Recombinant Human Cathepsin H Lacking the Mini Chain Is an Endopeptidase

Abstract

Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.

Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves

“…AALP is a predicted aminopeptidase similar to mammalian cathepsin-H. This protease cannot act as an endopeptidase because one side of the substrate binding groove is blocked by a minipeptide that originates from the prodomain and remains covalently bound through a disulphide bridge [63]. AALP shares these features and therefore probably acts on (neo) N-termini during the bulk protein degradation process in the vacuole.…”

Comparative genomic of the teleost cathepsin B and H and involvement in bacterial induced immunity of miiuy croaker

“…The occluding loop is present only in CTSB and absent in other cysteine proteases instead of the ERFNIN signature sequence in CTSH [41]. The mini-chain, an octapeptide (EPQNCSAT) originating from the CTSH propeptide [43,44], play important roles in the aminopeptidase activity of the enzyme [45], the attachment of carbohydrate residues to the body of the enzyme and the substrate recognition [46]. In addition, the glutamine oxyanion hole and active site cysteine, histidine and asparagine residues were conserved in both CTSB and CTSH among lower and higher vertebrates (Fig.…”

Processing and presentation of (pro)‐insulin in the MHC class II pathway: the generation of antigen‐based immunomodulators in the context of type 1 diabetes mellitus

“…In addition, CatG is directly involved in antigen processing [77]. Although CatH exhibits equally as an exoprotease and an endoprotease, the endoprotease activity is much lower, but it is capable of degrading the oxidized insulin β-chain at 13 EA 14 [87].…”