Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S

Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied proteomic identification of protease cleavage sites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P3' due to an occluding loop blocking access to the primed subsites. In P1', cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P1' phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues.     

Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries

Background

Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.

Methodology/Principal Finding

To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.

Conclusions

Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.     

Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry

To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime- and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography-tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.     

Factor Xa subsite mapping by proteome-derived peptide libraries improved using WebPICS, a resource for proteomic identification of cleavage sites

Proteomic identification of protease cleavage site specificity (PICS) is a recent proteomic approach for the easy mapping of protease subsite preferences that determines both the prime- and non-prime side specificity concurrently. Here we greatly facilitate user access by providing an automated and simple web-based data-analysis resource termed WebPics (http://clipserve.clip.ubc.ca/pics/). We demonstrate the utility of WebPics analysis of PICS data by determining the substrate specificity of factor Xa from P6-P6', an important blood coagulation protease that proteolytically generates thrombin from prothrombin. PICS confirms existing data on non-prime site specificity and refines our knowledge of factor Xa prime-site selectivity.     

Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin α, meprin β, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1' position because of a conserved positively charged residue in the active cleft subsite (S)1'. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2' or P3' leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1β, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing.     

A strategy to profile prime and non-prime proteolytic substrate specificity

A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4-P1 non-prime site substrate specificity. The data was used to synthesize biased donor-quencher positional scanning libraries to profile the P1'-P4' prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1'-P4' positional scanning libraries. From these libraries focused donor-quencher libraries incorporating P4-P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4-P4' substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.     

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.     

Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine–isoleucine or glycine–leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K_m similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.     

Human and mouse granzyme M display divergent and species-specific substrate specificities

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.     

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1′ were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R.     

HIV-1 Protease Uses Bi-Specific S2/S2′ Subsites to Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1′ proline. A P1′ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2–P1/P1′–P2′), with two complementary sets of rules. When P1′ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2′ amino acid interacts with a hydrophobic region of the S2′ subsite. When P1′ is not proline, the orientations of the P2 and P2′ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2′ amino acid usually engages hydrophilic amino acids in the S2′ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2′ subsites to accommodate the steric effects imposed by a P1′ proline on the orientation of P2 and P2′ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.     

Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1

Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.     

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites

We introduce human proteome-derived, database-searchable peptide libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used peptides in such libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P') sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6'. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.     

Definition of the extended substrate specificity determinants for beta-tryptases I and II.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.     

The extended cleavage specificity of human thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.     

Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16

Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.     

Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C(pro)), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C(pro), determined the crystal structure to 1.9-angstroms resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C(pro) adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4') and that FMDV 3C(pro) discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C(pro) enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C(pro) of an extended beta-ribbon that folds over the substrate binding cleft in other picornavirus 3C(pro) structures. Collectively, these results establish a valuable framework for the development of FMDV 3C(pro) inhibitors.     

Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.     

Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.     

Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.