The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

The reactive site of human inter-alpha-trypsin inhibitor is in the amino-terminal half of the protein

Human inter-alpha-trypsin inhibitor has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G. The protein was cleaved into two major fragments without loss of activity by incubation with Serratia marcescens metalloproteinase, and these were separated by ion-exchange chromatography. Inhibitory activity was found in only one of the fragments, the amino-terminal sequence of which was found to be identical with that of the native protein, as well as with that reported earlier for the urinary trypsin inhibitor. It may thus be concluded that the reactive site of the inter-alpha-trypsin inhibitor is located in the amino-terminal region.     

The purification of bovine cathepsin B1 and its mode of action on bovine collagens

1. Cathepsin B1 was isolated from bovine spleen by autolysis, (NH(4))(2)SO(4) fractionation and chromatography on Amberlite IRC-50. Two isoenzyme forms were purified to homogeneity by chromatography on CM-cellulose and DEAE-Sephadex. 2. A collagenolytic cathepsin was separated from cathepsin B1 during purification. The remaining collagenolytic activity of the purified cathepsin-B1 isoenzymes was no greater than 0.3 unit/unit of cathepsin B1 compared with about 5.0 unit/unit of cathepsin B1 in the autolysed spleen extracts. 3. The cathepsin B1 isoenzymes lowered the viscosity of gelatin at 37 degrees C. Optimum activity was at pH4-5. 4. At 28 degrees C the interchain cross-links in native tropocollagen were cleaved most effectively at pH4-5. Insoluble tendon collagen was digested at pH3.5 and 28 degrees C to yield mainly alpha-chain components, with the loss of a short N-terminal sequence. 5. Electron-microscope studies of collagen fibrils showed that cathepsin B1 caused longitudinal splitting and dissociation of the protofilaments. The effect was not general but occurred at selected sites. 6. The isoenzymes of cathepsin B1 cleaved the telopeptide region of calf skin tropocollagen between the lysine-derived cross-link and the triple helix. The CB1 peptide fragments obtained from enzyme-degraded alpha1 chains were hydrolysed at Gly(12)-Ile(13) and Ser(14)-Val(15). The residual alpha2 CB1 peptides were hydrolysed at Ala(8)-Asp(9) and Asp(9)-Phe(10).     

Cathepsin G binding to human platelets. Evidence for a specific receptor.

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.     

Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein

The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases.     

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.     

Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents.

A procedure is described for the purification of the individual major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 from HeLa cells. The salient feature of the method is the combined usage of antibodies against 2,2,7-trimethylguanosine (m3G) and 6-methyladenosine (m6A) for differential immune affinity chromatography of the snRNPs. While anti-m3G affinity columns allow the separation of snRNPs U1, U2 and U5 from U4/U6 RNPs, anti-m6A antibodies selectively react with snRNPs U2 and U4/U6. Our technique further incorporates immune affinity chromatography of snRNPs with antibodies against snRNP proteins in addition to ion exchange chromatography. The procedure avoids the usage of denaturing agents, so as to maintain the native structure of the particles. This is mainly provided for by the possibility of eluting the anti-m3G and anti-m6A bound snRNPs with excess of the respective nucleosides. We have so far identified 12 polypeptides as constituents of the major snRNPs U1 to U6. Seven proteins of approximate mol. wts 29 kd (B'), 28 kd (B), 16 kd (D), 15.5 kd (D'), 12 kd (E), 11 kd (F) and 9 kd (G) were present in each of the individual snRNPs U1, U2, U5 and U4/U6. In addition to the common proteins, U1 RNPs contain three unique polypeptides of mol. wts 70 kd, 34 kd (A) and 22 kd (C). U2 RNPs are characterized by the presence of a 33-kd and a 28.5-kd protein, denoted A' and B". We could not detect any unique polypeptide confined to the purified snRNPs U5 or U4/U6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Latent fibronectin-degrading serine proteinase activity in N-terminal heparin-binding domain of human plasma fibronectin

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.     

In vitro activity of the human neutrophil cathepsin G on Eimeria tenella sporozoites

The role of human neutrophil cathepsin G (Cat G) on Eimeria tenella sporozoites was studied in vitro. Sporozoites were incubated for 2 hr at 37 C in PO4 buffer, 0.9% NaCl (PBS), pH 7.6 in the presence of Cat G (50 micrograms/ml), diisopropyl fluorophosphate-inhibited Cat G (DFP-Cat G) (50 micrograms/ml) or PBS alone, prior to being inoculated into embryonated eggs. As judged by oocyst production on day 7 postinoculation, embryo mortality and the hemorrhage scores, both Cat G and DFP-Cat G demonstrated anticoccidial activity; greater activity was obtained with the DFP-Cat G. Sporozoites were exposed also to increasing concentrations of native and trypsin-digested DFP-Cat G (0-100 micrograms/ml) under the same conditions. Significant protection (37% and 49% for native and digested DFP-Cat G, respectively) was obtained with a low concentration (5 mu/ml), and higher concentrations resulted in 70% and 84% protection, respectively. The primary bactericidal domain of Cat G, the HPQYNQR peptide, at 3 concentrations (25, 50, and 100 micrograms/ml), reduced the oocyst production by 46%, 16%, and 15%, respectively. The anticoccidial activity of Cat G may involve a peptide fragment different from the antimicrobial domain of the enzyme.     

Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Synthesis of peptide fragments related to eglin c and examination of their inhibitory effect on human leukocyte elastase, cathepsin G and alpha-chymotrypsin

Various kinds of peptide fragments related to eglin c were prepared by the conventional solution method and their inhibitory effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Peptide (31-40) inhibited cathepsin G (Ki = 2.3 x 10(-4) M), peptide (41-49) potently inhibited cathepsin G and alpha-chymotrypsin (Ki = 4.2 x 10(-5) M and 2.0 x 10(-5) M, respectively), and peptide (60-63) inhibited leukocyte elastase (Ki = 1.6 x 10(-4) M), whereas, peptide (31-35) weakly inhibited both elastase and cathepsin G (Ki = 2.1 x 10(-3) M and 7.3 x 10(-4) M, respectively).     

Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.     

Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association

Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH

Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.     

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme

Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1-6), dynorphin (1-13), alpha-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid D-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1-13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1-6), alpha-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [delta1-10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10(-10) M range.     

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1alpha synthase (ST6GalNAc V)(1)

Cystatin C with the 11 N-terminal amino acids truncated shows a much lower affinity for cysteine proteinases than the intact inhibitor. Such truncation of cystatin C is recorded after action of glycyl endopeptidase and cathepsin L. Incubation of cystatin C with papain, cathepsin B or cathepsin H led to no changes in the cystatin C molecule. Isoelectric focusing of the cathepsin L and cystatin C mixture showed the formation of two new bands. One of them appeared whether E-64 or PMSF was added or not, evidently representing a cystatin C/cathepsin L complex. The other band is the truncated cystatin C molecule. N-terminal sequencing after separation by HPLC showed that cystatin C is cleaved by cathepsin L at the Gly11-Gly12 bond. The action of cathepsin L on cystatin C may be explained by the cleavage of the scissile bond in an inappropriate complex.     

Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase.

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of approximately 33 kDa and pI 5.1-5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7-15.0 nM), but poorly or not at all by stefin B (Ki > 250 nM) and L-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1' position, although the enzyme cleaved all P1' residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km approximately 5.0 x 103 M-1.s-1) were degraded approximately 25-fold less efficiently than the carboxypeptidase substrates (kcat/Km approximately 120.0 x 103 M-1.s-1).     

TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage

Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.