The slow-binding inhibition of cathepsin K by its propeptide.

A peptide corresponding to the full-length proregion (amino acids 16-114) of human cathepsin K was expressed and purified from Escherichia coli. This recombinant propeptide was investigated for its ability to inhibit the activity of three cysteine proteinases: cathepsins K, L, and B. Kinetic studies showed the propeptide to be a potent slow-binding inhibitor of its parent enzyme with a K(i) = 2. 61 nM at pH 6. This inhibition was pH-dependent, with a decrease in pH from 6 to 4 leading to a concomitant increase in K(i) to 147 nM. The propeptide also inhibited cathepsin L with a K(i) = 26.1 nM at pH 6, but showed little inhibition of cathepsin B at concentrations up to 400 nM.

Cathepsin B: an alternative protease for the generation of an aggrecan 'metalloproteinase' cleavage neoepitope.

Previously, only matrix metalloproteinases were believed capable of cleaving the cartilage proteoglycan, aggrecan, between Asn341 and Phe342, to yield a small G1 fragment terminating in the residues VDIPEN. We show that the combined endo- and exopeptidase activities of the cysteine protease, cathepsin B, also generate this epitope, suggesting that it should no longer be considered as an exclusive marker of metalloproteinase activity.

Immunolocalization of the cleavage of the aggrecan core protein at the Asn341-Phe342 bond, as an indicator of the location of the metalloproteinases active in the lysis of the rat growth plate.

In view of the extensive lysis of hyaline cartilage known to take place during endochondral bone formation, the current study was designed to test the hypothesis that metalloproteinases are the agents that mediate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn341-Phe342 bond, an immunohistochemical method has been developed to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavage of aggrecan by metalloproteinases is followed by the retention of the fragment known as G1, for it includes the G1 domain. Since the G1 fragment terminates in the amino acid residues ...FVDIPEN, we prepared an antiserum against FVDIPEN, confirmed its specificity, then applied it to the growth plate of 21-day-old rat tibia in the hope of localizing the G1 fragments. The antiserum specificity was shown by its recognition of the ...FVDIPEN sequence at the C-terminus of peptides and of G1 fragments produced by aggrecan cleavage. When the antiserum was applied to Western blots of guanidinium chloride extracts prepared from epiphyseal growth plate, it recognized two species (56 and 52 kDa), which differed only in the degree of glycosylation. These fragments were comparable in size to the G1 fragments generated by the action of recombinant metalloproteinase in vitro, thus confirming antiserum specificity for these fragments. Applying the antiserum to cryosections of 21-day-old rat tibiae revealed immunostaining at two intensities within the growth plate matrix: a strong staining was observed in a 1-5 microm-wide layer designated "peripheral" matrix, which borders the epiphyseal and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated "central" matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site where metalloproteinases exert their main lytic activity is a thin layer of matrix separating central from peripheral matrix.

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide.

Selected fragments of the 62-residue proregion (or residues 1p-62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p-51p and 26p-51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p-51p fragment, but remained in the 26p-43p and 28p-43p peptide fragments. Residues 23p-26p or TWQ25A in the N-terminal 1p-29p fragment did not show cathepsin B-induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p-51p peptide. Both the 1p-29p and 36p-51p fragments lack a common set of hydrophobic residues 30p-35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p-51p, 26p-51p, 28p-43p and 26p-43p, but the same residues induce the aggregation of peptides 16p-36p and 1p-36p. The peptide fragment 26p-43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p-43p or 26p-43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full-length cathepsin B proregion.

Crystallization of rat procathepsin B.

Rat procathepsin B has been expressed in the yeast Pichia pastoris. To facilitate crystallization of the proform two mutations were introduced: Cys29Ser to avoid self-processing and Ser115Ala to eliminate an N-glycosylation site. The recombinant protein was purified and crystallized by vapor diffusion against mother liquor containing 100 mM KSCN, 100 mM phosphate buffer, pH 6.5 and polyethylene glycol (PEG) 3350 as a precipitating agent. Crystal size was increased by multiple macroseeding. At a 16% PEG concentration trigonal crystals were obtained, with the space group P3(1)21 and a = 99.6, c = 141.4 A, gamma = 120 degrees. They diffract to 2.8 A resolution using a rotating-anode source. At a concentration of 11% PEG, rod-shaped crystals were grown. They are monoclinic, space group P2(1), a = 62.8, b = 67.9, c = 100.4 A, beta = 98.2 degrees and diffract to approximately 3.5 A.

Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing.

The cysteine protease papain is synthesized as a 40-kDa inactive precursor with a 107-amino-acid N-terminal pro region. Although sequence conservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp-36, papain precursor numbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate the function to this conserved motif, we have mutagenized at random each of the 4 residues individually within the pro region of the papain precursor. Precursor mutants were expressed in yeast, screened according to their ability to be processed through either a cis or trans reaction, into mature active papain. Three classes of mutants were found. Non-functional propapain mutants of the first class are completely degraded by subtilisin indicating that they are not folded into a native state. Mutants of the second class were neutral with respect to cis and trans processing. The third class included mutants that mostly accumulated as mature papain in the yeast vacuole. They had mutations that had lost the negatively charged Asp-36 residues and a mutation that probably introduces a positive charge, Phe-38His. The precursor of the Phe-38His mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe-38His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. These results indicate that the electrostatic status of the conserved motif participates in the control of intramolecular processing of the papain precursor.

E64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of S2 subsite interactions.

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) and benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) were synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if selectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter kinac./Ki and that the specificity of these analogues does not parallel that observed for substrates. This is particularly true in the case of the benzyl ester derivatives where the deviation from substrate-like behaviour is more important than with the benzyl amide derivatives. The results suggest that the amide proton of the benzyl amide group in HO-Eps-AA-NHBzl interacts in the S2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors. The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S2 subsite of the enzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the order of inactivation rates with chloromethane inhibitors (which are believed to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin substrates. In conclusion, the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E64 analogues in a rational manner.

Non-proteoglycan forms of biglycan increase with age in human articular cartilage.

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.