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Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M−1s−1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E−64, solved at 1.73 and 1.98 A, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E−64 and ananain explains the high inhibitory efficiency of E−64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

Summary Proteases are enzymes that specifically cleave the peptide backbone of their target proteins. As an important type of irreversible post-translational modification, protein cleavage underlies many key physiological processes. When dysregulated, proteases’ actions are associated with numerous diseases. Many proteases are highly specific, cleaving only those target substrates that present certain particular amino acid sequence patterns. Therefore, tools that successfully identify potential target substrates for proteases may also identify previously unknown, physiologically relevant cleavage sites, thus providing insights into biological processes and guiding hypothesis-driven experiments aimed at verifying protease–substrate interaction. In this work, we present PROSPERous, a tool for rapid in silico prediction of protease-specific cleavage sites in substrate sequences. Our tool is based on logistic regression models and uses different scoring functions and their pairwise combinations to subsequently predict potential cleavage sites. PROSPERous represents a state-of-the-art tool that enables fast, accurate and high-throughput prediction of substrate cleavage sites for 90 proteases. Availability and implementation http://prosperous.erc.monash.edu/ Supplementary information Supplementary data are available at Bioinformatics online.

A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis., Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine–containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S 2 subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola . An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P 2 proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P 2 proline, further highlighting the importance of Y69 for FhCatL2 P 2 proline acceptance. Finally, the P 1 –P 4 specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P 2, which is unique amongst Fasciola cathepsins examined to date.

SummaryPlasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.

The synthesis of fluorescence quenched peptide substrates of granzyme C is presented. These peptides which incorporate some unusual amino acids and have “difficult sequence” elements, in some cases could not be prepared by standard Fmoc-based SPPS. Application of three different contemporary strategies, namely the use of pseudoproline dipeptides, PEG-based solid supports and the application of microwave heating were able to provide for successful synthesis of our desired substrate peptides.

Victoria Peddie, Markus Pietsch, Karen M. Bromfield, Robert N. Pike, Peter J. Duggan, Andrew D. Abell

Thrombin (EC 3.4.4.13) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin.

Proteases play vital roles in a range of biological processes, such as cell cycle, cell growth and differentiation, apoptosis, haemostasis and signalling. Fundamental to our knowledge of protease action is an understanding of how the active site operates; this has been examined through extensive studies of the substrate specificity of the enzymes. Kinetic and structural analyses have shown that the binding of a particular substrate residue at a protease subsite can have either a positive or negative influence on the binding of particular residues at other subsites. This phenomenon has been termed subsite cooperativity and has been observed in a wide range of proteases, often between non-adjacent subsites. This review aims to highlight studies where subsite cooperativity has been observed, experimental techniques used in the past and potential methods that can be employed to comprehensively examine this phenomenon. Further understanding of how the protease active site recognises and chooses its substrates for cleavage will have a significant impact on the development of pharmaceuticals that target these enzymes.

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

The liver fluke,Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed inPichia pastorisand purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion byF. hepatica.

Inhibitors of procoagulant enzymes, such as factor Xa (fXa) and thrombin, are important for treating thrombosis. Thrombin has complex pro- and anti-coagulant roles and thus fXa is thought to represent an ideal target. Discrete kcat and Km values for cleavage of a library of fluorescence-quenched substrates by fXa were determined. The results highlighted the low selectivity of fXa at its prime sites, and its poor efficiency compared with thrombin, creating a challenge for the design of fXa-specific peptidic inhibitors. We hypothesized that Km rather than kcat/Km values may be better indicators of inhibitor potential for a peptidic sequence, leading us to design peptide sequences for both fXa and thrombin in three forms: fluorescence-quenched substrates, standard alpha-peptides and peptides containing a beta-homoarginine at the cleavage site. Kinetic and competitive inhibition assays with both fXa and thrombin showed the fluorescence-quenched substrates to be the best inhibitors, while the inhibitory effect of the beta-homoarginine peptides varied for the two proteases. Importantly, fXa was inhibited to a much greater extent by the beta-peptides than the corresponding alpha-peptides, resulting in an increased selectivity for fXa inhibition over thrombin for those peptides containing a beta-amino acid at the cleavage site.

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.

Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 A, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A beta-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90 degrees to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 +/- 0.9 x 10(6) m(-1) s(-)1) and cathepsin G (ka: 7.9 +/- 0.9 x 10(5) m(-1) s(-)1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4-P6' region of the RCL, the distal hinge, and the loop between strands 4B and 5B.

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2′-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine-specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA(cat), the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA(cat) or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aalpha-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bbeta-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene.

Factor Xa is a central protease in the coagulation cascade and the target for many anticoagulant compounds currently under development. The preferences of the enzyme for substrates incorporating residues N-terminal to the cleavage site (P1, P2, etc.) have been elucidated, but little is known of its preferences for residues C-terminal to the cleavage site (P1′, P2′, etc.). The preferences of bovine factor Xa for substrate residues in the P1′, P2′ and P3′ positions were mapped using fluorescence-quenched substrates. Bovine factor Xa, often used as a model for factor Xa, was most selective for the P2′ position, less selective at the P1′ position and almost completely non-selective at the P3′ position. It appears that while the prime side subsites of factor Xa impose some selectivity towards substrates, the influence of these sites on factor Xa cleavage specificity is relatively low in comparison to related enzymes such as thrombin.

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new “tethered ligand” N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca 2+ ] i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca + entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca 2+ ] i . Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca 2+ ] i followed by a sustained plateau; [Ca 2+ ] i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.

The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main β-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6°C) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0°C), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband’s plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.© 1998 by The American Society of Hematology.

Angiotensinogen, a plasma serpin, functions as a donor of the decapeptide angiotensin I, which is cleaved from the N-terminus by renin. To assess the contribution of the serpin framework to peptide cleavage we produced a chimaeric molecule of α 1 -antitrypsin carrying the angiotensinogen N-terminus and determined the kinetic parameters for angiotensin I release. The K m for plasma angiotensinogen was 18-fold lower than for the chimaeric protein while the catalytic efficiency was four-fold higher. We also show that Cys-18 participates in a disulphide bond and propose that constraints on the N-terminus profoundly affect the interaction with renin.

Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin-like activity which have been implicated as virulence factors in adult-onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis-derived proteinases are important virulence factors in the development of periodontal disease.

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M). Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3). alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.

Thrombin stimulates expression of interleukin 6 and cyclooxygenase 2 by osteoblasts, both of which enhance osteoblast-mediated osteoclast differentiation by increasing the ratio of receptor activator of nuclear factor κB ligand (RANKL) expression to that of osteoprotegerin (OPG) in osteoblasts. We hypothesised that thrombin would also increase this ratio and thereby stimulate osteoclast differentiation in mixed cultures of osteoblastic cells and osteoclast precursors. In primary mouse osteoblasts, but not in bone marrow stromal cells, thrombin increased the ratio of RANKL to OPG expression. Thrombin inhibited differentiation of osteoclasts, defined as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei, in mouse bone marrow cultures treated with osteoclastogenic hormones; this effect was not mediated by the major thrombin receptor, protease-activated receptor 1, nor did it require thrombin's proteolytic activity. Thrombin also caused a decrease in the number of TRAP-positive cells with fewer than three nuclei. Thrombin (active or inactive) also inhibited osteoclast differentiation and bone resorption, respectively, in cultures of mouse spleen cells and human peripheral blood mononuclear cells induced to undergo osteoclastogenesis by treatment with RANKL and macrophage colony-stimulating factor. Osteoclast differentiation in spleen cells was inhibited when they were exposed to thrombin from days 0 to 3 or 3 to 5 of culture but not days 5 to 7 when most fusion occurred. Thrombin inhibited expression of RANK by spleen cells. These observations indicate that, although thrombin stimulates production of osteoclastogenic factors by osteoblastic cells, it inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors that does not require thrombin's proteolytic activity.

Ragweed (Ambrosia artemisiifolia), the major cause of late summer hay fever (allergic rhinitis) in the United States and Canada, is clinically the most important source of the seasonal aeroallergens. A novel endopeptidase was extracted from the pollen of this plant and purified by a series of column chromatographic steps. It has a molecular mass of 82 kDa according to gel filtration and SDS-polyacrylamide gel electrophoresis and a pH optimum near 9.0, and its activity is unaffected by chelating or reducing agents. A 17-amino acid amino-terminal sequence of this protein showed no similarity with any other proteases. The enzyme was inhibited by diisopropyl fluorophosphate, a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone, a chymotrypsin-like proteinase inhibitor. Various synthetic substrates were efficiently cleaved with a strong preference for Phe in the P1 and P3 position and Pro in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. In addition to synthetic substrates, the neuropeptides, vasoactive intestinal peptide and substance P, which are required for normalized lung functions, were also rapidly hydrolyzed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor, which occurred through cleavage within the reactive site loop. These results indicate that the purified enzyme is a novel endopeptidase, which may be involved in both the degradation of neuropeptides and the inactivation of protective proteinase inhibitors during pollen-initiated allergic reactions.

African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and alpha2-macroglobulin effectively inhibited trypanopain-Tb, with the Ki values for cystatin C and low-molecular-mass kininogen (approximately 10(-11) M) predicting, that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or (a2-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats displayed no trypanopain-Tb-like activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.

The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate-4-morpholineethane sulfonic acid (MES)-Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate-MES-Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant, kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength. Km values, of ca. 5 microM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20-25 microM, specifically at pH 6.5 and I = 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L, kcat/Km declining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate-MES-Tris buffer. In Hanks' balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 were kcat, 18.9 s-1; Km, 13.5 microM; and kcat/Km, 1.4 x 10(6) M-1 s-1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks' balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).

Imbalances in GABA (γ-aminobutyric acid) homoeostasis underlie psychiatric and movement disorders. The ability of the 65 kDa isoform of GAD (glutamic acid decarboxylase), GAD65, to control synaptic GABA levels is influenced through its capacity to auto-inactivate. In contrast, the GAD67 isoform is constitutively active. Previous structural insights suggest that flexibility in the GAD65 catalytic loop drives enzyme inactivation. To test this idea, we constructed a panel of GAD65/67 chimaeras and compared the ability of these molecules to auto-inactivate. Together, our data reveal the important finding that the C-terminal domain of GAD plays a key role in controlling GAD65 auto-inactivation. In support of these findings, we determined the X-ray crystal structure of a GAD65/67 chimaera that reveals that the conformation of the catalytic loop is intimately linked to the C-terminal domain.

To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Arg-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10(-8) M), or human plasma incubated with RGP-1 (> 10(-9) M), induced VPE in a dose- and activity-dependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug. The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK). RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMWK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism.

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.

C1-inhibitor (serpin G1) is a 105 kDa inhibitor which functions as a major antiinflammatory protein in the body. It has its effects via inhibition of the proteases of the complement system and contact system of coagulation, as well as several direct effects mediated by its unique highly glycosylated N-terminal domain. The serpin controls a number of different proteases very efficiently and for some of these the function is augmented by the cofactor, heparin. Here, we describe the preparation of human plasma and recombinant C1-inhibitor and the basic methods required for their characterization, using the complement enzyme C1s as an example of a target enzyme.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals., Author Summary Extracellular proteases are produced by many bacterial pathogens and are commonly involved in the degradation of the host extracellular matrix, facilitating invasion and colonization. One such pathogen is Dichelobacter nodosus, the causative agent of ovine footrot, a disease of major economic significance to the international sheep industry. D. nodosus secretes a number of serine proteases, which are thought to cause the tissue damage associated with virulent footrot. Our study showed that a D. nodosus mutant lacking one of the three secreted proteases, AprV2, failed to cause virulent disease in sheep. We used x-ray crystallography to solve the structure of AprV2 and the closely related protease AprB2. Our structures revealed an unusual extended disulphide-tethered loop that is located next to, but does not form part of, the primary substrate binding site. Through targeted mutagenesis studies we were able to show that this loop functions as an exosite, mediating effective enzyme-substrate interactions. Bioinformatic analyses suggest that subtilases from other pathogenic bacteria may contain this loop and may therefore utilize a similar mechanism. Our multidisciplinary research approach has provided a comprehensive understanding of the functional role of extracellular proteases in the pathogenesis of ovine footrot.

Proteolytically active complexes of the proteinase cathepsin L, with an endogenous inhibitor of cysteine proteinases, were purified from sheep liver. The complexes were active against the synthetic substrate Z-Phe-Arg-NHMec and also the proteins azocasein and gelatin. The composition of the complexes was demonstrated by Western blotting, after reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with monospecific antibodies raised against purified sheep liver cathepsin L and purified sheep liver cysteine proteinase inhibitor (probably stefin B). Similar complexes could be formed in vitro, by coincubation of purified sheep liver cathepsin L with the purified sheep liver cystatin at a pH of 5.5 or higher.

The activity of single-chain cathepsin L was found to be markedly dependent on cysteine concentration, while a covalent, proteolytically active cathepsin L/cystatin complex was less cysteine-dependent. Cysteine levels and ionic strength did not affect the stability of either enzyme form and both enzyme forms were found to be stable for significant periods of time at or near physiological pH.

1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.

The complement system plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical complement and lectin pathways are controlled by SERPING1 [(C1 inhibitor) serpin peptidase inhibitor, clade G, member 1], which inactivates the components C1s and MASP-2 (mannan-binding lectin serine peptidase 2). GAGs (glycosaminoglycan) and DXS (dextran sulfate) are able to significantly accelerate SERPING1-mediated inactivation of C1s, the key effector enzyme of the classical C1 complex, although the mechanism is poorly understood. In the present study we have shown that C1s can bind to DXS and heparin and that these polyanions enhanced C1s proteolytic activity at low concentrations and inhibited it at higher concentrations. The recent determination of the crystal structure of SERPING1 has given rise to the hypothesis that both the serpin (serine protease inhibitor)–polyanion and protease–polyanion interactions might be required to accelerate the association rate of SERPING1 and C1s. To determine what proportion of the acceleration was due to protease–polyanion interactions, a chimaeric mutant of α1-antitrypsin containing the P4–P1 residues from the SERPING1 RCL (reactive-centre loop) was produced. Like SERPING1, this molecule is able to effectively inhibit C1s, but is unable to bind polyanions. DXS exerted a biphasic effect on the association rate of C1s which correlated strongly with the effect of DXS on C1s proteolytic activity. Thus, whereas polyanions are able to bind C1s and modulate its activity, polyanion interactions with SERPING1 must also play a vital role in the mechanism by which these cofactors accelerate the C1s–SERPING1 reaction.

Complement is a key component of the immune system, but can contribute to inflammatory diseases. The substrate specificity of C1s protease has been successfully investigated using a combinatorial approach, while a positional scanning method failed. The lack of success of the latter approach is possibly due to cooperativity in the active site, which could confound such analyses. With a panel of peptides devised using factorial design, we show pronounced cooperativity between the S4 and S1′ subsites in the active site of the enzyme, and weaker cooperativity between the S1′ and S3′ subsites. The use of factorial design has promise as a methodology for determining cooperativity in protease active sites.

Anti-peptide antibodies were raised against synthetic peptides selected from the sequences of human cathepsins B and L, porcine cathepsin D and human type IV collagenase. Sequences were selected from the active site clefts of the cathepsins in the expectation that these would elicit immunoinhibitory antibodies. In the case of type IV collagenase a sequence unique to this metalloproteinase subclass and suitable for immunoaffinity purification, was chosen. Antibodies against the chosen cathepsin B sequence were able to recognize the peptide but were apparently unable to recognise the whole enzyme. Antibodies against the chosen cathepsin L sequence were found to recognise and inhibit the native enzyme and were also able to discriminate between denatured cathepsins L and B on Western blots. Antibodies against the chosen cathepsin D sequence recognised native cathepsin D in a competition ELISA, but did not inhibit the enzyme. Native type IV collagenase was purified from human leukocytes by immuno-affinity purification with the corresponding anti-peptide antibodies.

Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor beta, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E(2) and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E(2) was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of synthetic prostanoids stimulated secretion of interleukin-6 with differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.

Osteopontin is a secreted glycoprotein expressed by many cell types including osteoblasts and lymphocytes; it is a constituent of the extracellular matrix (ECM) in bone, and a mitogen for lymphocytes. To investigate the role of osteopontin in muscle repair and development, expression of osteopontin by muscle cells in vivo and in vitro , and the effects of osteopontin on myoblast function in vitro were investigated. Osteopontin staining was weak in sections of muscle from normal mice, but associated with desmin-positive cells in areas of regeneration in muscles from mdx mice. In immunocytochemical, PCR and ELISA studies, cultured myoblasts were found to express osteopontin and secrete it into medium. Treatment of myoblast cultures with fibroblast growth factor-2, transforming growth factor β1, interleukin-1β or thrombin significantly increased osteopontin expression. Osteopontin-coated substrata promoted adhesion and fusion, but not proliferation or migration, of myoblasts. The effect of osteopontin on myoblast adhesion was RGD-dependent. In solution, osteopontin significantly increased proliferation and decreased fusion and migration of myoblasts. These results suggest that myoblasts are an important source of osteopontin in damaged muscle and that osteopontin released by myoblasts may assist in controlling both the myogenic and inflammatory processes during the early stages of muscle regeneration.

Cells responsible for the formation and maintenance of bone express thrombin-responsive members of the protease-activated receptor family of G protein-coupled receptors. Thrombin has been shown to elicit a number of functional responses in these cells, including proliferation and cytokine production in osteoblasts. Many, but not all, of the effects of thrombin on bone cells are initiated by activation of protease-activated receptor-1. A combination ofin vitroobservations and results ofin vivostudies in protease-activated receptor-1-null mice suggest that thrombin plays multiple roles in the early stages of bone healing.