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Cathepsin B (EC 3.4.22.1) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin (Z-Arg-Arg-NH-MEC), suggesting that procathepsin B is catalytically active. The activity-based probe DCG-04, which is an E-64-type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.

Cathepsin S is unique among mammalian cysteine cathepsins in being active and stable at neutral pH. We show that autocatalytic activation of procathepsin S at low pH is a bimolecular process that is considerably accelerated (approximately 20-fold) by glycosaminoglycans and polysaccharides such as dextran sulfate, chondroitin sulfates A and E, and dermatan sulfate through electrostatic interaction with the proenzyme. Procathepsin S is also shown to undergo autoactivation at neutral pH in the presence of dextran sulfate with t1/2 of approximately 20 min at pH 7.5. This novel property of procathepsin S may have implications in pathological conditions associated with the appearance of active cathepsins outside lysosomes.

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.

A wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Angstroms resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.

Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.

From human embrional lung fibroblasts mRNA was obtained and converted to cDNA. The procathepsin L coding region was amplified by PCR, inserted into pALTER and, after checking the nucleotide sequence, transferred into pET81F1+. Procathepsin L was expressed by induction of recombinant E. coli strain BL21[DE3](pLysS) with IPTG and was found to be deposited into inclusion bodies. These were isolated and solubilized in guanidinium hydrochloride. The soluble proteins were sulphonated and procathepsin L was obtained after gel filtration. Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected size and enzymatic activity against a fluorogenic substrate.