1

FINAL ACCEPTED VERSION: LCMP-00429-2004.R1

The Amino-terminal TACE Pro-domain Attenuates TNFR2

Cleavage Independent of the Cysteine Switch

Caitriona A. Buckley a

Farshid N. Rouhani a

Maryann Kaler a

Barbara Adamik a, b

Feras I. Hawari a, c

Stewart J. Levine a

a Pulmonary-Critical Care Medicine BranchNational Heart, Lung, and Blood Institute

National Institutes of HealthBuilding 10, Room 6D03, MSC 1590

Bethesda, Maryland 20892-1590

Running Title: The Amino-terminal TACE Pro-domain Inhibits TACE

Address Correspondence to:

Stewart J. Levine, M.D.Pulmonary-Critical Care Medicine Branch,National Heart, Lung, and Blood InstituteNational Institutes of HealthBuilding 10, Room 6D03, MSC 1590Bethesda, Maryland 20892-1590Phone: 301-402-1448Fax: 301-435-2883Email: levines@nhlbi.nih.gov

b Dr. Adamik is on scientific leave from the Department of Anesthesiologyand Intensive Therapy, Wroclaw Medical University, Poland.

c Dr. Hawari’s current address is the Division of Pulmonary and CriticalCare Medicine, King Hussein Cancer Center, Amman, Jordan.

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (March 4, 2005). doi:10.1152/ajplung.00429.2004

Copyright © 2005 by the American Physiological Society.

2

Abstract

TNF-α converting enzyme (TACE, ADAM17) cleaves membrane-associated

cytokines and receptors and thereby regulates inflammatory and immune events,

as well as lung development and mucin production. For example, the TACE-

mediated cleavage of the type II, 75-kDa TNF receptor (TNFR2) generates a

soluble TNF-binding protein that modulates TNF bioactivity. TACE is

synthesized as a latent pro-enzyme that is retained in an inactive state via an

interaction between its pro-domain and catalytic domain. Although, the formation

of an intramolecular bond between a cysteine in the pro-domain and a zinc atom

in the catalytic site had been thought to mediate this inhibitory activity, it was

recently reported that the cysteine switch motif is not required. Here, we

hypothesized that the amino-terminus of the TACE pro-domain might contribute

to the ability of the pro-domain to maintain TACE in an inactive state,

independent of a cysteine switch mechanism. We synthesized a 37-amino acid

peptide, corresponding to TACE amino acids 18 to 54 (N-TACE18-54), and

assessed whether it possessed TACE inhibitory activity. In an in vitro model

assay system, N-TACE18-54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc

substrate. Further, N-TACE18-54 inhibited constitutive TNFR2 shedding from a

human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain,

corresponding to TACE amino acids 30 to 48, demonstrated partial inhibitory

activity. In summary, we have identified a sub-domain within the amino-terminus

of the TACE pro-domain that attenuates TACE catalytic activity, independent of a

cysteine switch mechanism, which provides new insight into the regulation of

TACE enzymatic activity.

3

Introduction

TNF-α converting enzyme (TACE) or ADAM17, a member of the disintegrin and

metalloprotease family of zinc metalloproteases, is an important regulator of

inflammation, immune regulation, and cellular proliferation as a consequence of

its ability to process cell surface integral membrane proteins to soluble forms(2,

4, 24). TACE was originally identified as the enzyme that cleaves the

membrane-bound precursor of tumor necrosis factor-α (TNF-α), as well as the

type II, 75-kDa tumor necrosis factor receptor (TNFR2, TNFRSF1B, CD120b),

TGF-α, and L-selectin(3, 23, 24, 29). Other cell surface proteins that have been

identified as substrates for TACE include cytokines, chemokines, growth factors,

adhesion molecules, and cytokine and growth factor receptors, as well as the

cellular prion protein and the amyloid precursor protein(1, 6-8, 14, 17, 20, 29, 31-

33, 37, 42, 43, 47, 48).

TACE plays an important role in both lung development and the pathogenesis of

pulmonary disease. TACE is expressed by a variety of cells in the lung, including

alveolar macrophages, bronchial epithelial cells, and vascular smooth muscle

cells(13). Lungs from embryonic TACE-deficient mice display impaired

branching morphogenesis, inhibited epithelial cell proliferation and differentiation,

and delayed vasculogenesis, thereby demonstrating a role for TACE in normal

lung maturation(49). TACE also regulates mucin production by human airway

epithelial cells. Activation of TACE by phorbol ester, Pseudomonas aeruginosa,

or liposaccharide (LPS) catalyzes the cleavage of pro-TGF-α into soluble mature

TGF-α, which then binds to and induces the phosphorylation of the epidermal

growth factor receptor (EGFR), with resultant MUC5AC expression(39).

Cigarette smoke, via a process that may involve oxygen free radicals, also

activates TACE with resultant ligand-dependent EGFR phosphorylation and

MUC5AC production(38). Following activation, TACE undergoes stimulation-

dependent internalization, which may down-regulate catalytic activity at the

plasma membrane(11). This may be relevant to the pathogenesis of community

4

acquired pneumonia as epithelial lining fluid cells from infected lungs have down-

regulated cell surface TACE expression as compared to cells obtained from

uninvolved lungs(16).

ADAM family zinc metalloproteases, including TACE, have a conserved structure

that includes, from N- to C-terminus, a signal sequence, pro-domain,

metalloprotease domain, disintegrin domain, cysteine-rich domain containing a

epidermal growth factor-like repeat, a transmembrane domain and an

intracytoplasmic tail(3, 23, 24, 35, 36). An important function of the pro-domain

is to retain the pro-enzyme in an inactive state. The formation of an

intramolecular bond between a cysteine in the pro-domain and a zinc atom in the

catalytic site had been thought to mediate this inhibitory activity via a cysteine

switch mechanism. It was recently reported, however, that the cysteine switch

motif is not required for the inhibitory activity of the pro-domain(15). The TACE

pro-domain may also play an important role in protein folding, as TACE mutants

lacking the pro-domain are inefficiently synthesized in Sf9 cells, and possibly

undergo intracellular degradation(21). As found with other ADAM-family

members, cleavage of the TACE pro-domain typically occurs C-terminal to a

consensus proprotein-convertase sequence (RX(K/R)R)(12). Removal of the

TACE pro-domain is catalyzed by furin and other proprotein-convertases, such

as PC7, in the late Golgi compartment(5, 12, 28, 35).

In the present study, we hypothesized that the amino-terminal region of the

TACE pro-domain might contribute to the ability of the TACE pro-domain to

maintain TACE in an inactive state, independent of a cysteine switch

mechanism(15). We synthesized a 37-amino acid peptide that corresponds to

TACE amino acids 18 to 54 (N-TACE18-54), but does not contain the consensus

cysteine-switch motif (PKVCGY186)(21). N-TACE18-54 attenuated TACE-catalyzed

TNFR2 cleavage in a model assay system and constitutive TNFR2 shedding

from the human U937 monocytic cell line. A 19-amino acid, leucine-rich domain,

which corresponds to N-TACE amino acids 30 to 48, possessed partial TACE

5

inhibitory activity. Therefore, we propose that a sub-domain within the amino-

terminus of the TACE pro-domain attenuates TACE catalytic activity,

independent of the cysteine switch mechanism. This study provides new insight

into the ability of the TACE pro-domain to regulate TACE enzymatic activity.

6

Methods

Characterization of N-TACE18-54 Inhibitory Activity

A model assay system was developed to assess the ability of N-TACE18-54 to

attenuate TACE enzymatic activity. Recombinant human TACE (rhTACE), as

well as the recombinant human TNFR1:Fc (rhTNFR1:Fc) and TNFR2:Fc

(rhTNFR2:Fc) fusion proteins were purchased from R & D Systems (Minneapolis,

MN). Both TNFR1:Fc and TNFR2:Fc are recombinant human chimeric proteins

that encode the extracellular receptor domain, fused to a carboxy-terminal 6X-

histidine-tagged Fc region of human IgG1 via a linker peptide (IEGRMD).

Recombinant human TACE corresponds to the mature form after removal of the

pro-domain and has an apparent molecular size of 70 kDa. N-TACE18 – 54 was

synthesized by Sigma-Genosys (The Woodlands, TX). N-TACE18 – 54 truncation

mutants were also synthesized; an amino-terminal mutant corresponding to N-

TACE amino acids 18 – 29, a mid-domain mutant corresponding to N-TACE

amino acids 30 – 42, a carboxy-terminal mutant corresponding to amino acids 43

– 54, and an extended mid-domain mutant corresponding to N-TACE amino

acids 30 to 48 (N-TACE30-48). A scrambled peptide containing the N-TACE18 – 54

amino acids in a random order was also synthesized by Sigma-Genosys (The

Woodlands, TX). Chou-Fasman analysis was performed using MacVector

(Accelrys, Burlington, MA). Vasoactive intestinal peptide (VIP) and α-defensin

were purchased from Bachem (Torrance, CA). TAPI-2 was purchased from

Peptides International (Louisville, KY). 1,10-phenanthroline monohydrate and

zinc chloride were purchased from Sigma-Aldrich (St. Louis, MO).

Assays (50 µl) were performed in 50 mM Tris-HCl and 25 mM NaCl, pH 8.0 and

incubated at 30o C for 30 min. Proteins were separated by SDS-PAGE using 4%

- 12% Bis-Tris NUPAGE gels (Invitrogen, Carlsbad, CA) and visualized with the

SilverQuest Silver Staining Kit (Invitrogen, Carlsbad, CA). For Western blot

analysis, proteins were separated via SDS-PAGE, electroblotted onto

7

nitrocellulose membranes, and incubated overnight (4o C) with a murine IgG1

monoclonal antibody (200 ng/ml) directed against the 6X-histidine tag (Tetra-His,

Qiagen, Valenica, CA), which reacts with the C-terminus of the recombinant

human TNFR2:Fc fusion protein. A rabbit polyclonal antibody generated against

N-TACE amino acids 18 to 54 (Sigma-Genosys, The Woodlands, TX) was

utilized for Western blotting at a 1:1000 dilution. Detection was by

chemiluminescence using horseradish peroxidase-conjugated secondary

antibodies.

Quantification of TNFR2 Shedding

U937 cells were purchased from ATCC (Manassas, VA) were maintained in

RPMI-1640 medium with 10% fetal bovine serum. U937 cells were plated in 6-

well plates at a density of 2 x 106 cells/well in 1 ml of media. Release of TNFR2

into U937 cell culture medium during a 24-hour period was quantified utilizing a

sandwich ELISA (R & D Systems). Cellular apoptosis and necrosis were

measured using the TACS Annexin V-FITC Apoptosis Detection Kit (R & D

Systems, Minneapolis, MN) and a XL-MCL Flow Cytometer (Beckman-Coulter,

Miami, FL). Statistical analysis was performed by a paired Student's t test with a

Bonferroni correction for multiple comparisons and by single factor ANOVA.

Differences were considered significant at a P value < 0.05.

8

Results

N-TACE18-54 Attenuates TACE-Mediated TNFR2 Cleavage

Experiments were conducted to assess whether the amino-terminus of the TACE

pro-domain can regulate TACE catalytic activity, independent of the cysteine

switch mechanism. A peptide corresponding to amino acids 18 to 54 of the

TACE coding sequence (N-TACE18-54), which lacks the hydrophobic signal

peptide sequence and the cysteine switch consensus motif, was synthesized

(Figure 1). The N-TACE18-54 amino acid sequence was deduced from a RT-PCR

product generated from NCI-H292 human pulmonary epithelial cell line total RNA

and primers spanning the full-length TACE coding region. N-TACE18-54 has a

predicted molecular weight of 4186 daltons and pI of 4.61.

The inhibitory activity of N-TACE18-54 was assessed in an in vitro assay system

utilizing recombinant human TACE (rhTACE) and tumor necrosis factor chimeric

receptor fusion proteins as model substrates. Since rhTNFR1:Fc and

rhTNFR2:Fc each contain the entire extracellular domain of the receptor, we

reasoned that they might be susceptible to cleavage by rhTACE. As shown in

Figures 2A, rhTACE cleaved the rhTNFR2:Fc model substrate, generating two

predominant cleavage products, which were detected by silver staining. Further,

the TACE-catalyzed cleavage of the TNFR2:Fc model substrate was attenuated

by 80 µM N-TACE18-54. In contrast, rhTACE did not cleave the rhTNFR1:Fc

model substrate. Since both rhTNFR1:Fc and rhTNFR2:Fc encode the same

linker and Fc region of IgG1, we conclude that rhTACE cleaves TNFR2, but not

TNFR1 or the IgG1 chimera. Therefore, in subsequent experiments,

rhTNFR2:Fc was used as a substrate to assess the ability of N-TACE18-54 to

attenuate rhTACE activity. The ability of rhTACE to cleave the rhTNFR2:Fc

model substrate was also shown to be zinc-dependent, which is consistent with

the classification of TACE as a member of the ADAM family of zinc

metalloproteases. As shown in Figure 2B, incubation with the predominantly

9

zinc-specific chelator, 1,10-phenanthroline, significantly attenuated rhTACE-

mediated TNFR2:Fc cleavage, which was partially restored by the addition of 25

to 100 µM zinc chloride. As has been described for other zinc metalloproteases,

a further increase in ZnCl2 concentration resulted in a decline in enzyme

activity(10).

As shown by SDS-PAGE and silver staining (Figure 3A), N-TACE18-54 attenuated

the proteolytic cleavage of 0.95 µM rhTNFR2:Fc by 0.5 µM rhTACE in a

concentration-dependent fashion between 20 µM to 160 µM. The identity of

rhTNFR2:Fc and its cleavage products was confirmed by immunoblotting utilizing

an anti-6X-His antibody, which reacts with the C-terminal 6X-His tag of the

rhTNFR2:Fc chimeric protein (Figure 3B). Taken together, these experiments

demonstrate that N-TACE18-54 can attenuate TACE proteolytic activity towards

TNFR2.

We next performed experiments utilizing irrelevant peptides, α-defensin and

vasoactive intestinal peptide (VIP), to assess the specificity of N-TACE18-54

attenuation of TACE-catalyzed TNFR2 cleavage. As shown in Figure 4, neither

α-defensin nor VIP affected the ability of TACE to proteolytically cleave

rhTNFR2:Fc. In contrast, TACE-catalyzed rhTNFR2 cleavage was partially

inhibited by 80 µM N-TACE18-54 and completely inhibited by 25 µM TAPI-2, a

hydroxamic acid-based zinc metalloprotease inhibitor. These experiments are

consistent with the conclusion that the inhibitory activity of N-TACE18-54 is not a

non-specific peptide effect. We also assessed whether N-TACE18-54 is a

substrate for TACE catalytic activity. There was no decrease in the quantity of N-

TACE18-54 by immunoblotting after incubation with rhTACE for 4 hr (data not

shown), suggesting that N-TACE18-54 is not a substrate for TACE.

10

Characterization of N-TACE18-54 Inhibitory Activity

Experiments were next performed to characterize the N-TACE domains that

mediate its inhibitory activity. Truncation mutants were synthesized

corresponding to the amino-terminal (N), middle (M), and carboxy-terminal (C)

domains of N-TACE, not including the signal peptide. The amino-terminal mutant

corresponded to TACE amino acids 18 – 29, the mid-domain mutant

corresponded to TACE amino acids 30 – 42, and the carboxy-terminal mutant

corresponded to amino acids 43 – 54. As shown in Figure 5, none of these

truncation mutants (80 µM) attenuated the ability of TACE to proteolytically

cleave rhTNFR2:Fc. This demonstrates that these N-TACE18-54 truncation

mutants do not possess TACE inhibitory activity. Further, since these truncation

mutants were synthesized in a fashion identical to N-TACE18-54, this experiment

also demonstrates that the ability of N-TACE18-54 to function as a TACE inhibitor

is not an artifact related to its synthesis and/or purification.

To characterize further the structural requirements for inhibitory activity, another

truncation mutant corresponding to N-TACE amino acids 30 to 48 (N-TACE30-48)

was synthesized. N-TACE30-48 is predicted to have a helical structure by Chou-

Fasman analysis and is leucine-rich, which may be important for its ability to

attenuate TACE activity. As shown in Figure 6, both N-TACE18-54 and N-TACE30-

48 attenuated the TACE-catalyzed proteolytic cleavage of rhTNFR2:Fc. The

inhibitory activity of N-TACE30-48, however, was less than that of N-TACE18-54.

These experiments demonstrate that the domain corresponding to amino acids

30 to 48 of N-TACE partially mediates the TACE inhibitory activity of N-TACE18-

54.

N-TACE18-54 Attenuates Constitutive TNFR2 Shedding from U937 Cells

We next assessed whether N-TACE18-54 was capable of attenuating TNFR2

shedding in a cell-based system. The U937 monocytic cell line was incubated

11

with either N-TACE18-54 (0.04 to 40 µM) or the amino-terminal mutant,

corresponding to N-TACE amino acids 18 – 29 (40 µM), for 24 hrs. As shown in

Figure 7, the quantity of sTNFR2 present in medium from cells treated with N-

TACE18-54 was significantly reduced in a concentration-dependent fashion.

Further, 40 µM N-TACE18-54 significantly attenuated TNFR2 shedding by 42% as

compared to cells treated with media alone (107.3 + 3.3 pg/ml vs. 184.6 + 2.2

pg/ml, n = 6, P < 10-8). In contrast, the amino-terminal mutant had no effect on

TNFR2 shedding as compared to cells treated with medium alone (186.5 + 1.8

pg/ml vs. 184.6 + 2.2 pg/ml, n = 6, P = NS). The ability of N-TACE18-54 to

decrease TNFR2 shedding was not a consequence of either apoptosis or

necrosis, as assessed by Annexin V binding and propidium iodide uptake (data

not shown). Additional experiments were performed utilizing a scrambled 37-

amino acid peptide that contained the N-TACE18-54 amino acids in a random

order to confirm that the inhibition of TNFR2 shedding by N-TACE18-54 is

dependent upon its amino acid sequence. The scrambled peptide did not

attenuate TNFR2 shedding, but instead was associated with a 4% increase in

constitutive TNFR2 shedding as compared to cells treated with medium alone

(115.7 + 0.6 pg/ml vs. 111.4 + 1.7 pg/ml, n = 6, P = 0.038). These data

demonstrate that N-TACE18-54 significantly attenuates constitutive TNFR2

shedding from U937 cells.

Experiments were also performed to assess whether N-TACE30-48 inhibits TNFR2

shedding from U937 cells. Treatment with 40 µM N-TACE30-48 inhibited TNFR2

shedding by 16% as compared to cells treated with media alone (160.8 + 3.4 vs.

190.4 + 2.9 pg/ml, n = 6, P < 10-4). This suggests that although N-TACE30-48

partially attenuates TNFR2 shedding, N-TACE amino acids 18 to 54 are required

for a maximal effect in this cell-based system.

12

Discussion

TACE can regulate inflammatory responses via the proteolytic cleavage and

shedding of TNFR2 to function as a soluble TNF binding protein(29). The

important role that soluble TNFR2 (sTNFR2) plays in regulating TNF bioactivity is

exemplified by virally encoded soluble TNF binding proteins that function as

highly effective modulators of innate immune responses(9). For example, both

the Shope fibroma and myxoma viruses express T2 proteins, which are

structurally similar to TNFR2 and are secreted by infected cells to subvert TNF-

dependent host defenses(40, 46). Similarly, a soluble human TNFR2-Ig fusion

protein is utilized clinically to attenuate TNF bioactivity and disease severity in

patients with inflammatory arthritides and psoriasis(26). Further, sTNFR2 may

modulate pulmonary inflammatory responses in the acute respiratory distress

syndrome, asthma, sarcoidosis, bacterial pneumonia, and tuberculosis(18, 25,

27, 30, 44, 45).

Regulation of TACE enzymatic activity is important to prevent excessive or

unanticipated cleavage of target proteins. TACE is synthesized as a latent pro-

enzyme that is retained in an inactive state via an interaction between its pro-

domain and catalytic domain. Although this interaction was thought to be

mediated via a cysteine switch mechanism, it has recently been shown that the

pro-domain cysteine switch motif is not required for this inhibitory activity(15).

This is based upon the finding that a TACE pro-domain variant, containing a

cysteine to alanine substitution at position 184, showed the same inhibitory

activity towards a recombinant TACE catalytic domain construct as the wild-type

TACE pro-domain(15).

Here, we report that the amino-terminus of the TACE pro-domain also possesses

TACE inhibitory activity that is independent of the cysteine switch mechanism.

We synthesized a 37-amino acid peptide that corresponded to the amino-

terminus of the TACE pro-domain, but did not include the consensus cysteine

13

switch motif. This peptide, termed N-TACE18-54, comprises amino acids 18 to 54

of the TACE protein and was demonstrated to attenuate TACE-catalyzed

cleavage of TNFR2 in vitro. This inhibitory activity appeared to be specific, as

neither truncation mutants corresponding to the amino-terminal (N), middle (M),

and carboxy-terminal (C) domains of N-TACE nor irrelevant small proteins (VIP

and α-defensin) possessed TACE inhibitory activity. N-TACE18-54 also

attenuated by 42% constitutive TNFR2 shedding from the U937 monocytic cell

line, which suggests that N-TACE may partially attenuate the activity of native,

cell-associated TACE. This is consistent with a role for TACE in constitutive

TNFR2 shedding, as was described in HEK293 cells expressing a dominant

negative TACE(41). Neither the amino-terminal truncation mutant nor a

scrambled peptide inhibited constitutive U937 cell TNFR2 shedding, which

suggests that N-TACE18-54 mediates this inhibitory activity in a sequence-specific

fashion. Our findings, however, do not establish whether the ability of N-TACE18-

54 to attenuate constitutive TNFR2 shedding in intact cells is specific for TACE

alone, as N-TACE18-54 could conceivably inhibit other enzymes that also function

as TNFR2 sheddases. Taken together, we propose that the amino-terminal

region of the TACE pro-domain can attenuate TACE catalytic activity

independent of the cysteine switch mechanism.

Interestingly, the TACE disintegrin/cysteine-rich domain has been reported to

diminish the inhibitory potency of the pro-domain for the catalytic domain(15).

While the full-length TACE pro-domain was a potent inhibitor of a recombinant

TACE catalytic domain construct (IC50 = 70 nM), its inhibitory activity was

significantly less against a construct that contained both the TACE catalytic and

disintegrin/cysteine-rich domains (IC50 > 2 µ M) (15). Further, the

disintegrin/cysteine-rich domain appeared to decrease the ability of the pro-

domain to stably bind the catalytic domain(15). Thus, it is possible that in our

study, the disintegrin/cysteine-rich domain impaired the ability of N-TACE18-54 to

inhibit TACE catalytic activity, since rhTACE corresponds to the mature TACE

ectodomain. Further, this may in part explain why micromolar concentrations of

14

N-TACE18-54 were required to inhibit rhTACE-mediated TNFR2:Fc cleavage, as

well as constitutive TNFR2 shedding from U937 cells.

TNFR1 has been reported to represent a substrate for TACE based upon the

demonstration of increased TNFR1 shedding following reconstitution of TACE-

deficient cell lines(31). In our model system, rhTNFR2, but not rhTNFR1, served

as a substrate for rhTACE enzymatic activity. Similarly, TACE has been reported

to have no detectable activity against a TNFR1 model peptide substrate

corresponding to the known TNFR1 cleavage site(22). The inability of rhTACE

to cleave rhTNFR1:Fc raises the question as to whether TNFR1 serves as a

substrate for TACE in vivo or alternatively, whether there is a requirement for

either or both proteins to be membrane-anchored or whether additional

regulatory proteins are required.

In conclusion, we have identified that a sub-domain within the amino-terminus of

the TACE pro-domain attenuates TACE catalytic activity toward TNFR2. We

propose that the ability of N-TACE18-54 to inhibit TACE activity in vitro, as well as

constitutive TNFR2 shedding in a cell-based system, provides a new insight into

the mechanism by which the activity of a disintegrin metalloprotease might be

attenuated by its pro-domain, independent of a cysteine switch mechanism.

15

Acknowledgements

The authors thank Drs. Martha Vaughan and Joel Moss for their helpful advice

and critical review of the manuscript. Research funding was provided by the

Division of Intramural Research, NHLBI.

Abbreviations

1,10-Phe: 1,10-Phenanthroline

ADAM: a Disintegrin and Metalloprotease

s: soluble

TACE: TNF-α Converting Enzyme

TNF: Tumor Necrosis Factor

TNFR1: 55-kDa, Type I TNF Receptor

TNFR2: 75-kDa, Type II TNF Receptor

VIP: Vasoactive Intestinal Peptide

16

Figure Legends

Figure 1. Characterization of N-TACE18-54. A. TACE protein structure. The

TACE pro-domain is comprised of TACE amino acids 18 – 214 and encodes a

consensus cysteine-switch motif. B. N-TACE18-54 amino acid sequence. The

synthesized N-TACE18-54 peptide corresponds to TACE amino acids 18 – 54.

The signal peptide (underlined), encoded by TACE amino acids 1 – 17, is not

included in the N-TACE18-54 sequence. The 19 amino acid, leucine-rich inhibitory

domain is denoted by the double underline.

Figure 2. A. rhTACE catalyzes the cleavage of rhTNFR2, but not rhTNFR1.

rhTACE (0.5 µM) was incubated with 0.95 µM rhTNFR1:Fc (left panel) or

TNFR2:Fc (right panel) alone or in combination with 80 µM N-TACE18-54 for 30

min at 30o C. Samples were separated by SDS-PAGE and proteins were

visualized by silver staining. Positions of protein standards (kDa) are on the left.

Diagrams of the rhTNFR:Fc chimeric substrates are at the bottom. The entire

extracellular domain of human TNFR1 (Met 1 – Thr 211)(34) or TNFR2 (Met 1 –

Asp 257)(19, 40) was fused to the Fc region of human IgG1 (Pro 100 – Lys 330),

via a linker peptide (IEGRMD). Both chimeric fusion proteins contain a 6X

histidine tag at the carboxy-terminus. B. Cleavage of TNFR2:Fc by rhTACE is

zinc-dependent. rhTACE (0.5 µM) was incubated with 1,10-phenanthroline

(1,10-Phe) for 30 min before the addition of the indicated concentration of zinc

chloride for 30 min at 24o C. The rhTNFR2:Fc substrate (0.95 µM) was added for

an additional 30 min at 30o C. Proteins were separated by SDS-PAGE and

rhTNFR2:Fc cleavage products were identified by Western blotting utilizing the

antibody against the carboxy-terminal 6X histidine tag of TNFR2:Fc. Positions

of protein standards (kDa) are on the right.

Figure 3. N-TACE18-54 inhibits cleavage of TNFR2 by TACE. rhTACE (0.5 µM)

was incubated with 0.95 µM rhTNFR2:Fc alone, or with increasing concentrations

of N-TACE18-54 (20 µM to 160 µM) for 30 min at 30o C. Proteins were separated

17

by SDS-PAGE before silver staining (Panel A) or immunoblotting with an

antibody against the carboxy-terminal 6X histidine tag of TNFR2:Fc (Panel B).

Positions of protein standards (kDa) are on the left.

Figure 4. Specificity of inhibition of TACE-catalyzed cleavage of TNFR2 by N-

TACE18-54. rhTACE (0.5 µM) was incubated with 0.95 µM TNFR2:Fc alone, or in

combination with 80 µM N-TACE18-54, 25 µM TAPI-2, 80 µM α-defensin, or 80

µM vasoactive intestinal peptide (VIP) for 30 min at 30o C. Proteins were

separated by SDS-PAGE before silver staining. Positions of protein standards

(kDa) are on the left.

Figure 5. Effect of N-TACE truncation mutants on TACE-catalyzed TNFR2

cleavage. rhTACE (0.5 µM) was incubated with 0.95 µM TNFR2:Fc alone, or in

combination with 80 µM N-TACE or proteins corresponding to the amino-

terminus (N: represents N-TACE amino acids 18 – 29), middle (M: represents N-

TACE amino acids 30 – 42), or carboxy-terminus (C: represents amino acids 43 -

54) of N-TACE18-54 for 30 min at 30o C. Proteins were separated by SDS-PAGE

before silver staining. Positions of protein standards (kDa) are on the left.

Figure 6. Effect of N-TACE30-48 on TACE-catalyzed TNFR2 cleavage. rhTACE

(0.5 µM) was incubated with 0.95 µM TNFR2:Fc alone, or in combination with 80

µM N-TACE18-54 or N-TACE30-48 for 30 min at 30o C. Proteins were separated by

SDS-PAGE before silver staining. Positions of protein standards (kDa) are on the

left.

Figure 7. Effect of N-TACE18-54 on TNFR2 shedding from U937 cells. U937 cells

were incubated for 24 hr with 0.04 to 40 µM N-TACE18-54 or 40 µM of the

truncation mutant corresponding to the amino-terminus of TACE (N). sTNFR2 in

cell culture medium was quantified by ELISA. N-TACE18-54 inhibited TNFR2

shedding in a concentration-dependent fashion (n=6, P < 0.05 as compared to

control, single factor ANOVA). * P < 0.05 vs. untreated cells (Control).

18

References

1. Althoff K, Reddy P, Voltz N, Rose-John S, and Mullberg J. Shedding ofinterleukin-6 receptor and tumor necrosis factor alpha. Contribution of the stalksequence to the cleavage pattern of transmembrane proteins. Eur J Biochem267: 2624-2631, 2000.

2. Black RA. Tumor necrosis factor-α converting enzyme. Int J Biochem CelBiol 34: 1-5., 2002.

3. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, WolfsonMF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N,Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ,March CJ, and Cerretti DP. A metalloproteinase disintegrin that releasestumour-necrosis factor-alpha from cells. Nature 385: 729-733, 1997.

4. Blobel CP. Metalloprotease-disintegrins: links to cell adhesion andcleavage of TNF alpha and Notch. Cell 90: 589-592, 1997.

5. Borroto A, Ruiz-Paz S, De La Torre TV, Borrell-Pages M, Merlos-Suarez A, Pandiella A, Blobel CP, Baselga J, and Arribas J. ImpairedTrafficking and Activation of Tumor Necrosis Factor-{alpha}-converting Enzymein Cell Mutants Defective in Protein Ectodomain Shedding. J Biol Chem 278:25933-25939, 2003.

6. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, CumanoA, Roux P, Black RA, and Israel A. A novel proteolytic cleavage involved inNotch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5:207-216, 2000.

7. Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ,Johnson RS, Castner BJ, Cerretti DP, and Black RA. Evidence that tumornecrosis factor alpha converting enzyme is involved in regulated alpha-secretasecleavage of the Alzheimer amyloid protein precursor. J Biol Chem 273: 27765-27767, 1998.

8. Contin C, Pitard V, Itai T, Nagata S, Moreau JF, and Dechanet-MervilleJ. Membrane-anchored CD40 is processed by the TNF-alpha-convertingenzyme: Implications for CD40 signaling. J Biol Chem 278: 32801-32809., 2003.9. Cunnion KM. Tumor necrosis factor receptors encoded by poxviruses.Mol Genet Metab 67: 278-282, 1999.

10. Demaegdt H, Laeremans H, De Backer JP, Mosselmans S, Le MT,Kersemans V, Michotte Y, Vauquelin G, and Vanderheyden PM. Synergisticmodulation of cystinyl aminopeptidase by divalent cation chelators. BiochemPharmacol 68: 893-900, 2004.

19

11. Doedens JR and Black RA. Stimulation-induced down-regulation oftumor necrosis factor-alpha converting enzyme. J Biol Chem 275: 14598-14607,2000.

12. Endres K, Anders A, Kojro E, Gilbert S, Fahrenholz F, and Postina R.Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation.Eur J Biochem 270: 2386-2393, 2003.

13. Ermert M, Pantazis C, Duncker HR, Grimminger F, Seeger W, andErmert L. In situ localization of TNFalpha/beta, TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue. Cytokine 22: 89-100,2003.

14. Garton KJ, Gough PJ, Blobel CP, Murphy G, Greaves DR, DempseyPJ, and Raines EW. Tumor necrosis factor-alpha-converting enzyme (ADAM17)mediates the cleavage and shedding of fractalkine (CX3CL1). J Biol Chem 276:37993-38001, 2001.

15. Gonzales PE, Solomon A, Miller AB, Leesnitzer MA, Sagi I, and MillaME. Inhibition of the tumor necrosis factor-alpha-converting enzyme by its prodomain. J Biol Chem 279: 31638-31645, 2004.

16. Greene C, Lowe G, Taggart C, Gallagher P, McElvaney N, and O'NeillS. Tumor necrosis factor-alpha-converting enzyme: its role in community-acquired pneumonia. J Infect Dis 186: 1790-1796, 2002.

17. Hansen HP, Dietrich S, Kisseleva T, Mokros T, Mentlein R, Lange HH,Murphy G, and Lemke H. CD30 shedding from Karpas 299 lymphoma cells ismediated by TNF-alpha-converting enzyme. J Immunol 165: 6703-6709, 2000.

18. Hino T, Nakamura H, Shibata Y, Abe S, Kato S, and Tomoike H.Elevated levels of type II soluble tumor necrosis factor receptors in thebronchoalveolar lavage fluids of patients with sarcoidosis. Lung 175: 187-193,1997.

19. Kohno T, Brewer MT, Baker SL, Schwartz PE, King MW, Hale KK,Squires CH, Thompson RC, and Vannice JL. A second tumor necrosis factorreceptor gene product can shed a naturally occurring tumor necrosis factorinhibitor. Proc Natl Acad Sci U S A 87: 8331-8335, 1990.

20. Merlos-Suarez A, Ruiz-Paz S, Baselga J, and Arribas J.Metalloprotease-dependent protransforming growth factor-alpha ectodomainshedding in the absence of tumor necrosis factor-alpha-converting enzyme. JBiol Chem 276: 48510-48517, 2001.

20

21. Milla ME, Leesnitzer MA, Moss ML, Clay WC, Carter HL, Miller AB, SuJL, Lambert MH, Willard DH, Sheeley DM, Kost TA, Burkhart W, Moyer M,Blackburn RK, Pahel GL, Mitchell JL, Hoffman CR, and Becherer JD.Specific sequence elements are required for the expression of functional tumornecrosis factor-alpha-converting enzyme (TACE). J Biol Chem 274: 30563-30570, 1999.

22. Mohan MJ, Seaton T, Mitchell J, Howe A, Blackburn K, Burkhart W,Moyer M, Patel I, Waitt GM, Becherer JD, Moss ML, and Milla ME. The tumornecrosis factor-alpha converting enzyme (TACE): a unique metalloproteinasewith highly defined substrate selectivity. Biochemistry 41: 9462-9469, 2002.

23. Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, ChenWJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH,Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G,Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Becherer JD, and etal. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385: 733-736, 1997.

24. Moss ML, White JM, Lambert MH, and Andrews RC. TACE and otherADAM proteases as targets for drug discovery. Drug Discov Today 6: 417-426,2001.

25. O'Grady NP, Preas HL, Pugin J, Fiuza C, Tropea M, Reda D, BanksSM, and Suffredini AF. Local inflammatory responses following bronchialendotoxin instillation in humans. Am J Respir Crit Care Med 163: 1591-1598,2001.

26. Olsen NJ and Stein CM. New drugs for rheumatoid arthritis. N Engl JMed 350: 2167-2179, 2004.

27. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F, 2nd,Park DR, Pugin J, Skerrett SJ, Hudson LD, and Martin TR. Cytokine balancein the lungs of patients with acute respiratory distress syndrome. Am J RespirCrit Care Med 164: 1896-1903, 2001.

28. Peiretti F, Canault M, Deprez-Beauclair P, Berthet V, Bonardo B,Juhan-Vague I, and Nalbone G. Intracellular maturation and transport of tumornecrosis factor alpha converting enzyme. Exp Cell Res 285: 278-285, 2003.

29. Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, LeeDC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N,Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ,

21

and Black RA. An essential role for ectodomain shedding in mammaliandevelopment. Science 282: 1281-1284, 1998.

30. Petelin M, Naruishi K, Shiomi N, Mineshiba J, Arai H, Nishimura F,Takashiba S, and Murayama Y. Systemic up-regulation of sTNFR2 and IL-6 inPorphyromonas gingivalis pneumonia in mice. Exp Mol Pathol 76: 76-81, 2004.

31. Reddy P, Slack JL, Davis R, Cerretti DP, Kozlosky CJ, Blanton RA,Shows D, Peschon JJ, and Black RA. Functional analysis of the domainstructure of tumor necrosis factor-alpha converting enzyme. J Biol Chem 275:14608-14614, 2000.

32. Rio C, Buxbaum JD, Peschon JJ, and Corfas G. Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem275: 10379-10387, 2000.

33. Rovida E, Paccagnini A, Del Rosso M, Peschon J, and Dello SbarbaP. TNF-alpha-converting enzyme cleaves the macrophage colony-stimulatingfactor receptor in macrophages undergoing activation. J Immunol 166: 1583-1589, 2001.

34. Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GHW, GatanagaT, Granger GA, Lentz R, Raab H, Kohr WJ, and Goeddel DV. Molecularcloning and expression of a receptor for human tumor necrosis factor. Cell 61:361-370, 1990.

35. Schlondorff J, Becherer JD, and Blobel CP. Intracellular maturation andlocalization of the tumour necrosis factor alpha convertase (TACE). Biochem J347 Pt 1: 131-138, 2000.

36. Schlondorff J and Blobel CP. Metalloprotease-disintegrins: modularproteins capable of promoting cell-cell interactions and triggering signals byprotein-ectodomain shedding. J Cell Sci 112 ( Pt 21): 3603-3617, 1999.

37. Schlondorff J, Lum L, and Blobel CP. Biochemical and pharmacologicalcriteria define two shedding activities for TRANCE/OPGL that are distinct fromthe tumor necrosis factor alpha convertase. J Biol Chem 276: 14665-14674,2001.

38. Shao MX, Nakanaga T, and Nadel JA. Cigarette smoke inducesMUC5AC mucin overproduction via tumor necrosis factor-alpha-convertingenzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell MolPhysiol 287: L420-427, 2004.

22

39. Shao MX, Ueki IF, and Nadel JA. Tumor necrosis factor alpha-convertingenzyme mediates MUC5AC mucin expression in cultured human airwayepithelial cells. Proc Natl Acad Sci U S A 100: 11618-11623, 2003.

40. Smith CA, Davis T, Anderson D, Solam L, Beckman MP, Jerzy R,Dower SK, Cosman D, and Goodwin RG. A receptor for tumor necrosis factordefines an unusual family of cellular and viral proteins. Science 248: 1019-1023.,1990.

41. Solomon KA, Pesti N, Wu G, and Newton RC. Cutting edge: a dominantnegative form of TNF-alpha converting enzyme inhibits proTNF and TNFRIIsecretion. J Immunol 163: 4105-4108, 1999.

42. Sunnarborg SW, Hinkle CL, Stevenson M, Russell WE, Raska CS,Peschon JJ, Castner BJ, Gerhart MJ, Paxton RJ, Black RA, and Lee DC.Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermalgrowth factor receptor ligand availability. J Biol Chem 277: 12838-12845, 2002.

43. Thathiah A, Blobel CP, and Carson DD. Tumor necrosis factor-alphaconverting enzyme/ADAM 17 mediates MUC1 shedding. J Biol Chem 278: 3386-3394, 2003.

44. Tillie-Leblond I, Pugin J, Marquette CH, Lamblin C, Saulnier F,Brichet A, Wallaert B, Tonnel AB, and Gosset P. Balance betweenproinflammatory cytokines and their inhibitors in bronchial lavage from patientswith status asthmaticus. Am J Respir Crit Care Med 159: 487-494, 1999.

45. Tsao TC, Hong J, Li LF, Hsieh MJ, Liao SK, and Chang KS.Imbalances between tumor necrosis factor-alpha and its soluble receptor forms,and interleukin-1beta and interleukin-1 receptor antagonist in BAL fluid ofcavitary pulmonary tuberculosis. Chest 117: 103-109, 2000.

46. Upton C, Macen JL, Schreiber M, and McFadden G. Myxoma virusexpresses a secreted protein with homology to the tumor necrosis factor receptorgene family that contributes to viral virulence. Virology 184: 370-382, 1991.

47. Vincent B, Paitel E, Saftig P, Frobert Y, Hartmann D, De Strooper B,Grassi J, Lopez-Perez E, and Checler F. The disintegrins ADAM10 and TACEcontribute to the constitutive and phorbol ester-regulated normal cleavage of thecellular prion protein. J Biol Chem 276: 37743-37746, 2001.

48. Zhang Y, Jiang J, Black RA, Baumann G, and Frank SJ. Tumornecrosis factor-alpha converting enzyme (TACE) is a growth hormone bindingprotein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for(PMA-induced) GH receptor proteolysis and GHBP generation. Endocrinology141: 4342-4348, 2000.

23

49. Zhao J, Chen H, Peschon JJ, Shi W, Zhang Y, Frank SJ, andWarburton D. Pulmonary hypoplasia in mice lacking tumor necrosis factor-alphaconverting enzyme indicates an indispensable role for cell surface proteinshedding during embryonic lung branching morphogenesis. Dev Biol 232: 204-218, 2001.

24

Figure 1.

25

Figure 2.

26

Figure 3.

27

Figure 4.

28

Figure 5.

29

Figure 6.

30

Figure 7.