Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.     

Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.     

Use of anti-neoepitope antibodies for the analysis of degradative events in cartilage and the molecular basis for neoepitope specificity

Degradation of the cartilage proteoglycan, aggrecan, is an essential aspect of normal growth and development, and of joint pathology. The roles of different proteolytic enzymes in this process can be determined from the sites of cleavage in the aggrecan core protein, which generates novel termini (neoepitopes). Antibodies specific for the different neoepitopes generated by such cleavage events provide powerful tools with which to analyse these processes. The same approach can be used to differentiate the processed, active forms of proteases from their inactive pro-forms. Since the proteolytic processing of these enzymes requires the removal of the inhibitory pro-region, it also results in the generation of N-terminal neoepitopes. Using the newborn rat long bone as a model system, it was shown that the active form of ADAMTS-4 [ADAM (a disintegrin and metalloproteinase) with thrombospondin motifs-4], but not ADAMTS-5, co-localizes with the aggrecan cleavage neoepitopes known to be produced by this metalloproteinase. Thus, in long bone growth, aggrecan turnover seems to be dependent on ADAMTS-4 activity. To demonstrate the molecular basis of the specificity of anti-neoepitope antibodies, the Fv region of a monoclonal antibody specific for a neoepitope generated by the ADAMTS-4-mediated cleavage of aggrecan has been modelled and the binding of the peptide epitope simulated. In the docked structure, the N-terminus of the peptide antigen is clearly buried in the binding-site cavity. The absence of an open cleft makes it impossible for the intact substrate to pass through the binding site, providing a rationale for the specificity of this class of antibodies.     

Bifunctional thrombin inhibitors based on the sequence of hirudin45-65

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Mg X ATP2-dependent interaction of the inhibitor protein of the cAMP-dependent protein kinase with the catalytic subunit

The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase has been investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was shown to be competitive versus peptide substrate and uncompetitive versus Mg X ATP2-. This, together with the observations of Gronot et al. (Gronot, J., Mildvan, A.S., Bramson, H. N., Thomas, N., and Kaiser, E.T. (1981) Biochemistry 20, 602-610) and those given in the accompanying paper (Whitehouse, S., Feramisco, J.R., Casnellie, J.E., Krebs, E.G., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3693-3701), would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg X ATP complex. The Ki for this interaction at saturating Mg X ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5'-adenylimidodiphosphate (AMP X PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP X PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit, and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 approximately 60 nM for ATP; this value is two orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP but may simply be a reflection of the formation of a catalytic subunit-Mg X ATP X inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.     

Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.     

Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.     

Exosite binding tethers the macromolecular substrate to the prothrombinase complex and directs cleavage at two spatially distinct sites

The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg(323)-Ile(324), followed by cleavage at Arg(274)-Thr(275). We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaDeltaF1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIaDeltaF1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K(m). This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIaDeltaF1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIaDeltaF1 to the enzyme. Specific recognition of mIIaDeltaF1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.     

The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen

Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.     

Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions

Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.     

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor.

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.     

TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K(m) in the 10 microm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.     

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods

To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results

Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion

We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance

Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.