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Previous Article | Next Article 
Molecular and Cellular Biology, September 2003, p. 6315-6326, Vol. 23, No. 17
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.17.6315-6326.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Medicine,1 Department of Dermatology,2 Department of Cell Biology and Anatomy, School of Medicine, Johns Hopkins University, Baltimore, Maryland 212053
Received 21 October 2002/ Returned for modification 30 December 2002/ Accepted 30 May 2003
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ABSTRACT |
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INTRODUCTION |
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While there is significant evidence to implicate granzymes in target cell cytolysis, the mechanisms involved remain incompletely understood. Granzyme B (GrB), one of the most abundant proteases found in cytotoxic granules (42), catalyzes the cleavage and activation of several downstream caspases, inducing apoptotic changes in target cells (46). In addition, GrB can initiate caspase-independent pathways of cell death (31, 47, 54) through direct proteolysis and activation of molecules such as DFF45/ICAD (56) as well as cleavage of several substrates involved in cellular homeostasis (4, 13, 23). GrB is unique among mammalian serine proteases for its strict requirement for aspartic acid in the substrate P1 position. By use of combinatorial substrate libraries, the amino acid preferences at additional subsites have been defined for human GrB (58) and rat GrB (24) and are very similar for the two proteases. Rat and human GrB proteases are also quite similar at the structural level, with the major difference residing in the Asp-specific S1 subsite, which is significantly larger in the human enzyme (44, 61).
Adenoviruses are DNA viruses that have adopted several strategies to prevent the apoptosis of infected target cells (27, 62). These include (i) forced degradation of Fas and TRAIL receptor 1 by RID (previously named E3-10.4K/14.5K) (59, 60), (ii) direct interaction with and inhibition of members of the Fas signaling complex (E1B19K and E314.7) (15, 28, 39), (iii) binding with p53 and inhibition of p53-induced apoptosis (E1B55K and E4orf6) (19, 35, 48, 55, 63, 64), and (iv) expression of the antiapoptotic bcl-2 homologue E1B19K (11, 16, 38). Andrade et al. recently defined the 100,000-molecular-weight (100K) assembly protein of human adenovirus type 5 (Ad5-100K) as a potent and specific inhibitor of human GrB. This inhibitory effect is absolutely dependent on Asp48 in the AD5-100K protein, which is found within the GrB consensus motif Ile-Glu-Gln-Asp48 (2). The AD5-100K protein is a nonstructural protein which has essential functions in the adenovirus life cycle, including virus assembly and activation of late viral protein synthesis (14, 25, 43), and is present and well conserved in all members of the Adenoviridae family (18).
In these studies, we show that although human, mouse, and rat GrB proteases are well conserved in terms of structure, substrate specificity, and function, Ad5-100K has inhibitory activity exclusively against the human protease. Biochemical analysis demonstrates that the specificity for human GrB inhibition by Ad5-100K is the result of a unique cleavage preference for the site at Asp48 in Ad5-100K by the human but not the mouse or rat protease, the presence of Pro49 at the P1' position, and the requirement for an additional inhibitor-enzyme interaction outside the enzyme catalytic site (i.e., an extended macromolecular recognition domain, or exosite). This additional component of the inhibitory mechanism may provide a critical target for the development of highly potent and specific inhibitors of GrB, which may be useful therapeutic tools for chronic human diseases in which GrB likely plays a role (e.g., graft rejection and autoimmunity) (3, 13, 36).
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MATERIALS AND METHODS |
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Cell culture, viral infection, and cell transfection. NIH 3T3 and 293T cells were cultured by standard procedures. Confluent NIH 3T3 cell monolayers were infected with murine adenovirus type 1 (MAV-1) at 40 PFU/cell, followed by incubation at 37°C in a 5% CO2 humidified incubator for 48 h. Biochemical analysis was performed after these procedures. 293T cells were transfected by using Lipofectamine 2000 (Gibco BRL) as recommended by the manufacturer.
GrB cleavage of endogenous substrates in cell lysates. Cell lysates were prepared as described previously (4). Lysates were incubated with 5 mM iodoacetamide for 10 min at 4°C to inactivate endogenous caspases and then incubated at 37°C for 60 min in the presence or absence of purified GrB. Equimolar amounts of human, mouse, and rat GrB proteases were confirmed based upon equal proteolytic activities against macromolecular substrates (i.e., NuMA, PARP, and Mi-2). The reactions were stopped by boiling in sodium dodecyl sulfate (SDS) sample buffer, and samples were electrophoresed on SDS-10% polyacrylamide gels containing 0.058% bisacrylamide. Intact autoantigens and their specific GrB cleavage fragments were visualized by immunoblotting as described previously (13).
Expression vectors, 100K protein cloning, and purification.
Ad5-100K was cloned and purified as described previously (2). For cloning of the 100K protein of MAV-1 (MAV1-100K), total RNA was purified from MAV-1-infected NIH 3T3 cells 48 h postinfection with Trizol (Gibco BRL) as described by the manufacturer. First-strand cDNA synthesis was performed with SuperScript II (Gibco BRL) according to the manufacturer's directions. Two microliters of this cDNA was used as a template in a PCR with Pfu and Taq DNA polymerases (1:1) and with oligonucleotides containing restriction enzyme adapters. The PCR-generated cDNA fragment containing the entire MAV1-100K open reading frame was cloned into pcDNA3.1(+) (Invitrogen). 100K protein hybrids and deletion mutants were generated by either site-directed mutagenesis (Stratagene) or PCR. All of the constructs, as well as green fluorescent protein (GFP) cDNA from pEGFP-N3, were cloned into prokaryotic expression vector pET28(+) (Novagen), generating N-terminal His6-T7-tagged fusion proteins that were further purified as previously described (2). The purity of the preparations was 
70 to 90%, as assessed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining (data not shown).
GrB cleavage of substrates generated by coupled IVTT. [35S]methionine-labeled substrates were generated by in vitro transcription-translation (IVTT; Promega), and cleavage reactions were performed with ICE buffer (10 mM HEPES-KOH, 2 mM EDTA, 1% NP-40 [pH 7.4]). After incubation at 37°C for 30 min, reactions were terminated and samples were electrophoresed on SDS-10% polyacrylamide gels. Radiolabeled proteins and their fragments were visualized by fluorography. To better resolve the difference between full-length Ad5-100K and its 95-kDa human GrB cleavage fragment, N-terminal His6-T7-tagged Ad5-100K (which retards the migration of Ad5-100K to an apparent molecular mass of 110 kDa) was used as a substrate for the human, mouse, and rat GrB cleavage reactions.
Analysis of GrB inhibition by purified 100K protein and its mutants. A fixed amount of purified GrB (25 nM) was preincubated with increasing amounts of purified recombinant Ad5-100K (rAd5-100K) or its mutants in ICE buffer at 37°C for 10 min. The residual proteolytic activity of GrB was determined by adding [35S]methionine-labeled human Mi-2 and incubating the mixture for an additional 15 min at 37°C. As a control, the radiolabeled substrate was incubated with buffer alone. The reactions were terminated by boiling in SDS sample buffer. Samples were analyzed by electrophoresis on SDS-8% polyacrylamide gels, and radiolabeled proteins and their fragments were visualized by fluorography.
Calculation of catalytic constant values.
Catalytic constant (kcat/Km) values were determined by using subsaturating substrate concentrations. Substrate and product bands on autoradiograms were scanned by densitometry to calculate the percentages of substrate cleavage. To calculate kcat/Km, these values were fitted to the following first-order rate equation: 
. In this equation, [E] is the concentration of enzyme.
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RESULTS |
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104 M-1 s-1). Interestingly, as previously observed for NuMA and PARP, the patterns of Mi-2 cleavage by the three granzymes were different. In the presence of equimolar amounts of rAd5-100K and human GrB, the cleavage of Mi-2 was decreased by 90% (Fig. 1B, compare lanes 6 and 7). In contrast, rAd5-100K did not inhibit the mouse (Fig. 1B, compare lanes 2 and 3) and rat (Fig. 1B, compare lanes 4 and 5) proteases, even when a 20-fold molar excess of the inhibitor was used. These data therefore demonstrate that even though Ad5-100K is a potent and specific inhibitor of human GrB, this molecule has no effect on the activities of the mouse GrB and rat GrB orthologues. In addition, these data highlight the striking observation that even though GrB proteases from different species have similar structures, their cleavage specificities for macromolecular substrates appear quite distinct. While structural differences in GrB proteases (e.g., different glycosylation patterns or free versus complexed with serglycin) that relate to the different sources of the enzymes used (i.e., native versus recombinant from baculovirus or P. pastoris) may influence protease substrate-inhibitor interactions, the cleavage specificities of human GrB (native or recombinant) and mouse GrB (native or recombinant) were determined exclusively by the species of origin and were independent of the source of purification (data not shown).
Ad5-100K is differentially cleaved by human GrB, mouse GrB, and rat GrB.
It was previously shown that 100K protein inhibition of human GrB is dependent on Asp48 in Ad5-100K, which is found within a classic GrB consensus motif (IEQD48-P) (2). Ad5-100K cleavage at Asp48 by human GrB is very efficient and generates a prominent 95-kDa fragment (Fig. 2A, lanes 10 and 11) which is abolished when Asp48 is changed to Ala (2). Since human GrB, mouse GrB, and rat GrB cleave the macromolecular substrates NuMA, PARP, and Mi-2 at different sites (see above), we addressed whether Ad5-100K was differentially cleaved by the human, mouse, and rat proteases. When [35S]methionine-labeled Ad5-100K was incubated in the presence of increasing concentrations of GrB, Ad5-100K was cleaved by the rat protease with an efficiency similar to that of the human protease (kcat/Km values, 1.5 x 104 and 3 x 104 M-1 s-1, respectively). In contrast, the cleavage of Ad5-100K by mouse GrB was approximately 1 order of magnitude less efficient than that by human GrB (kcat/Km value, 3 x 103 M-1 s-1). Interestingly, Ad5-100K cleavage at Asp48 by either mouse or rat GrB was very inefficient. The most prominent fragments generated by mouse GrB and rat GrB were 65 and 38kDa (Fig. 2A, lanes 2 to 4 and lanes 6 to 8, respectively); an additional 30-kDa fragment resulted from rat GrB cleavage (Fig. 2A, lanes 6 to 8). It is therefore likely that the efficient recognition of the IEQD48-P motif by different granzymes plays a role in the species selectivity of inhibition by Ad5-100K.
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P49 (arrow, cleavage site) was mutated to IEQD48A49 in Ad5-100K (i.e., Ad5-100K-P49A), neither its cleavage by mouse GrB and rat GrB nor its inhibitory activity against these proteases was changed (data not shown). However, Ad5-100K-P49A lost all inhibitory activity against the human protease and was very efficiently cleaved (Fig. 2B, compare lanes 4 and 6). Thus, these data strongly suggest that proline in the P1' position of the 100K protein does not play a role in defining the species selectivity of Ad5-100K but is essential for the inhibition of human GrB. Interestingly, this inhibitory cleavage site (IEQD48P49) alone is not sufficient to inhibit human GrB, since deletion mutants of Ad5-100K containing this motif but lacking the C terminus were poor inhibitors of this protease (see below and Fig. 6); these results suggest that additional interactions (outside the cleavage site) are critical for the precise recognition and inhibition of human GrB by Ad5-100K. In addition, it is likely that these further species-specific enzyme substrate-inhibitor interactions outside the substrate binding pockets of the active site influence the cleavage specificity of each GrB for macromolecular substrates (e.g., NuMA, PARP, and Mi-2).
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83 and 80 kDa. Since MAV1-100K was expressed at low levels in transfected cells (data not shown), we determined whether mouse GrB inhibitory activity was present in lysates from MAV-1-infected NIH 3T3 cells by analyzing the cleavage of endogenous mouse NuMA and Mi-2. No inhibitory activity against human, mouse, or rat GrB was observed in lysates from infected cells (Fig. 4A, lanes 5 to 8) compared to lysates from mock-infected cells (Fig. 4A, lanes 1 to 4).
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Evolution of Ad5-100K as an inhibitor of human GrB.
The 100K protein is a multifunctional protein with essential functions in the adenovirus life cycle. Sequence analysis of several adenovirus subgenera distinguished a major conserved region of 600 to 700 amino acids located centrally in the protein (central conserved region [CCR]) and a nonconserved region located at each terminus of the molecule (Fig. 3A). Interestingly, the NTR (which is unique to each adenovirus subgenus) contains the human GrB inhibitory motif in Ad5-100K. To better understand the importance of the Ad5-100K NTR and CCR in human GrB inhibition, we constructed a chimeric molecule (Ad5-MAV100K) containing the NTR of Ad5-100K (amino acids 1 to 93) and both the CCR and the C-terminal region (CTR) of MAV1-100K (amino acids 42 to 743) (Fig. 5A). Similarly, we also addressed whether the NTR of Ad5-100K alone can inhibit human GrB by fusing this region to GFP (Ad5-GFP) (Fig. 5A).
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The human GrB inhibitory activities of the different molecules then were determined by using purified components in vitro and assaying GrB activity by quantifying Mi-2 cleavage. Equimolar amounts of rAd5-100K inhibited GrB significantly; a fivefold molar excess of the inhibitor completely abolished GrB activity (Fig. 5C, lanes 9 and 10). Interestingly, recombinant Ad5-MAV100K (rAd5-MAV100K) demonstrated a similarly potent inhibitory activity against human GrB (Fig. 5C, lanes 14 to 16). When similar studies were performed with mouse GrB and rat GrB, no inhibitory activity was observed, even with a 20-fold molar excess of the inhibitor (data not shown). Of note, although rMAV1-100K did not show inhibitory activity against the mouse and rat proteases, even when present at a molar excess (Fig. 4B), inhibitory activity against human GrB was observed at a 20-fold molar excess of the inhibitor (Fig. 5C, lane 13). In contrast, recombinant GFP (Fig. 5C, lanes 17 and 18) or recombinant Ad5-GFP (rAd5-GFP) (Fig. 5C, lanes 19 and 20) had no inhibitory effects on human GrB. Taken together, these data demonstrate that elements within both the NTR of Ad5-100K and the CCR and CTR are required for efficient and potent inhibition of human GrB.
Identification of a C-terminal domain in the 100K protein that is required for efficient inhibition and stable complex formation between Ad5-100K and human GrB.
To determine whether an extended macromolecular recognition domain, in addition to the reactive-site loop (containing IEQD48-P), in Ad5-100K is required for human GrB inhibition, we generated a series of Ad5-100K deletion mutants (Fig. 6). Increasing amounts of wild-type rAd5-100K or deletion mutants were titrated against a fixed amount of purified human GrB, and the residual protease activity was determined by cleavage of [35S]methionine-labeled Mi-2. All deletion mutants that included the NTR of Ad5-100K showed some inhibitory activity against human GrB, although their potencies varied greatly (Fig. 6). Interestingly, the most potent inhibitors were those in which amino acids 688 to 781 at the C terminus (CT688-781) were included (except for 100K-277-CT, in which the NTR is absent). Indeed, deletion mutant r100K
5, which contains only the NTR plus CT688-781, was as efficient as wild-type rAd5-100K in inhibiting GrB. Thus, equimolar amounts of rAd5-100K or r100K5 inhibited GrB significantly, and a fivefold molar excess of the inhibitor completely abolished GrB activity (Fig. 7A, lanes 3 and 4 and lanes 7 and 8, respectively).
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5) was cleaved as efficiently as the full-length protein (Fig. 7B, lanes 17 to 20 and lanes 1 to 4, respectively) and was equally potent as an inhibitor of GrB (Fig. 7A). These data were somewhat surprising, in light of previous observations demonstrating that IEQD48 within the NTR is the major cleavage site within the inhibitor and likely interacts with the active site of the protease (2). These results therefore support the idea that the efficiency of cleavage at IEQD48 is improved by an interaction with the CT688-781 domain.
Since extended macromolecular interactions outside the active site (exosite) in serine proteases frequently contribute significant binding energy and specificity to the interaction, we used coimmunoprecipitation analysis to address whether the CT688-781 domain in the 100K protein might be required for the establishment of a stable inhibitory complex with GrB. GrB and Ad5-100K or several deletion mutants were coincubated and immunoprecipitated with a polyclonal antibody that is directed against the 100K protein and that recognizes wild-type and mutant proteins. The 100K proteins were visualized by immunoblotting of N-terminal epitope tag T7, which is present on all of the 100K proteins, and GrB was visualized by immunoblotting with a monoclonal antibody against GrB. Full-length Ad5-100K formed a stable complex with human GrB (Fig. 7C, lane 2) but not mouse GrB (data not shown), as demonstrated by coprecipitation of both the inhibitor and the protease. In contrast, deletion mutant 100K-NT-245 did not coprecipitate human GrB (Fig. 7C, lane 3). Mutant 100K5 did coprecipitate GrB (Fig. 7C, lane 4), demonstrating that the stable interaction of the 100K protein with the protease is species specific and requires two distinct elements: (i) interaction of the reactive-site loop with the active site of the protease and (ii) an additional interaction involving the CT688-781 domain with an as-yet-unidentified region on GrB. Both interactions are critical for stable binding of the protease and the inhibitor, as well as for efficient cleavage and potent inhibition.
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DISCUSSION |
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This species-specific interaction of GrB with its substrate or inhibitor has also been observed for other serpin inhibitors of GrB. Thus, the murine serpin SPI6 has potent inhibitory activity against murine GrB (33, 34) but is a relatively inefficient inhibitor of human GrB (50). In contrast, the human homologue of SPI6 (PI-9) has potent inhibitory activity against the human protease (52). Interestingly, GrB cleavage of PARP, as well as the activation of caspase 3 and Bid, does not appear to be species specific, since interspecies enzymes and substrates have been broadly used (i.e., rat, human, and mouse enzymes all cleave human, mouse, bovine, and monkey substrates) (1, 17, 23, 32, 53), although their cleavage efficiencies have not been addressed.
Although the 100K protein from mouse adenovirus does not inhibit mouse or rat GrB (even at a 20-fold molar inhibitor excess), it has some inhibitory activity against human GrB. Interestingly, fusion of the NTR of Ad5-100K with MAV1-100K (but not GFP) increased the inhibitory activity of MAV1-100K against human GrB by approximately 20-fold, demonstrating that there are at least two critical regions which define efficient 100K protein inhibition against human GrB: (i) a unique sequence within the reactive-site loop [(P1)Asp48-(P1')Pro49] in Ad5-100K which interacts with the active site and (ii) the presence of an additional inhibitor-enzyme interaction likely outside the catalytic site. Indeed, by analyzing the inhibitory effects of different Ad5-100K deletion mutants, we defined a small region (amino acids 688 to 781) located toward the end of Ad5-100K which is crucial for the efficient inhibition of human GrB, likely through stabilizing the protease-inhibitor interaction (see below). This requirement for an extended interaction outside the GrB catalytic site is not exclusive for the Ad5-100K inhibitor but is likely required for all efficient GrB substrate interactions. This notion is supported by prior studies showing that the hydrolysis of synthetic substrates catalyzed by GrB is highly dependent on the length and sequence of the substrates; in addition, even a peptide comprising an optimized P4-P2' sequence resulted in a molecule that was not cleaved efficiently by GrB, indicating that substrate recognition involves features beyond this recognition motif (24, 30).
All protein proteinase inhibitors prevent access of substrates to the proteinase catalytic site through steric hindrance. Members of one class of inhibitors (e.g., most serpins) achieve this goal through binding of a peptide segment directly to the catalytic site in a substrate- or product-like manner; the selectivity of inhibition is achieved through the utilization of the substrate recognition sites in the proteinase. Although these inhibitors make additional contacts with their cognate proteinases, these secondary contacts are in most cases not very specific and apparently not important for the tightness of binding (10). In contrast, other inhibitors (e.g., hirudin and related inhibitors) bind mainly through extended macromolecular interaction sites (exosites) adjacent to the catalytic residues of their cognate proteinases, utilizing substrate binding and other sites (10). Since Asp48 within the NTR of Ad5-100K is directly cleaved by GrB and the motif Asp48Pro49 is absolutely required for protease inhibition, it is likely that the consensus sequence containing IEQD48P49 is bound to the GrB active site. Interestingly, deletion mutants containing the N-terminal reactive-site loop but lacking the CT688-781 region are inefficiently cleaved by human GrB and are poor inhibitors of the protease. We therefore propose that the CT688-781 region in Ad5-100K interacts with human GrB through an exosite (likely specific for human GrB but not mouse or rat GrB). The binding site on GrB for CT688-781 is not yet known, nor is the mechanism whereby this additional interaction participates in the establishment of rapid and potent inhibition of the protease. Since CT688-781 alone neither has inhibitory activity against human GrB nor stably binds this protease (data not shown) but is required for potent inhibition by the NTR and since both domains are required for stable complex formation with GrB, these data support the proposal that the function of CT688-871 is to stabilize the interaction between the protease and the inhibitor, allowing stable complex formation and protease inhibition at the active site. Although the proline in the P1' position likely disfavors GrB cleavage at IEQD through steric hindrance of the scissile bond by the proline secondary amide, this proline is essential for human GrB inhibition. Interestingly, the domain containing IEQD-P alone is insufficient for inhibition and requires the presence of CT688-871. It is therefore possible that this docking site (CT688-871) serves to enhance the recognition or cleavage of this suboptimal site. Thus, the optimal tetrapeptide sequence upstream of the cleavage site allows occlusion of the active site, while the P1' proline disfavors a conformation which allows for efficient cleavage, dramatically slowing down the catalytic activity of the protease (kcat, 5.2 x 10-5/s) (2).
Since exosite interactions define the affinity and binding specificity within enzyme-substrate complexes (7), different exosite specificities among human GrB, mouse GrB, and rat GrB could explain the differences in cleavage site recognition for the same substrate. These differences in specificity of the proteases in different species raise significant obstacles for the use of GrB-deficient mice as a model from which to infer the role of GrB in human inflammatory and autoimmune pathology.
Despite their distinct catalytic mechanisms, GrB and the caspase family members share a novel and stringent specificity for aspartic acid in the P1 position (24, 57, 58). The GrB optimal tetrapeptide recognition motif (P4-P1) is also similar to that of caspases involved in the initiation of apoptosis (caspases 6, 8, 9, and 10), presumably reflecting their common role as initiators of the caspase-driven proteolytic cascade (24, 58). Despite these similarities, there are also striking differences in the substrate recognition properties of the two families of apoptotic proteases. For example, caspases cleave tetrapeptides and macromolecules with similar, high efficiencies (58). In contrast, GrB cleaves tetrapeptides and synthetic substrates comprising an optimized P4-P2' sequence poorly relative to macromolecules, supporting the concept that substrate recognition involves features beyond the limited cleavage motif (13, 24, 30, 51). The data presented here define the substrate- and/or inhibitor-specific component of such extended macromolecular recognition. The definition of the corresponding exosite in GrB will reveal novel methods for selective inhibitor design, which may be useful for therapy of the inflammatory and autoimmune processes in which GrB has been implicated.
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ACKNOWLEDGMENTS |
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F.A. holds the Mariana De Garay endowed chair for research on rheumatoid arthritis and related conditions and is supported by the Lupus Foundation of America. L.A.C.-R. is supported by NIH grant AR-44684. A.R. is supported by NIH grant DE-12354 and a Burroughs Wellcome Foundation translational research award.
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FOOTNOTES |
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