Primer
1. The 4-epimer of Glucose once it is 2-N-acetylated is called?2. If the SNFG shape of a monosaccharide is a diamond it is an ___?3. Which of the 11 common mammalian monosaccharides is:
a) a pentose?b) a deoxyhexose?c) never incorporated as a sugar nucleotide?
4. If a GT uses a one-step mechanism it is retaining or inverting?5. Name:
6. Shape/Color:
Gal GlcNAc Neu5Gc Xyl GlcA
O-Glycosylation
Ser/Thr
GalNAc
Mucins
Ser/Thr
Man*
Yeast mannoproteinsα-dystroglycan
cadherins
Ser/Thr
Fuc*
NotchCoagulation FactorsFibrinolytic Factors
Ser/Thr
GlcNAc*
Nuclear ProteinsCytoplasmic Proteins
After Esko, J
Ser/Thr
Glc*
NotchThrombospondin
Factor IX
ALSO: Proteoglycans, Hydroxyproline/Hydroxylysine Glycosylation
* SEPARATE LECTURES
O-Glycosidic Linkage
α
Ser
GalNAc
After Esko, J
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
β-elimination
O
OH
H
H
HO
H
O
NAcHH
OH
NH2CHC
H2C
O
GalNAc
α
Glycan synthesisin a cellular context
Most O-Glycosylatedproteins are synthesizedin the secretory pathway
O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
Mucin-Type O-GalNAc Glycans• Major extracellular vertebrate
O-glycan• Begins in cis-Golgi (ERGIC) by
attachment of GalNAc in α-linkage to specific Ser/Thr residues
• Assembly is simpler than N-linked chains - no lipid intermediate is used
• Always involves nucleotide sugars
• Always occurs by addition to non-reducing terminus or by branching
β4
β3
β4
β3
β4
α3
α3
α3
Ser/Thr
β3 β6
After Esko, J
Polypeptide GalNAc Transferases
• >20 ppGalNAcT family members• Share structural features in active site• Some have lectin (ricin) domain
Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R).
After Esko, J
Core 1 and Core 2 Synthesis
After Esko, J
T (TF)Antigen
TnAntigen
Core 2GlcNAcT
Core 1GalT(cis)
Ser/Thr
β3 β6
Ser/Thr
β3
Ser/Thr
α6
α3
ST6GalNAc1(trans)
Ser/Thr
SialylTn
AntigenSer/Thr
β3
Ser/Thr
β3 α6Disialyl
TAntigen
From: Tongzhong Ju and Richard D. Cummings
Core 4GlcNAcT
Core 3GlcNAcT
Ser/Thr
β3 β6
Ser/Thr
β3
Ser/Thr
Core 3 and Core 4 Synthesis
After Esko, J
Core 3
Ser/Thr
β3
Core 4
Ser/Thr
β3 β6
Ser/Thr
β3
Core 1
Ser/Thr
β3 β6
Core 2
Core 7
Ser/Thr
α6
Core 6?
Ser/Thr
β6
Core 5
Ser/Thr
α3
Core 8
Ser/Thr
α3
Unusual Core O-Glycan Structures
After Esko, J
Mucins are Heavily O-glycosylated
• Apomucin contain tandem repeats (8-169 amino acids) rich in proline, threonine, and serine (PTS domains)
• Glycosylation constitutes as much as 80% of mass and tends to be clustered - bottle brush
• Expressed by epithelial cells that line the gastrointestinal, respiratory, and genito-urinary tracts
After Esko, J
Lung Epithelium
Goblet cells in intestinal crypts
Mucin Production
After Esko, J
Mucins: Protective Barriers for Epithelial Cells
• Lubrication for epithelial surfaces• Modulate infection:
– Receptors for bacterial adhesins– Secreted mucins can act as decoys
• Barrier against freezing:– Antifreeze glycoproteins– [Ala-Ala-Thr]n≤40 with Core 1
disaccharides
After Esko, J
Antigenic epitopes found in mucins
β4
β3
β4
β3
β4
α3
α3
α3
Ser/Thr
β3 β6
Type-3 H, A, and B antigens that form the O (H), A, and B blood group determinants on N- and O-glycans
Chapter 13, Figure 6Essentials of Glycobiology
Second Edition
A1 and A2 blood group antigens
Chapter 13, Figure 9Essentials of Glycobiology
Second Edition
Type-1 and -2 Lewis determinants
Chapter 13, Figure 10Essentials of Glycobiology
Second Edition
Lewis blood groups
Chapter 13, Figure 11Essentials of Glycobiology
Second Edition
Terminal GlcNAc residues are usually galactosylated
Chapter 13, Figure 2Essentials of Glycobiology
Second Edition
Poly-N-acetyllactosamine chains
Chapter 13, Figure 3Essentials of Glycobiology
Second Edition
i and I antigen synthesis
Chapter 13, Figure 4Essentials of Glycobiology
Second Edition
Synthesis and structure of the HNK-1 epitope
Chapter 13, Figure 21Essentials of Glycobiology
Second Edition
Structure and synthesis of the Galα1-3Gal antigen
Chapter 13, Figure 13Essentials of Glycobiology
Second Edition
NOT IN HUMANS!!!
Synthesis of glycoproteins bearing terminal α2-3 sialic acid transferred by the ST3Gal family of sialyltransferases
Chapter 13, Figure 17Essentials of Glycobiology
Second Edition
Synthesis of the human Sda or mouse CT antigen
Chapter 13, Figure 16Essentials of Glycobiology
Second Edition
Synthesis of α2-6 sialic acid on O-glycans and glycolipids by the ST6GalNAc family of sialyltransferases
Chapter 13, Figure 18Essentials of Glycobiology
Second Edition
Complex O-GalNAc glycans with different core structures
Chapter 9, Figure 2Essentials of Glycobiology
Second Edition
Questions
• What is the function of multiple polypeptide GalNAc transferases?
• How is tissue specific expression of transferases regulated?
• How does competition of transferases for substrates determine the glycoforms expressed by cells and tissues?
• What roles do chaperones play?
After Esko, J
O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
O-Fuc
• One of the clearest examples of glycosylation (Fringe) modulating signal transduction
• What other proteins carry O-Fuc and how does glycosylation modulate activity?
• How is glycosylation regulated?
• Separate Lecture (Haltiwanger)
O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
Shao, L. et al. Glycobiology 2002 12:763-770; doi:10.1093/glycob/cwf085
O-Glc Pathway
Rumi is OGluT
KDEL Retention Signal
Temp. Sensitive Mutation
O-Glc• Always a trisaccharide?• Glc & Xyl (except for proteoglycans) rarely
used on mammalian glycoproteins--why both here? Many of the same proteins as O-Fuc modifed, why?
• Role in Modulating Signaling? Regulated by enzymes or sugar nucleotide availability?
• Separate Lecture (Haltiwanger)
O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
POMT1in the ER
In Complex
WithPOMT2
Uses Dol-ManAs Donor
Alpha-Dystroglycan
Muscular Dystrophies associated with glycosylation of α – DG (oversimplified)
Disease Species Affected
Gene Biochemical
Lesion Biochemical Phenotype Walker - Warburg Syndrome Human POMT1 O - Man addition to
Ser/Thr Decreased protein O - mannosylation
Muscle - Eye - Brain D isease Human POMGnT1 Addition of GlcNac
β 2 to O - Man Underglycosylated α - DG, uncapped O - Man
Fukuyama - type MDC Human Fukutin Glycosyltransferase -
like protein Underglycosylated α - DG
Limb - Girdle and MDC 1C Human
Fukutin - Related Protein
Glycosyltransferase - l ike Golgi protein Underglycosylated α - DG
Myodystrophy, myd Mouse
MDC 1D Human LARGE Glycosyltransferase -
like Golgi protein Underglycosylated α - DG
Other Disease causing genes: B4GAT1, POMGnT2, DPM1-3, ISPD, TMEM5 ….
Mammalian O-Mannosylation
POMGnT1 POMGnT1+GnTVb POMGnT2
The ECM binding O-Man Structure (also the receptor for certain clades of arenaviruses including Lassa)
Praissman J L et al. eLife Sciences 2014;3:e03943
Other O-Mannosylated Proteins:
1. The Cadherin Family of Proteins -also involved in adhesion
2. RPTPβ-Receptor protein tyrosine receptor
3. OTHERS???
O-Man
• O-Man is clearly involved in CMD• What mammalian proteins (especially in the
brain) are O-Man modified besides α-DG?• What are the roles for M1, M2, and M3
glycans?• What is relationship between O-Man and O-
GalNAc?
• Separate Lecture (Wells)
O-Glycosylation
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
-6-P
-6-P
-6-P
-6-P
-1-P
UDP-
GlycogenPPP Glycolysis Hexosamine Biosynthetic Pathway
(HBP)
UDP-
- -O
UDP-
CMP-
Complex Glycosylation
A.
B.
Key:
= Glc
= Fru
= GlcN
= GlcNAc= GalNAc= Neu5Ac
Features of O-GlcNAc• NOT elongated• Nucleocytoplasmic Proteins• Dynamic & Inducible• Enzymes for its Addition (OGT) and
Removal (O-GlcNAcase) are Nucleocytoplasmic
• OGT Knock-Out is Lethal at the ES cell stage– X-linked OGT, Females Heterozygotes are NOT mosaic
• Reciprocity with Phosphorylation (Yin-Yang) on Some Proteins
Classes of Proteins Modified By O-GlcNAc ���>1,000 Proteins Identified to Date
❖ Cytoskeletal ComponentsTau, Vinculin
❖ Hormone ReceptorsERα&β
❖ Kinases & Other Signaling Molecules CKII, eNOS, IRS-2
❖ Nuclear Pore ProteinsNUP62, NUP155
❖ Oncogenes & Tumor Suppresors
p53, Rb
❖ Transcription Factorsc-myc, Sp1, Pax6
❖ Metabolic EnzymesGAPDH, Pyruvate Kinase
❖ Transcriptional and Translational Machinery
RNA Pol II, EIF4A
❖ Viral ProteinsSV40 T Antigen, v-Erb-a
❖ Heat-Shock ProteinsHSP90, α-crystallin
O-GlcNAc Challenges• Assigning function to specific site on particular protein
(mapping sites)– Recent data has demonstrated this– Great example CAMKIV (O-GlcNAc in active site)– Another Example: Fox01 repression of gluconeogenic
enzymes in liver– Another Example: O-GlcNAc of Histone required for
mono-Ub modification• Regulation of OGT/OGA (PTM, protein:protein
associations)
• Separate Lecture (Wells)
Ser/Thr
GalNAc
Ser/Thr
Man
Ser/Thr
Fuc
Ser/Thr
GlcNAc
Ser/Thr
Glc
Hyl HypSer
Gal Xyl
β
A few more O-glycans……
O-Xyl….precursor for GAGs…Separate Lecture (Bergmann)
O-GlcNAc (nucleocytoplasmic….separate lecturenote: recent report of O-GlcNAc on Notch extracellular domain (different enzyme (eOGT) than the intracellular one (OGT))
O-Glycosylation of Hyl
O-Glycosylation of Hyl
• Found on Collagen and Adiponectin (which has a “collagen-like” domain)
• Glycosylation Essential for Basement Membrane Formation in Tissues
• Modulates Collagen Cross-linking?• Other proteins with modification?
After Esko, J
The Glycosaminoglycans
���O-Glycosylation���
-less studied (until recently?)-tools to study are underdevelopment-in many cases, clear functional effect