©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Granzyme B Is Inhibited by the Cowpox Virus Serpin Cytokine Response Modifier A(*)

Long T. Quan (1)(§), Antonio Caputo (3)(¶), R. Chris Bleackley (3)(**), David J. Pickup (2), Guy S. Salvesen (1)(§§)

From the (1) Departments of Pathology and (2) Microbiology, Duke University, Durham, North Carolina 27710 and the (3) Biochemistry Department, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ability of cytolytic cells to cause apoptosis in target cells is in part due to the action of the serine proteinase granzyme B. We demonstrate that granzyme B is inhibited, with an association rate constant of 2.9 10 M s, by the cowpox viral serpin cytokine response modifier A (CrmA). Previously we have shown CrmA to be an inhibitor of the cysteine proteinase interleukin-1-converting enzyme (ICE). Thus the mechanism of CrmA involves the unusual ability to efficiently inhibit proteinases from two distinct catalytic classes, in this case serine and cysteine proteinases. Granzyme B and ICE are both used to combat viral infection, and we propose that cowpox virus uses CrmA to evade the contribution of these two proteinases. Thus, through CrmA, the virus may influence two of the pathways normally used to kill virus-infected cells: acting on endogenous proteinases such as ICE and on exogenous proteinases delivered by cytotoxic lymphocytes to infected cells.


INTRODUCTION

The granzymes are a family of serine proteinases present in the lytic granules of cytotoxic T-lymphocytes and natural killer cells (1) . They consist of a number of enzymes, with various primary substrate specificities, that play a role in apoptosis of susceptible target cells (2, 3, 4, 5) . Granzymes must be delivered from an effector cell to the target cell, with the help of the pore-forming protein perforin to produce their apoptotic effect (6) . The granzyme shown to cause the most rapid kinetics of cell death is granzyme B (2) , which has an unusual preference for Asp in the primary substrate site (7, 8) . Of the hundreds of characterized eukaryotic proteinases, the only other enzymes that share a specificity for Asp are interleukin-1-converting enzyme (ICE),() which cleaves the Asp-Ala bond in prointerleukin-1 (9, 10) , and a proteolytic activity designated prICE that cleaves poly(ADP-ribose) polymerase between Asp and Gly in an in vitro model of apoptosis (11) . The shared primary specificity of these proteinases is an unlikely coincidence and implies that proteins involved in cell death pathways include key substrates that are activated by cleavage at Asp residues.

If apoptotic proteinases can share primary substrate specificities it is possible that they will be inactivated by the same inhibitors. Currently the only known natural inhibitor of these Asp-specific proteinases is the cowpox virus serpin CrmA that is able to rapidly inhibit ICE (12, 13, 14) . This is consistent with the ability of the virus to inhibit the host's inflammatory response (13, 15) . The interaction between CrmA and ICE is unusual because ICE is a cysteine proteinase and CrmA is from a family of inhibitors of serine proteinases. The inhibitory site of CrmA consists of a stretch of primary structure that mimics a good substrate for ICE; indeed this sequence contains an Asp residue strategically placed to bind to the primary substrate site of proteinases (12) . Since ICE and GraB share primary substrate specificity, we investigated whether CrmA could inhibit GraB and whether this could constitute part of the virus' offensive strategy. GraB is a reasonable target for inhibition by CrmA for at least two reasons. First, it is a serine proteinase of the type to which serpins such as CrmA are normally directed. Second, GraB is secreted into the cytosolic compartment of virally infected target cells, where CrmA exerts its biological role.


MATERIALS AND METHODS

GraB was purified from 3 10 YT cells by cavitation, differential salt extraction, and ion exchange chromatography essentially as described (16) . Enzymatic activity was routinely determined by using Boc-Ala-Ala-Pro-Asp- pNA (where pNA is p-nitroanilide) at 0.5 mM final concentration. GraB/CrmA association kinetics were determined using the more sensitive substrate Boc-Ala-Ala-Asp-SBzl (where SBzl is S-benzyl) with detection of released thiobenzene by 5,5`-dithiobis(nitrobenzoic acid) (7) . Substrate hydrolyses were recorded using a Molecular Devices V plate reader or a Shimadzu spectrophotometer. All assays were performed in reaction buffer consisting of 20 mM Hepes buffer, pH 7.4, containing 0.1 M NaCl and 0.05% Nonidet P-40.

For expression of CrmA in vitro or in Escherichia coli the full-length crmA gene was ligated to a linker that encoded an N-terminal Met-His fusion to facilitate purification. The coding sequence started with the initiator methionine, followed by six histidines, a serine, and then the entire coding region of CrmA. The parent plasmid for in vitro translation was pTM1 (17) , and the parent for E. coli expression was based on the isopropyl-1-thio--D-galactopyranoside-inducible pFLAG (IBI). Coupled transcription/translation was performed with the TNT kit from Promega according to the manufacturer's recommendations. Briefly, 0.5 µg of plasmid DNA was incubated for 1 h at 31 °C in a total volume of 50 µl containing the kit reagents and 20 µCi of translation grade [S]Met. Once translated, the reaction mix was either used immediately or stored at -20 °C until needed.

Expression and Purification of CrmA from E. coli

E. coli TG1 containing the CrmA construct was induced with isopropyl-1-thio--D-galactopyranoside for 3 h and harvested, and the cells were lysed by sonication and pelleted by centrifugation. The supernatant containing soluble CrmA was filtered through a 0.22-µm filter and then loaded onto a 2-ml Ni-nitrilotriacetate column (Qiagen) and washed with 50 mM Tris, pH 8.0, containing 0.5 M NaCl. CrmA was eluted with 50 mM Tris, 50 mM imidazole, pH 8.0, containing 0.1 M NaCl. This material was diluted with 9 volumes of 20 mM Hepes, pH 7.4, containing 2 mM dithiothreitol and applied to a 2-ml column of DEAE-Sepharose. This column was developed with a linear gradient of 0-1 M NaCl in 20 mM Hepes buffer, pH 7.4, and CrmA was eluted at approximately 0.4 M NaCl to produce a yield of 4 mg of protein from 6 liters of culture fluid. The material was greater than 95% pure as estimated by Coomassie Blue staining and was stored at 4 °C until use. In all experiments using this material the CrmA was treated with 2 mM dithiothreitol for 5 min, followed by 4 mM NaAsO to block excess dithiothreitol. This procedure resulted in CrmA with the highest inhibitory activity.

Polyacrylamide Gel Electrophoresis (PAGE)

CrmA protein samples were analyzed by non-denaturing PAGE or reduced SDS-PAGE in gels of 5-15% linear acrylamide gradients (18) . For radioactive samples, the gels were dried and then subjected to autoradiography using a phosphorescent image scanner (PhosphorImager 410A and densitometer, Molecular Dynamics, Sunnyvale, CA); otherwise, protein bands were visualized by R250 Coomassie staining.


RESULTS AND DISCUSSION

We analyzed the interaction between human GraB and CrmA using an in vitro expression system developed for rapid screening of serpin/proteinase interactions (19) . Preliminary experiments using lysates of COS cells that express mouse GraB revealed the formation of SDS-stable complexes with CrmA and active enzyme but not when the proenzyme was used (not shown). We performed the rest of our studies using human GraB because it can be isolated in active pure form, in milligram quantities, from the human YT (natural killer cell leukemia) cell line.() The majority of in vitro translated CrmA formed a complex with GraB that did not migrate in native gel electrophoresis due to the highly basic composition of GraB (Fig. 1 A, top). This complex is resolved on SDS-PAGE, a phenomenon typical of serpin/proteinase interactions (Fig. 1 A, bottom).


Figure 1: Specificity of the interaction of GraB with CrmA. PanelA, complex formation between CrmA and GraB. [S]Met-labeled CrmA was incubated with dilutions of unlabeled purified human GraB for 30 min at 37 °C in a total volume of 20 µl. The reactions were divided and then run on native PAGE ( top) and reduced SDS-PAGE ( bottom), and the products were quantitated by PhosphorImaging. Because the complex does not migrate into a native gel, the formation of the CrmAGraB complex can be seen as the disappearance of CrmA ( arrowhead). However, SDS-PAGE allows visualization of the CrmAGraB complex ( arrow) since all components migrate in the gel when denatured. The intermediate band seen in some lanes is probably partially degraded complex. PanelB, activity of GraB. Purified human GraB (85 pmol) was allowed to react with a 3-fold molar excess of recombinant CrmA in a total volume of 50 µl and then run on reducing SDS-PAGE, followed by staining with Coomassie Blue. Lane1, GraB alone; lane2, the same amount of GraB reacted with CrmA; lane3, CrmA alone. Virtually all of the GraB shifted to form an SDS-stable complex with CrmA, indicating that the enzyme is fully active. The lowerband in the CrmA sample probably represents a small amount of cleaved inhibitor present in the preparation.



PhosphorImager quantitation of the radioactive bands indicated that about 95% of CrmA was able to form a complex with GraB. Similarly, virtually all of the purified GraB formed a complex with CrmA (Fig. 1 B). To characterize this apparent inhibition, we titrated GraB activity with CrmA produced in E. coli (Fig. 2) and observed linear concentration-dependent inhibition of GraB, indicative of a tight binding interaction. Under the conditions used here, the binding to CrmA was too tight to measure equilibrium, so we explored the reaction in more detail by observing the rate of inhibition (Fig. 2). This was linear over at least three half-lives of the reaction, indicating standard bimolecular kinetics. Thus we demonstrate for the first time that CrmA can efficiently inhibit a serine proteinase. Previously we showed that CrmA is an efficient inhibitor of ICE, a cysteine proteinase (14) . Our current data now firmly establish that CrmA, since it is able to inhibit a serine proteinase and a cysteine proteinase, is a cross-class inhibitor. This unusual property would be explained by ICE having a substrate binding geometry that more closely resembles serine proteinases than other cysteine proteinases.


Figure 2: Kinetics of the interaction of GraB with CrmA. PanelA, GraB titrated by CrmA. Purified GraB was incubated with the indicated concentrations of recombinant CrmA at 37 °C in reaction buffer. After 15 min Boc-Ala-Ala-Pro-Asp- p-nitroanilide was added to determine the residual GraB activity. The active enzyme concentration was calculated based on the concentration of CrmA determined as described before (13). PanelB, determination of the rate of inhibition of GraB by CrmA. CrmA was incubated with equimolar GraB (1.7 10 M) at 37 °C in reaction buffer. Portions were removed at timed intervals and assayed for residual GraB activity via a coupled assay with Boc-Ala-Ala-Asp-SBzl (where SBzl is S-benzyl) and 5,5`-dithiobis(nitrobenzoic acid). The plot of reciprocal residual enzyme concentration against time was linear, indicating a bimolecular reaction, with a slope of 2.9 10 M s.



The rate of inhibition of GraB by CrmA, 2.9 10 M s, is certainly fast enough to be of physiologic significance. For example it exceeds the rate of inhibition of plasma kallikrein by the serpin C1-inhibitor (20) . However, the rate is almost 2 orders of magnitude slower than the inhibition of ICE by CrmA, which at 1.7 10 M s is among the fastest of serpin/target proteinase interactions. A comparison of the rates of inhibition predicts that ICE would compete for binding to CrmA about 60 times better than would GraB, and we tested this by incubating CrmA with a mixture of the two proteinases at different molar ratios (Fig. 3). The ICECrmA complex is not stable to SDS but is observed in native gels (Fig. 3 A). Conversely the GraBCrmA complex is stable to SDS, so the resulting competition can be distinguished by observing complexes in native and SDS gels. The equivalence point is in lane4, representing a GraB/ICE ratio of 100:1. This is in agreement with the rate determinations and demonstrates that CrmA, though it is an adequate GraB inhibitor, will preferentially inhibit ICE.


Figure 3: Competition between GraB and ICE for binding to CrmA. [S]Met-labeled CrmA was reacted with buffer ( lane1) or serial 3-fold dilutions of ICE ( lanes2-7) in a total volume of 20 µl ( A). About 35% of the CrmA formed a complex with ICE, and the rest was turned over as described previously for this reaction (14). CrmA was allowed to react with GraB (42.5 pmol) in the presence of the serial dilutions of ICE in a total volume of 30 µl ( B). The reactions were divided and then run on native PAGE and reduced SDS-PAGE. The reactions were carried out in 0.1 M NaCl, 10% sucrose, 0.1% Chaps, and 50 mM Hepes buffer, pH 7.4, at 37 °C for 30 min. After PAGE the gels were dried and then analyzed by PhosphorImaging.



Our findings have implications for the mechanism of proteinase inhibition by serpins and the biological role of CrmA. Previously we had shown that CrmA is an inhibitor of the cysteine proteinase ICE, and now we show that it is also an inhibitor of the serine proteinase GraB. Since both proteinases compete for binding to CrmA they most likely react with the same inhibitory site. Thus this serpin can inactivate structurally unrelated proteinases, provided they share the same primary substrate specificities. How CrmA accomplishes this is still a puzzle, but this adaptability has important implications for other members of the ICE family, including Ced-3 (21) , CPP32 (22) , and Ich-1 (23) (the human ortholog of Nedd-2 (24) ), that have been implicated as positive regulators of apoptosis. The substrate specificity of these putative proteinases has yet to be reported, though they contain the residues that impart Asp specificity to ICE, and it is predicted that they will cleave protein substrates required for progression of the death pathway inherent to a number of cell types. Thus they are candidates for the CrmA-inhibitable mediator(s) of programmed cell death implicated in a number of recent studies based on cell transfection strategies (21, 25, 26) .

Commitment to cell death is an irreversible process, and it is reasonable to utilize proteinases since they generate irreversible events. The proteinases identified in the endogenous cell death pathway have a characterized or suspected preference for cleaving substrates at Asp, a rare specificity compatible with tight regulation. Because GraB is delivered from cells whose main function is to kill virally infected targets (reviewed by Berke (6) ), its inhibition by CrmA is consistent with the biology of the cowpox virus. Variants of the virus containing deletions in the crmA gene have an attenuated phenotype consistent with an inability of the variants to inhibit ICE-mediated processing of prointerleukin-1. Our data on GraB inhibition suggest a second role for CrmA. Since virally infected cells can be cleared from the body by cytolytic cells using their perforin/granzyme system, an additional function of CrmA may be to prevent the activation of apoptosis via the GraB pathway. Thus we raise the possibility that, through the CrmA serpin, the virus has acquired the ability to manipulate the cell death program by inhibiting endogenous and exogenous mediators.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM38860 and AI32982 and by the Medical Research Council and National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
National Institutes of Health predoctoral fellow.

Postdoctoral fellow of the Alberta Heritage Foundation for Medical Research.

**
Medical scientist of the Alberta Heritage Foundation for Medical Research.

§§
To whom correspondence should be addressed: Box 3712, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2864; Fax: 919-684-8883; E-mail: gss@galactose.mc.duke.edu.

The abbreviations used are: ICE, interleukin-1-converting enzyme; CrmA, cytokine response modifier A; GraB, granzyme B; PAGE, polyacrylamide gel electrophoresis; Boc, t-butoxycarbonyl; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

C. Froelich and W. Hanna, personal communication.


ACKNOWLEDGEMENTS

We thank Bill Hanna and Chris Froelich for providing advice and the YT cell line, and Ed Madison and our colleagues at Duke for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.