ten
t p
er
well (
RL
U)
0
200
400
600
800
1000
1200
1400
1600
Control 50 100 200 10050 200
rOvUS (μg/ml) OVA (μg/ml)
**
***
†
109
Figure 4-3. Lack of cytotoxic effect of recombinant ovine uterine serpin (rOvUS) on PC-3 cells
was measured by the release of lactate dehydrogenase. Ovalbumin (OVA) was
used as a control protein. Data represent least-squares means ± SEM.
Perc
en
t c
yto
tox
icit
y
0
5
10
15
20
25
30
0
5
10
15
20
25
30
100 200 10050 200
OVA (μg/ml)rOvUS (μg/ml)
Control 50
110
Figure 4-4. Representative photomicrographs of PC-3 cells labeled using the TUNEL procedure
after 48 h of culture with either 100 (A) or 200 µg/ml (B) of rOvUS or 200 μg/ml of
the control protein ovalbumin. Cells in panel D were treated with DNAse as a
positive control.
C
E F
D
111
Figure 4-5. Effect of recombinant ovine uterine serpin (rOvUS) on DNA fragmentation
(apoptosis) of PC-3 cells. Results show the percent of TUNEL positive cells at 24
(A) and 48 (B) h after addition of the treatments. Data represent least-squares
means ± SEM. Ovalbumin (OVA, 200 μg/ml) was used as control protein.
Pe
rce
nt
TU
NE
L p
os
itiv
e c
ells
0
5
10
15
20
rOvUS (μg/ml)
100 OVA50 200Control0
5
10
15
A
B
112
molecule that inhibits PC-3 cell proliferation, soy isoflavones, also reduces IL-8 secretion
(Handayani et al. 2006). As shown in Figure 4-6, there was, however, no effect of rOvUS on
accumulation of IL-8 into conditioned cultured medium. Thus, rOvUS does not block PC-3 cell
proliferation through inhibition of IL-8 secretion.
Cell Cycle Dynamics
Dynamics through the different phases of the cell cycle were affected by the treatment of
PC-3 cells with rOvUS. Representative DNA histograms after treatment with vehicle or 200
μg/ml rOvUS are shown in Figures 4-7A and 4-7B while least-squares means ± SEM for for
results at 12 and 24 h after treatment are shown in Figures 4-7C and 4-7D, respectively. At 12 h
after addition of treatments, rOvUS decreased (P<0.01) the percent of cells in S phase and
increased (P<0.1 and P<0.05 for 100 and 200 μg/ml of rOvUS, respectively) the percent of cells
in the G2/M phase (Figure 4-7C). There was no effect of rOvUS on the percent of cells in G0/G1.
At 24 h after addition of treatment, 200 μg/ml rOvUS increased the percent of cells in G0/G1
(P<0.001), decreased the percent of cells in S phase (P<0.01), and did not affect the percent of
cells in G2/M phase (Figure 4-7D). Control of the cell cycle dynamics by rOvUS was also
evaluated in a second cell type - the peripheral blood lymphocyte. Representative DNA
histograms for PHA-treated lymphocytes are shown for control cells and cells treated with 200
μg/ml rOvUS in Figures 4-8A and 4-8B, respectively while least-squares means ± SEM are
shown in Figures 4-8C and 4-8D. At both 72 (Figure 4-8C) and 96 h (Figure 4-8D) after
addition of PHA, rOvUS increased (P<0.001) the proportion of lymphocytes in the G0/G1 phase
and decreased (P<0.05) the proportion of cells in the S phase. In contrast, there was no effect of
the control protein (OVA) on the distribution of cells in the cell cycle. These results indicate that
OvUS block cell proliferation through cell cycle arrest in both PC-3 and lymphocytes. The
differences in specific stages at which the cell cycle was blocked between PC-3 cells and
113
Figure 4-6. Effect of recombinant ovine uterine serpin (rOvUS) on interleukin (IL) – 8
concentration in cell culture supernatants of PC-3 cells. Ovalbumin (OVA) was
used as control serpin. Data represent least-squares means ± SEM.
Protein concentration (μg/ml)
0 50 100 150 200 250
IL-
8 c
on
cen
tra
tio
n (
pg
/ml)
0
5000
10000
15000
20000
25000
rOvUS
OVA
114
Figure 4-7. Cell cycle dynamics of PC-3 cells as affected by recombinant ovine uterine serpin
(rOvUS). Controls included vehicle (control) and ovalbumin (OVA).
Representative DNA histograms for analysis at 12 h after treatment with vehicle or
200 μg/ml rOvUS are shown in panels A and B, respectively. The least-squares
means for results of three separate assays are shown in panels C and D for analysis
at 12 h (C) and 24 h (D) after treatment. Bars with different superscripts differ (†
P<0.10, others at P<0.05 or less).
0
10
20
30
40
50
60
0
10
20
30
40
50
60
G0/G1 G2/M S
Perc
en
t o
f cell
s
10
20
30
40
50
0
10
20
30
40
50
0 Veh
icleO
VA
200rO
vUS 100
rOvU
S 200
Veh
icleO
VA
200rO
vUS 100
rOvU
S 200
Veh
icleO
VA
200rO
vUS 100
rOvU
S 200
D
C
S 14%
B
G2/M 25%
G0/G1 61%
rOvUS-12h
A
G2/M 20%
G0/G1 57%
S 23%
Control-12h a a a a
a
a a
a
a
a
a
a
a
a aa
ab†
b† b
b
b
bb
115
lymphocytes is likely to be caused by differences in activation and regulation pathways for these
two cell types. Unlike PC-3 cells, lymphocytes are arrested at G0 until proliferation is induced
by addition of PHA. Inhibitions at points in the cell cycle other than G0/G1 are less likely to be
seen since few cells progress to later stages of the cell cycle. In addition, it is possible that
genetic mutations in PC-3 cells compromise some potential regulatory mechanisms. In
particular, unlike lymphocytes, PC-3 cells lack functional p53 (Isaacs et al. 1991) which causes
cell cycle arrest at G1/S by inducing p21cip1
that inhibits cyclin dependent kinases (Harper et al.
1993, Duliæ et al. 1994).
The mechanism by which OvUS inhibits cell cycle dynamics is not understood. One serpin
has been identified which can affect cell cycle regulatory proteins. This serpin, myeloid and
erythroid nuclear termination stage-specific protein (MENT), is a nuclear protein that inhibits
cell proliferation through interactions with a nuclear protein with papain-like cysteine proteinase
activity (Irving et al. 2002a). Inhibition of the proteinase prevents degradation of the cell cycle
protein Rb although antiproliferative effects may depend more on other actions of MENT to
mediate euchromatin condensation in an Rb-independent manner (Irving et al. 2002a). In any
case, OvUS, is apparently without proteinase inhibitory activity and is an extracellular protein
that is unlikely to achieve a nuclear localization. The antipepsin activity of OvUS is probably
not biologically significant. Ovine US is a very weak inhibitor of pepsin C [a 35 and 8 fold
molar excess of OvUS was required to inhibit pepsin A and C (Mathialagan & Hansen 1996)]
and the binding of OvUS to pepsin is electrostatic (Peltier et al. 2000a). Moreover, pepsin
shows an acidic pH optimum and is unlikely to be involved in cell proliferation under the
conditions utilized.
116
Figure 4-8. Cell cycle dynamics of lymphocytes as affected by recombinant ovine uterine serpin
(rOvUS). Controls included vehicle (control), phytohemagglutinin (PHA) and
ovalbumin (OVA). Representative DNA histograms for analysis at 72 h after
treatment with PHA or 200 μg/ml rOvUS are shown in panels A and B,
respectively. The least-squares means for results of four separate assays are shown
in panels C and D for analysis at 72 h (C) and 96 h (D) after treatment. Bars with
different superscripts differ (P<0.05 or less).
Perc
en
t o
f C
ells
0
20
40
60
80
100
120
0
20
40
60
80
S
Veh
icle
OVA
200rO
vUS 200
Veh
icle
OVA
200rO
vUS 200
Veh
icle
OVA
200rO
vUS 200
G0/G1 G2/M
PH
A-C
ontro
l
PH
A-C
ontro
l
PH
A-C
ontro
l
C
D
S 25%
A
G0/G1 75%
Control-PHA 72h
B
G2/M 2%
G0/G1 91%
S 7%
PHA-rOvUS-72h
G2/M 0%
a a
b
b
a
b
b
b
b
c
b
a
b
c
aaaa
bc
a
b b
a
117
A major point in the cell cycle regulated by OvUS is transition from G0/G1 to S phase:
rOvUS decreased the proportion of cells in S phase in all experiments and increased the
proportion of cells in G0/G1 at 24 h after treatment for PC-3 cells and at both times examined for
lymphocytes. Ovine uterine serpin can bind to cell membranes (Liu et al. 1999) and, perhaps,
OvUS inhibits proliferation by activation of signal transduction systems that inhibit transition
from G0/G1 to S phase or prevents pro-proliferative molecules in culture medium from binding
their receptors. Experiments with Rp-8-Cl-cAMPS, a selective inhibitor of cAMP-dependent
type-I protein kinase A, indicated that effects of OvUS on proliferation of PHA-stimulated
lymphocytes are not dependent on this kinase (Tekin et al. 2005b). Studies to determine
activation of other anti-proliferative signal transduction systems by OvUS are warranted.
Taken together, the present study indicates that the mechanism by which OvUS inhibits
proliferation of PC-3 cells and lymphocytes involves cell cycle arrest and not, at least for PC-3
cells, apoptosis, cytotoxicity or inhibition of IL-8 secretion.
118
CHAPTER 5
CHANGES IN EXPRESSION OF CELL-CYCLE RELATED GENES IN PC-3 PROSTATE
CANCER CELLS CAUSED BY OVINE UTERINE SERPIN
Introduction
Uterine serpins (US), also known as uterine milk proteins (UTMP), are members of the
serine proteinase inhibitor (serpin) superfamily (Ing & Robets 1989, Mathialagan & Hansen
1996) and are designated as SERPINA14. These progesterone-induced glycoproteins are
secreted in large quantities into the uterus of a restricted group of mammals during pregnancy
(Malathy et al. 1990, Leslie et al. 1990, Tekin et al. 2005a). The best studied of the US is the
protein found in the sheep. Ovine uterine serpin (OvUS), which is the most abundant protein in
uterine secretions of the pregnant sheep (Moffatt et al. 1897, Hansen et al. 1987a), is an example
of a serpin that has gained a new function while apparently losing proteinase inhibitory activity
characteristic of serpins. Other examples include the heat shock protein 47 (Nagata 1998),
corticosteroid and thyroxine binding globulin (Pemberton et al. 1998) and angiotensinogen
(Morgan et al. 1996).
Inhibitory serpins inactivate their target proteinases by an irreversible suicide substrate-like
mechanism after the proteinase binds to the reactive center loop (RCL) (Silverman et al. 2001).
Usually, inhibitory serpins are recognized by a consensus sequence in the hinge region which is
localized within the RCL of the serpin (Irving et al. 2000) but the hinge region of OvUS is not
conserved with inhibitory serpins (Tekin et al. 2005a, Irving et al. 2000). Ovine US does not
inhibit cathepsins B, D and E (Mathialagan & Hansen 1996), dipeptidyl proteinase IV (Liu &
Hansen 1995), trypsin, chymotrypsin, plasmin, thrombin, elastase and plasminogen activator
(Ing & Robets 1989). While OvUS inhibits the aspartic proteinases pepsin A and C, this
inhibition is atypical for serpins since an excess of 35- and 8-fold molar of OvUS was required
for a 50% inhibition of pepsin A and C, respectively (Mathialagan & Hansen 1996).
119
The role of OvUS during pregnancy has been linked to the protection of the allogeneic
conceptus against the maternal immune system (Hansen 1998). It exerts this role in large part by
inhibiting the proliferation of activated-lymphocytes (Hansen et al. 1987b, Peltier et al. 2000b).
The antiproliferative effect of OvUS is also exerted on some other cell types including mouse
lymphoma (P388D1), canine primary osteogenic sarcoma (D-17) and human prostate cancer
(PC-3) cell lines (Tekin et al. 2005a, 2006).
The mechanism by which OvUS inhibits cell proliferation is poorly understood. Ovine US
did not cause cytotoxic or apoptotic effects on lymphocytes or PC-3 cells (Tekin et al. 2005b,
Skopets & Hansen 1993, Padua & Hansen 2008). It was recently determined that OvUS blocks
cell cycle progression in mitogen-stimulated lymphocytes and increases the number of cells at
the G0/G1 stage at 96 h after addition of the protein (Padua & Hansen 2008). Ovine US also
blocks the progression of the cell cycle of PC-3 cells in a manner that leads to an accumulation
of cells at G2/M at 12 h after addition of the protein and at G0/G1 at 24 h after treatment (Padua
& Hansen 2008). The objective of the present study was to understand the mechanism by which
OvUS blocks cell cycle progression in PC-3 cells by determining cell-cycle related genes whose
expression is altered by OvUS.
Materials and Methods
Materials
The FreeStyleTM 293 expression medium, Dulbecco’s Modified Eagle Medium Nutrient
Mixture F-12 (DMEM-F12) and 0.25% Trypsin-EDTA were purchased from Gibco-Invitrogen
(Carlsbad, CA). The G418 disulfate (geneticin) was purchased from Research products
international (Mount Prospect, IL), nickel Sepharose chromatography medium (high
performance) from Amersham Biosciences (Piscataway, NJ), Precast Tris-HCl gradient Ready
gels® were obtained from BioRad (Richmond, CA) and Centricon® filter devices were from
120
Millipore Corporation (Bedford, TX). The human prostate cancer (PC-3) cell line was from
ATCC (Rockville, MD), [3H]thymidine (6.7 Ci/mmol) was from ICN (Irvine, CA) and fetal
bovine and horse sera from Atlanta Biologicals (Norcross, GA). Other reagents were obtained
from either Fisher (Pittsburg, PA) or Sigma-Aldrich (St. Louis, MO).
Purification of rOvUS
Human embryonic kidney (HEK)-293F (Gibco-Invitrogen, Carlsbad, CA) cells transfected
with a plasmid construct containing the gene for OvUS (Tekin et al. 2006) were cultured
continuously in selective medium [FreeStyleTM 293 expression medium containing 700 μg/ml of
geneticin] at 37oC in a humidified incubator containing a gas environment of 8% (v/v) CO2 in air.
The rOvUS was purified by using immobilized metal ion (nickel) exchange chromatography as
described by Padua & Hansen, 2008. Briefly, rOvUS was eluted with 20 mM phosphate buffer,
500 mM imidazole, 0.3 M NaCl, pH 8.0, concentrated and buffer-exchanged into Dulbecco’s
phosphate buffered saline (DPBS) using Centricon plus-20® concentration devices. Sodium
dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions using 4-15%
polyacrylamide Tris-HCl gradient gels and Coomassie Blue were used to assess the purity of the
rOvUS. After filter-sterilization of rOvUS using 0.22 μm micro centrifuge devices, the
concentration of the protein was determined by Bradford assay (Bradford 1976) using bovine
serum albumin as standard.
PC-3 Cell Culture
The PC-3 cell line was cultured into 75 cm2
flasks continuously in complete medium
[Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM-F12) supplemented with
10 % (v/v) heat-inactivated fetal bovine serum, 200 U/ml penicillin and 2 mg/ml streptomycin]
at 37oC in a humidified incubator with a gas environment 5% (v/v) CO2 in air. Cells were then
trypsinized after reaching 50-70% of confluence, centrifuged at 110 x g for 5 min and
121
resuspended in fresh medium. The viability of the cells was assessed by trypan blue exclusion
and cell concentration was adjusted according to each experiment.
Proliferation Assay
PC-3 cells were cultured in 96 well plates in a volume of 100 μl and at a final concentration
of 1 x 104 cells/ml. After 24 h in culture, cells were treated with either rOvUS, ovalbumin
(OVA, a control serpin dissolved in DPBS) or vehicle (DPBS). The vehicle was added to control
wells at an equivalent volume (6-20 μl) as for the rOvUS and OVA. Additional culture medium
was added to all wells to bring the final volume to 200 µl. The final concentration of rOvUS and
OVA was 200 μg/ml. After 48 h of culture, an aliquot of 10 µl of culture medium containing 0.1
µCi [3H]thymidine was added to the wells. Cells were collected on fiber glass filters by using a
cell harvester (Brandel, Gaithersburg, MD) 24 h after [3H]thymidine addition. Radioactivity on
the filters was counted by scintillation spectrometry (Beckman Coulter Inc., Fullerton, CA). The
experiment was replicated on five different occasions using a total of four different batches of
rOvUS. For each replicate, each treatment was tested in triplicate.
Cell Culture for RNA Extraction
PC-3 cells were cultured in 4 well plates at a final concentration of 4 x 105 cells/ml in 100
μl. After 24 h of culture, treatments and complete medium were added to achieve a final
concentration of 200 μg/ml rOvUS or an equivalent volume of DPBS as experimental control in
a final volume of 400 μl. At 12 or 24 h after treatment addition, medium was removed from the
plates and cell lysed for total RNA cell extraction as described below. The experiment was
replicated four times, with a different batch of rOvUS for each replicate.
RNA Extraction
Total RNA was extracted using the RNeasy® plus micro kit (Qiagen Inc, Valencia, CA)
following the manufacturer’s instructions. Briefly, PC-3 cells were lysed in wells for 5 min by
122
repeat pipetting using 350 μl of lysis buffer supplied in the kit. Cell lysates were transferred into
microcentrifuge tubes, vortexed for 1 min and placed into gDNA eliminator columns to remove
genomic DNA. After mixing with 70% (v/v) ethanol, samples were transferred RNeasy
MinElute spin columns, washed and total RNA eluted with RNase-free water. RNA
concentration and quality was determined by the Agilent 2100 BioAnalyzer (Agilent
Technologies, Santa Clara, CA) at the Gene Expression Core Laboratory of the Interdisciplinary
Center of Biotechnology Research, University of Florida. High-quality RNA was used for the
RT-PCR array experiments (RNA integrity numbers ≥ 8.0).
cDNA Synthesis and Real Time-PCR Array
The cDNA for each RNA sample was obtained by using the Super Array RT2 First Strand
kit (SABiosciences Corporation, Frederick, MD) according to the manufacturer’s instructions.
Briefly, after genomic DNA elimination, the reverse transcription reaction was performed at
42oC for 15 min and then heated at 95
oC for 5 min to inactivate the enzyme. The cDNA was
mixed with the RT2 SYBR green/ROX q PCR master mix (SABiosciences Corporation) and 25
µl aliquots were loaded into each well of the RT2 Profiler PCR Array (SA Biosciences
Corporation, catalog number PAHS-020A). The PCR array was designed to study the profile of
84 human cell-cycle related genes (Table 5-1). The PCR array experiments were performed on
an ABI 7300 instrument (Applied Biosystems, Foster City, CA). The conditions for
amplification were as follows: 1 cycle of 10 min at 95oC followed by 40 cycles of 15 sec at 95
oC
and 1 min at 60oC.
Statistical Analysis
The General Linear Models procedure of SAS (SAS System for Windows, Version 9.0;
SAS Institute, Cary, NC, USA) was used to analyze the data from the proliferation experiments
by the least-square means analysis of variance. All main effects were considered fixed and the
123
Table 5-1. Cell cycle related genes screened using the RT2 Profiler
TM PCR Array.
1House keeping genes.
2Human Genomic DNA contamination control.
3Reverse Transcription control.
4Positive PCR control.
ABL1 ANAPC2 ANAPC4 DIRAS3 ATM ATR BAX BCCIP BCL2 BIRC5 BRCA1 BRCA2
CCNB1 CCNB2 CCNC CCND1 CCND2 CCNE1 CCNF CCNG1 CCNG2 CCNH CCNT1 CCNT2
CDC16 CDC2 CDC20 CDC34 CDK2 CDK4 CDK5R1 CDK5RAP1 CDK6 CDK7 CDK8 CDKN1A
CDKN1B CDKN2A CDKN2B CDKN3 CHEK1 CHEK2 CKS1B CKS2 CUL1 CUL2 CUL3 DDX11
DNM2 E2F4 GADD45A GTF2H1 GTSE1 HERC5 HUS1 KNTC1 KPNA2 MAD2L1 MAD2L2 MCM2
MCM3 MCM4 MCM5 MKI67 MNAT1 MRE11A NBN PCNA RAD1 RAD17 RAD51 RAD9A
RB1 RBBP8 RBL1 RBL2 RPA3 SERTAD1 SKP2 SUMO1 TFDP1 TFDP2 TP53 UBE1
B2M1 HPRT1 RPL13A1 GADPH1 ACTB1 HGDC2 RTC3 RTC3 RTC3 PPC4 PPC4 PPC4
124
model included effects of treatments and batch of rOvUS. Differences between levels of a
treatment were determined by the pdiff mean separation test of SAS.
The PCR array data were analyzed by the ∆∆Ct method. Genes with Ct values greater than
35 cycles were considered as non-detectable and assigned a value of 35. The average of four
house keeping genes [Beta-2-microglobulin (B2M), Hypoxanthine phosphoribosyltransferase 1
(HPRT1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB)] was used
to obtain the ∆Ct value for each gene of interest. The ∆∆Ct value for each gene was calculated
by the difference between the ∆Ct of the treated and the ∆Ct of the control groups. The fold-
change for each gene was calculated by 2- ∆∆Ct
and the statistical analysis to determine
differences between treatments was performed using the RT2 Profiler PCR Array Data Analysis
web-based software (SABiosciences Corporation).
Results
Inhibition of PC-3 Cell Proliferation by OvUS
The inhibitory effect of OvUS on the proliferation of PC-3 cells is shown in Figure 5-1.
Incorporation of [3H]thymidine into the DNA of PC-3 cells was reduced (p<0.05) by rOvUS. In
contrast, the control serpin OVA did not affect [3H]thymidine incorporation.
Cell-Cycle Related Gene Expression Profile at 12 h after Treatment with rOvUS
The mRNA expression of 17 genes was significantly altered by rOvUS (Table 5-2). Three
genes involved in cell cycle checkpoint and arrest were up-regulated. These genes were
CDKN1A (p21cip1
), CCNG2 (cyclin G2), and CDKN2B (p15ink
). In addition, the mRNA for 14
genes was decreased by rOvUS. Among these are 3 genes (MCM3, MCM5 and PCNA), whose
gene products are required at the S phase of the cell cycle for DNA synthesis and replication.
Others are genes involved in the regulation and progression at the M phase [CDC2, CKS2,
CCNH (cyclin H), BIRC5 (survivin), MAD2L1, MAD2L2] and at the G1 phase (CDK4, CUL1,
125
Figure 5-1. Inhibition of [3H]thymidine incorporation of PC-3 cells by 200 μg/ml of the
recombinant ovine uterine serpin (rOvUS). Ovalbumin (OVA) was used as control
serpin and Dulbecco’s phosphate buffered saline (DPBS) as vehicle. Data represent
least-squares means ± SEM. Bars with different letters differ (p<0.05).
[3H
]th
ym
idin
e in
co
rpo
rati
on
(d
pm
)
0
5000
10000
15000
20000
25000
Vehicle(DPBS)
rOvUS OVA(200 μg/ml)
aa
b
(200 μg/ml)
[3H
]th
ym
idin
e in
co
rpo
rati
on
(d
pm
)
0
5000
10000
15000
20000
25000
Vehicle(DPBS)
rOvUS OVA(200 μg/ml)
aa
b
(200 μg/ml)
126
Table 5-2. Regulation of cell-cycle related genes of PC-3 cells after 12 h of treatment with 200
µg/ml recombinant ovine uterine serpin.
a CDK2-associated dual specificity phosphatase
b Mitotin (S. cerevisiae)
c Cell division cycle 46 (S. cerevisiae)
Gene
Symbol Description AVG∆Ct ± SE
Fold
Change 95%CI P-value
Up-regulated Control Treat
CCNG2 Cyclin G2 7.545 ± 0.29 6.134 ± 0.31 2.6585 (1.12, 4.20) < 0.05
CDKN1A CDK inhibitor 1A ( p21, Cip1) 4.078 ± 0.05 2.727 ± 0.24 2.5502 (1.70, 3.40) < 0.01
CDKN2B CDK inhibitor 2B , p15 8.25 ± 0.36 7.037 ± 0.32 2.3184 (0.81, 3.83) < 0.05
Down-regulated
BIRC5 Survivin 6.885 ± 0.11 7.514 ± 0.16 0.6465 (0.47, 0.82) < 0.05
CCNH Cyclin H 3.783 ± 0.03 4.042 ± 0.06 0.8354 (0.76, 0.91) < 0.01
CDC2 Cell division cycle 2 1.45 ± 0.07 1.844 ± 0.06 0.7608 (0.67, 0.85) < 0.01
CDK4 Cyclin-dependent kinase 4 3.45 ± 0.06 3.942 ± 0.08 0.7111 (0.61, 0.81) < 0.01
CDKN3 Cyclin-dependent kinase inhibitor 3a 1.265 ± 0.05 1.519 ± 0.07 0.8384 (0.74, 0.94) < 0.05
CKS2 CDC28 protein kinase regulatory subunit 2 1.13 ± 0.07 1.437 ± 0.06 0.8084 (0.71, 0.91) < 0.05
CUL1 Cullin 1 4.375 ± 0.07 4.629 ± 0.06 0.8384 (0.74, 0.94) < 0.05
MAD2L1 MAD mitotic deficient-like 1 3.06 ± 0.06 3.622 ± 0.17 0.6774 (0.51, 0.85) < 0.05
MAD2L2 MAD mitotic deficient-like 2 4.593 ± 0.06 4.944 ± 0.09 0.7836 (0.66, 0.90) < 0.05
MCM3 Minichromosome maintenance deficient 3b 2.563 ± 0.17 3.632 ± 0.25 0.4765 (0.28, 0.67) < 0.05
MCM5 Minichromosome maintenance deficient 5c 5.965 ± 0.16 6.604 ± 0.05 0.642 (0.50, 0.79) < 0.05
PCNA Proliferating Cell Nuclear Antigen 2.225 ± 0.20 2.909 ± 0.08 0.6223 (0.44, 0.80) < 0.05
RAD1 RAD1 homolog (S. pombe) 7.41 ± 0.06 7.719 ± 0.07 0.807 (0.71, 0.91) < 0.05
RBBP8 Retinoblastoma Binding Protein 8 4.233 ± 0.15 4.927 ± 0.15 0.618 (0.44, 0.80) < 0.05
127
CDKN3). The last two genes that were down-regulated were DNA damage checkpoint and
repair genes RBBP8 (retinoblastoma binding protein 8) and RAD1.
Cell-Cycle Related Gene Expression Profile at 24 h after Treatment with rOvUS
Treatment of PC-3 cells with rOvUS for 24 h caused down-regulation of 16 genes (Table 5-
3). Some of them (BIRC5, CDK4, CDKN3, CKS2, MAD2L2 and RAD1) were also down-
regulated at 12 h. The others are genes related to the regulation and progression through the M
(CCNB1, CDK5RAP1, CDC20 and E2F4) and G1 (CDK4 and TFDP2). Likewise, rOvUS
down-regulated three DNA damage checkpoint related genes (RAD17, KPNA2 and BRCA1)
whose activation blocks cell cycle progression at all stages of the cell cycle. The expression of
BCCIP (BRCA2 and CDKN1A interacting protein) a gene involved in DNA repair, spindle
formation and cytokinesis was also down-regulated by rOvUS. In addition, rOvUS caused the
down-regulation of MKI67. The gene product of MKI67 (ki-67) is a marker of cell proliferation.
Discussion
It was previously shown that OvUS inhibited cell proliferation of PC-3 cells by disrupting
cell cycle progression (Padua & Hansen 2008). The results presented in this study corroborate
those findings and provide an overview of the changes in gene expression that are associated
with alterations in the cell cycle. In particular, the inhibition of the cell cycle progression is
initially associated with increased expression of genes that block cell cycle and decreased
expression of genes needed for progression through G1, S and M phases. After more prolonged
treatment, the inhibition of expression of genes required for cell cycle progression is extended to
a wider range of genes.
In an earlier study, OvUS caused accumulation of PC-3 cells at the G2/M phase at 12 h
128
Table 5-3. Down-regulation of human cell cycle-related genes of PC-3 cells after 24 h of
treatment with 200 µg/ml recombinant ovine uterine serpin.
Gene
Symbol Description AVG∆Ct ± SE
Fold
change 95%CI P-value
Down-regulated Control Treat
BCCIP BRCA and CDKN1A interacting protein 2.63 ± 0.16 3.17 ± 0.10 0.6881 (0.51, 0.87) < 0.05
BIRC5 Survivin 6.21 ± 0.09 7.32 ± 0.29 0.4643 (0.27, 0.65) < 0.05
BRCA1 Breast cancer 1, early onset 6.41 ± 0.16 7.29 ± 0.30 0.5445 (0.29, 0.80) < 0.05
CCNB1 Cyclin B1 1.10 ± 0.06 2.00 ± 0.30 0.537 (0.31, 0.76) < 0.01
CDC20 Cell Division Cycle 20 homolog 0.53 ± 0.20 1.55 ± 0.17 0.4916 (0.32, 0.67) < 0.01
CDK4 Cyclin-dependent kinase 4 2.88 ± 0.11 3.41 ± 0.12 0.6941 (0.54, 0.85) < 0.05
CDK5RAP1 CDK5 regulatory subunit associated protein 1 9.07 ± 0.19 9.65 ± 0.13 0.6704 (0.46, 0.88) < 0.05
CDKN3 Cyclin-dependent kinase inhibitor 3a 1.02 ± 0.01 1.68 ± 0.24 0.6354 (0.43, 0.84) < 0.05
CKS2 CDC28 protein kinase regulatory subunit 2 0.91 ± 0.08 1.64 ± 0.20 0.6042 (0.43, 0.78) < 0.05
E2F4 E2F transcription factor 7.14 ± 0.14 7.62 ± 0.07 0.7148 (0.57 0.86) < 0.05
KPNA2 Karyopherin α 2 (RAG cohort 1, importin α1) 1.97 ± 0.23 2.89 ± 0.25 0.5269 (0.29, 0.77) < 0.05
MAD2L2 MAD2 mitotic arrest deficient-like 2 3.99 ± 0.08 4.69 ± 0.04 0.6148 (0.54, 0.69) < 0.001
MKI67 Antigen identified by monoclonal antibody Ki-67 3.35 ± 0.24 4.48 ± 0.34 0.4579 (0.20, 0.72) < 0.05
RAD1 RAD1 homolog (S. pombe) 6.96 ± 0.06 7.33 ± 0.13 0.7741 (0.62, 0.92) < 0.05
RAD17 RAD17 homolog (S. pombe) 6.38 ± 0.06 6.56 ± 0.02 0.8831 (0.80, 0.96) < 0.05
TFDP2 Transcription factor Dp-2 7.21 ± 0.09 7.49 ± 0.04 0.8211 (0.71, 0.93) < 0.05 a CDK2-associated dual specific phosphatase.
129
after treatment (Padua & Hansen 2008). It is clear from examination of Figure 5-2A that
different phases of the cell cycle are disrupted by OvUS at 12 h.
Ovine US caused an increase in mRNA expression of CDKN1A (p21cip1
), CDKN2B
(p15ink
) and CCNG2 (cyclin G2), all of which are involved in cell cycle checkpoint and arrest. It
is very likely that the upregulation of expression of these genes is the proximal cause for the
inhibition of cell cycle progression. CDKN1A for example, belongs to the cyclin dependent
kinase inhibitor (CDKI) family and inhibits cell cycle progression by inhibiting CDK2 and
CDK4 and by blocking DNA replication and repair by binding to PCNA (Harper et al. 1993,
Waga et al. 1994, Li et al. 1994, Cayrol et al. 1998). This inhibitor of cell cycle progression
causes arrest at G1, S and G2 phases (Harper et al. 1993, Krug et al. 2002). CDKN2B also
belongs to the CDKI family and binds to CDK4 and CDK6 to prevent their association with
cyclin D, thereby blocking the cell cycle at G1 (Krug et al. 2002). Finally, CCNG2 (cyclin G2) a
non-typical cyclin whose expression is independent of p53 (Bates et al. 1996) blocks cell cycle
progression at the G1/S phase by association with the active protein phosphatase 2A (Bennin et
al. 2002). The CCNG2 gene is also expressed at the late S and G2 phases (Le et al. 2007).
At 12 h, OvUS caused down-regulation of genes involved in DNA replication (PCNA,
MCM3 and MCM5) at the S phase and those implicated in the regulation and progression of the
cell cycle at M (CDC2, CCNH, MAD2L1, MAD2L2) and G1 (CDK4, CUL1, CDKN3) phases
(Figure 5-2A). As an example, CCNH (cyclin H) is the regulatory subunit of the cdk-activating
kinase (CAK) and is distinct from mitotic cyclins because is expressed constantly through the
cell cycle. The function of cyclin H is related to the phospholylation of different cyclin-
dependent kinases (CDKs) and components of the transcriptional machinery (Kaldis 1999). At
130
Figure 5-2. Points in the cell cycle where genes were differentially regulated by ovine uterine
serpin at 12 and 24 h are represented in panel A and B, respectively. Up-regulated
genes are in green and down-regulated genes in red. Genes that block cell cycle
progression are underlined. Genes that were regulated at 12 and 24 h are shown
with an asterisk (*). Genes involved in DNA repair are in the center of the cycle to
represent that many of these are involved in several stages of the cell cycle.
G1
G2
S
M
S-phase
entry
M-phase
entry
MKI67
Anaphase
TelophaseMAD2L2*
BIRC5*
CDC20
CDK4*
CDKN3*
MKI67
CKS2*
CCNB1
CDK5RAP1
E2F4
MKI67
TFDP2
BCCIP G0
B
RAD1*
RAD17
BRCA1
KPNA2
BCCIP
G1
G2
S
M
S-phase
entry
M-phase
entry
MKI67
Anaphase
TelophaseMAD2L2*
BIRC5*
CDC20
CDK4*
CDKN3*
MKI67
CKS2*
CCNB1
CDK5RAP1
E2F4
MKI67
TFDP2
BCCIP G0
B
RAD1*
RAD17
BRCA1
KPNA2
BCCIP
G1
G2
S
M
S-phase
entry
M-phase
entry
CCNG2
CDKN1A
PCNA
MCM3
MCM5
Anaphase
TelophaseMAD2L1
MAD2L2*
BIRC5*
CDKN1A
CDKN2B
CDK4*
CDKN3*
CUL1
CDKN1A
CDC2
CCNH
CKS2*CCNG2
CDKN1A
CCNG2
A
PCNA
RAD1*
RBBP8
G1
G2
S
M
S-phase
entry
M-phase
entry
CCNG2
CDKN1A
PCNA
MCM3
MCM5
Anaphase
TelophaseMAD2L1
MAD2L2*
BIRC5*
CDKN1A
CDKN2B
CDK4*
CDKN3*
CUL1
CDKN1A
CDC2
CCNH
CKS2*CCNG2
CDKN1A
CCNG2
A
PCNA
RAD1*
RBBP8
131
24 h, downregulation of cell cycle related genes became more widespread (Figure 5-2B). Some
genes whose mRNA were decreased at 12 h remained so at 24 h (BIRC5, CDK4, CDKN3,
CKS2, MAD2L2 and RAD1). Additional genes were also decreased at 24 h (CCNB1,
CDK5RAP1, CDC20, E2F4, TFDP2, RAD17, KPNA2 and BRCA1, BCCIP and MKI67).
Down-regulation of genes such as CCNB1, BIRC5, CDK4, CDC20 and TFDP2 would impede
progression through the G1, S and G2/M phases (Figure 5-2B). For example, CDC20 is the
activator of the anaphase-promoting complex/cyclosome (APC/C) required for the metaphase-
anaphase progression during mitosis (Baker et al. 2007). It was inhibited by 50% by OvUS.
The down-regulation of MKI67 at 24 h suggests that a proportion of PC-3 cells entered the
resting state (G0) since the gene product for MKI67 (ki-67) is a cell marker linked to
proliferation and is present in all stages of the cell cycle with the exception of the G0 stage
(Gerdes et al. 1984). This is consistent with the global decrease in gene expression observed at
24 h and also with earlier observations where OvUS caused an increase in the proportion of PC-3
cells at G0/G1 phase at 24 h after treatment (Padua & Hansen 2008).
Upregulation of expression of CDKN1A (p21cip
), CDKN2B (p15ink
) and CCNG2 (cyclin
G2) at 12 h is likely to be a cause for the down-regulation of genes at 12 and 24 h. It has been
shown that high levels of expression of CDKN1A (p21cip
) down-regulates the expression of
BIRC5 (survivin) in other cells (Lühr & Müritz 2003, Xiong et al. 2008). Also, it is possible that
the down-regulation of expression of some genes is the cause for the inhibition of transcription
of other genes. As an example, the down-regulation of CKS2 causes a reduction in the
transcription of CCNB1 (cyclin B1) and CDC2 (CDK1) (Martinsson-Ahlzén et al. 2008). The
activity of CDK1 is required for the regulation of some DNA repair pathways (Aylon et al. 2004,
Branzei & Foiani 2008).
132
The lack of functional p53 on PC-3 cells (Isaacs et al. 1991) probably affects feedback
loops in response to OvUS. This protein is an effector molecule in the DNA repair pathway and
lack of p53 abolishes the DNA checkpoints response and apoptosis (Sancar et al. 2004, Gatz &
Wiesmüller 2006). Moreover, cells with disrupted p53 can overcome controls at the G2/M
checkpoint and fail to maintain a sustained arrest at this stage (Bunz et al. 1998).
All genes studied that are related to DNA damage checkpoints or repair (RBBP8, RAD1,
RAD17, BRCA1, KPNA2, PCNA and BCCIP) were down-regulated either at 12 or 24 h after
OvUS treatment. The products of these genes are induced in response to incomplete DNA
replication or damage at specific or several points of the cell cycle (Waga et al. 1994, Sancar et
al. 2004, Sartori et al. 2007, Teng et al. 2003, Lu et al. 2005). Thus, OvUS disrupts the
transcription of some genes involved in nucleotide excision, mismatch, homologous
recombination repair and translesion synthesis pathways in addition to the sensor (RAD1 and 17)
and mediator molecules (BRCA1) of the DNA damage checkpoint. The failure of OvUS to
induce apoptosis in PC-3 cells (Tekin et al. 2005b, Padua & Hansen 2008) despite these changes
in gene expression, could reflect the lack of p53 or an as yet undescribed anti-apoptotic action of
OvUS.
Ovine US is one of the few serpins identified that alters cell-cycle dynamics. The other is
the intracellular protein MENT which also has a very basic isoelectric point (9 versus 5-6.5 for
other serpins) (Silverman et al. 2001). MENT inhibits the enzymatic activity of the nuclear
cysteine proteinase SPase, a cathepsin L-like proteinase involved in the degradation of the
phosphorylated form of the retinoblastoma (Rb) protein, a known regulator of the cell cycle
(Irving et al. 2002a). Another intracellular serpin, PAI-2, can protect Rb from degradation by an
independent anti-proteinase mechanism (Croucher et al. 2008). Unlike MENT and PAI-2, OvUS
133
is an extracellular protein that can bind to cell membranes (Liu et al. 1999). Nonetheless, it is
possible that OvUS inhibits cell proliferation by being internalized. Other extracellular serpins
can become internalized and affect cell function. An example is α1-antitrypsin, which enters and
resides in the cytoplasm of a mouse insulinoma cell line and protects against apoptosis through
inhibition of caspase-3 activation (Zhang et al. 2007).
Alternatively, OvUS blocks cell proliferation through blockage or induction of signal
transduction systems. In lymphocytes, OvUS inhibits proliferation of phorbol myristol acetate
stimulated lymphocytes, suggesting that the protein blocks downstream actions of the protein
kinase (PK) C pathway (Peltier et al. 2000b). Like OvUS, transforming growth factor (TGF)-β
causes cell cycle arrest by upregulation of CDKN2B (p15ink
), CDKN1A (p21cip1
) and CCNG2
(cyclin G2) (Horne et al. 1997, Gartel &d Tyner 2002). Interferon (IFN)-γ also induces
CDKN1A (p21cip1
) expression, independent of p53 by the STAT-1 pathway (Gartel & Tyner
2002). Both, TGF-β and IFN-γ signaling can cause transactivation of p21 by a p53-independent
mechanism, where p21 protects cells against p53-independent apoptosis which is induced by
these signals (Gartel & Tyner 2002). Signal transduction pathways affected by OvUS have not
been determined, but OvUS may regulate components of signaling pathways shared with TGF-β
or IFN-γ pathways.
In summary, OvUS inhibits proliferation of PC-3 cells through disruption of the cell cycle
dynamics. Disruption involves increased expression of cell-cycle checkpoint and arrest genes
CDKN1A (p21cip1
), CDKN2B (p15ink
) and CCNG2 (cyclin G2) and down-regulation of genes
involved in cell-cycle progression.
134
CHAPTER 6
GENERAL DISCUSSION
The serpin genes are an ancient origin gene family, being identified in bacteria, fungus,
nematodes, archaea and virus (Irving et al. 2000, 2002b, Steenbakkers et al. 2008). Evidence
presented in this dissertation strongly indicates that one of the subclades in this superfamily, the
uterine serpins, evolved only within a restricted group of mammals, the Laurasiatheria
superorder of eutherian mammals. The uterine serpins are thus an example of a new gene arising
from gene duplication and selection for sequence divergence (Louis 2007) that plays a particular
role in pregnancy in a group of mammals with unique attributes for gestation (an epitheliochorial
placenta).
The distribution of known uterine serpins as well as orders where a uterine serpin was not
identified is shown in Figure 2-9. All the identified uterine serpins are in Ruminatia (sheep,
goat, water buffalo and cow), Suidae (pig), Perisodactyla (horse) or Carnivora (dog) orders of
Laurasiatheria. An important question is whether uterine serpin genes are in the orders within
the Laurasiatheria superorder (Cetacea, Hippopotamidae, Pholidota, Chiroptera,
Erinaceomorpha, Scalopus and Talpa). Most of these orders have epitheliochorial placenta
although the Chiroptera, Erinaceomorpha and Talpa orders have either endotheliochorial or
hemochorial type of placentation. Experiments in Chapter 2 strongly suggested that the uterine
serpin gene is modified (dog) or become a pseudogene (cat) in carnivore species with
endotheliochorial placenta. Examination of the situation in species of the remaining orders of
the Laurasiatheria superorder with either endotheliochorial or hemochorial placenta would allow
determination of whether similar changes in uterine serpin genes are occurring within these
orders. If the gene is either lost or modified in species of the Chiroptera, Erinaceomorpha and
135
Talpa orders, the idea that uterine serpins play a specific role required for pregnancy in species
with epitheliochorial type of placentation would be strengthened.
As indicated in Figure 2-9, there is an additional order of animals having epitheliochorial
placenta that is not within Laurasiatheria, the lemurs and lorises of the order Lemuriformes
within the Euarchontoglires superorder. Identification of the uterine serpin gene in this species
would indicate that the genes predate the diversion of Laurasiatheria from Euarchontoglires
superorder and that epitheliochorial placentation is absolutely dependent upon the uterine serpin
genes. Peltier et al. (2000c) estimated that uterine serpin genes diverged from CBG before the
divergence of mammals so much a result remains possible. It is more likely, however, that
uterine serpins will not be found in Lemuriformes since, even if the uterine serpin gene is older
than the common ancestor of Laurasiatheria and Euarchontoglires, the gene would be likely be
lost in the ancestors of lemurs and lorises that did not have epitheliochorial placenta.
The results from the positive (Darwinian) selection study reported in Chapter 2 strongly
suggest that there is pressure on the evolution of the uterine serpin gene to affect its
functionality. Positive selection has occurred at the RCL region of the inhibitory serpins, in
particular at the P1 site proximal to the scissile bond of the RCL, which changes the selectivity
for the target proteinase (Brown 1987, Hill & Hastie 1987, Goodwin et al. 1996, Zang & Maizels
2001, Barbour et al. 2002). The uterine serpins apparently lack anti-proteinase activity (Ing &
Roberts 1989, Liu & Hansen 1995, Mathialagan & Hansen 1996, Peltier et al. 2000a; see also
Chapter 2 for lack of conservation at residues important for anti-proteinase activity). There is
also evidence that the uterine serpin gene has evolved different functions within mammals with
the gene. It is possible that the uterine serpin gene in species with epitheliochorial placentation
has been retained as a secretory protein while the gene in species with endotheliochorial
136
placentation has either been changed to become an intracellular protein (dog) or to be lost (cat).
If this idea is correct, uterine serpins play an important role in the uterine lumen of species with
epitheliochorial placentation that is not required for species with endotheliochorial placentae.
Even within species with epitheliochorial placenta, there might be different functions. The
PoUS-1 and -2, for example, regulate iron stability in uteroferrin and may be involved in iron
transport to the fetus (Baumbach et al. 1989, Roberts & Bazer 1988) while the OvUS can inhibit
proliferation of lymphocytes and certain other cell types (Tekin et al. 2005b, Padua & Hansen
2008; also see Chapters 3, 4 and 5).
Inhibition of lymphocyte proliferation has been interpreted as signifying a role for uterine
serpins in prevention of rejection of conceptus tissue by the maternal immune system (Hansen
1998). The sheep protein OvUS for example, has been shown to inhibit proliferation of mitogen-
stimulated lymphocytes, NK cell activity and a variety of cancer cells (Segerson et al. 1984,
Hansen et al. 1987b, Stephenson et al. 1989b, Skopets & Hansen 1993, Skopets et al. 1995,
Padua & Hansen 2008). In addition, the limited invasiveness of trophoblast tissue in species
with epitheliochorial placentatation could be achieved, at least in part, by inhibition of
trophoblast proliferation by uterine serpin. It may also be that uterine serpins participate in the
process of trophoblast binucleate cell formation, a phenomenon characterized by nuclear
endoreduplication without proper cytokinesis, which occurs in sheep, goat, cattle and horse
(Hoffman & Wooding 1993).
Ovine uterine serpin acts to inhibit cell proliferation by inducing changes in gene
expression of proteins involved in the cell cycle and, most notably, the up-regulation of cell
cycle inhibitors such as CDKN1A (p21cip
), CDKN2B (p15ink
) and CCNG2 (cyclin G2) (Chapter
5). These cell cycle inhibitors have been related to differentiation of stromal cells into decidual
137
cells, formation of polyploidy stromal cells as well as terminal differentiation and apoptosis of
luminal and stromal cells during decidualization (Tan et al. 2002, Yao et al. 2003, Yue et al.
2005, Li et al. 2008).
Ovine US is one of the few serpins identified that inhibits cell proliferation by blocking
cell-cycle progression (Chapter 4). In PC-3 cells, OvUS decreased the percent of cells in S phase
and increased the percent of cells in the G2/M phase at 12 h after treatment addition. At 24 h,
OvUS increased the percent of cells in G0/G1 and decreased the percent of cells in S phase. In
the mitogen (PHA) stimulated lymphocyte, at both 72 and 96 h after stimulation, OvUS
increased the proportion of lymphocytes in the G0/G1 phase and decreased the proportion of cells
in the S phase (Chapter 4). There are two other serpins that can alter cell cycle progression. One
is the intracellular protein MENT which also inhibits cell proliferation by blocking cell cycle
progression, inhibiting the enzymatic activity of the nuclear cysteine proteinase SPase, a
cathepsin L-like proteinase involved in the degradation of the phosphorylated form of the Rb
protein, a known regulator of the cell cycle (Irving et al. 2002a). The other is the intracellular
serpin, PAI-2 that can protect Rb from degradation by an independent anti-proteinase mechanism
(Croucher et al. 2008).
The signal transduction pathway activated or down-regulated by OvUS remains to be
determined. Unlike MENT and PAI-2, OvUS is an extracellular protein that can bind to cell
membranes (Liu et al. 1999). Ovine US could either regulate signal transduction pathways by
binding to a specific membrane receptor or by becoming internalized into the cell and interacting
with intracellular binding partners (Figure 6-1). It is possible that OvUS inhibits cell
proliferation by being internalized through endocytosis. Other extracellular serpins can become
internalized and affect cell function. An example is α1-antitrypsin, which enters and resides in
138
Figure 6-1. Possible pathways by which OvUS could block cell proliferation. A) OvUS
could bind to its own receptor or to another ligand’s receptor and activate a
pathway that blocks cell cycle progression. B) OvUS could block the binding
site of a ligand needed for cell cycle progression so that the pathway for
activation is not stimulated. C) OvUS could become internalized and block
cell cycle progression after internalization by binding too and disrupting
intracellular binding partners that are the effectors of the cell cycle arrest.
Yellow circles represent the phosphorylated status of the proteins in the signal
transduction pathway.
?
?
?
Nucleus
G1/S
Cytoplasm
OvUS
?
?
AB
C
p21p15
Cyclin G2
G2/M
?
?
?
Nucleus
G1/S
Cytoplasm
OvUS
?
?
AB
C
p21p15
Cyclin G2
G2/M
139
the cytoplasm of a mouse insulinoma cell line and protects against apoptosis through inhibition
of caspase-3 activation (Zhang et al. 2007). Alternatively, OvUS could bind to a specific cell
surface receptor or compete with other molecules for a receptor binding site (Figure 6-1), like
α1-antitrypsin which competes with diferric transferrin for the trasnferrin receptors inhibiting the
proliferation of human skin fibroblasts (Graziadei et al. 1988). In lymphocytes, OvUS inhibits
proliferation of phorbol myristol acetate stimulated lymphocytes, suggesting that the protein
blocks downstream actions of the PKC pathway (Peltier et al. 2000b). OvUS could regulate
components of signaling pathways shared with TGF-β or IFN-γ pathways. Like OvUS, TGF-β
causes cell cycle arrest by upregulation of CDKN2B (p15ink
), CDKN1A (p21cip1
) and CCNG2
(cyclin G2) (Horne et al. 1997, Gartel & Tyner 2002). Interferon-γ also induces CDKN1A
(p21cip1
) expression by the STAT-1 pathway (Gartel & Tyner 2002). Thus, it is possible that
uterine serpins are involved in the process of trophoblast binucleate cell formation and/or uterine
luminal and stromal cell differentiation.
In the dog (Chapter 2) as well as in the cow (Khatib et al. 2007), the uterine serpin gene
was recently identified to be expressed in the ovary. The presence of uterine serpin in other
reproductive tissues distinct to the uterus raises the possibility that perhaps uterine serpins may
have different functions according to the cell type and stage of differentiation. One possibility is
that uterine serpin functions in the ovary to regulate follicular growth by binding to activin.
Activin improves follicular growth and granulosa cell proliferation (Knight & Glister 2006).
OvUS can also this member of the TGF-β family (McFarlane et al. 1999). However, the binding
affinity of activin for OvUS is much lower than activin affinity towards follistatin.
It is also possible that OvUS is participating in the differentiation of granulose cells into
luteal cells after ovulation. In the ovary, granulosa cells luteinize under the influence of
140
luteinizing hormone. The expression of cell cycle inhibitors has been involved in the inhibition
of proliferation of differentiating granulosa cells in mice, specifically the CDK inhibitors
p21Cip1/Waf1
and p27Kip1
which are up-regulated throughout this period (Burns et al. 2001,
Jirawatnotai et al. 2003). Likewise, p15ink4b
, another CDK inhibitor has been linked with
luteinization and granulosa cell differentiation (Burns et al. 2001). Results presented in this
dissertation showed that OvUS blocked cell cycle progression and up-regulated the expression of
the CDK inhibitors CDKN1A (p21cip1
) and CDKN2B (p15ink
) and also caused the down-
regulation of MKI67 (Chapter 5). The gene product of MKI67 (Ki-67) is a marker of cell
proliferation and is present in all stages of the cell cycle with the exception of the G0 stage
(Gerdes et al. 1984).
In summary, the uterine serpin gene is present only in a restricted group of species within
the Laurasiatheria superorder of eutherian mammals and likely evolved under positive selection
which suggests diversifying functionality of the protein within these species. The finding that
the uterine serpin has been retained as a secretory protein in most species with epitheliochorial
placentation within the Laurasiatheria superorder also suggests that the protein has an important
role during pregnancy in species with this kind of placenta. The finding that OvUS inhibits cell
proliferation by blocking cell cycle progression, specifically by the upregualtion of cell cycle
inhibitors suggests other possible functions for uterine serpins in reproductive tissue, in
particular trophoblast binucleate cell formation, uterine cell differentiation and granulosa cell
differentiation in the ovary.
141
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BIOGRAPHICAL SKETCH
Maria Beatriz Padua was born in 1968 in Caracas, Venezuela. She graduated from El
Carmelo High School in the same city in 1985 and enrolled the next year in the School of
Agronomy at the University Centroccidental Lisandro Alvarado in Barquisimeto, Lara State,
Venezuela where she received her Bachelor of Science degree in Agricultural Engineering in
1994. In 1995, she worked as sales representative for the veterinary health supply firm Grupo
Catalina C.A. in Caracas, Venezuela. From 1996 to 2000, she was a pharmacist assistant in
Celbefar C.A. Farmacia El Roble in the same city. She participated in an English language
program at the University of Florida in 2001 and she enrolled in the Animal Molecular and
Cellular Biology Graduate Program at the University of Florida under the supervision of Dr.
Peter J. Hansen in April, 2002. She received her Master of Science degree in 2004 and her thesis
was elected as the best thesis for the Department of Animal Sciences. She is currently a Doctor
of Philosophy candidate. Upon completion of her degree, she will pursue a postdoctoral
position.
