A wild-type human procathepsin B was expressed, crystallized in two crystal° forms and its crystal structure determined at 3.2 and 3.3 A 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 enzymaticaily 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.

Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K s and k cat values of 2.1 þ 0.9 W WM and 0.12 þ 0.02 s 31 , respectively. Autoactivation is substantially accelerated in the presence of active cathepsin B molecules, indicating that mature cathepsin B is the catalytic species in the process. Proenzyme is cleaved without significant conformational changes as judged by circular dichroism, suggesting that propeptide unfolding occurs only after the cleavage. Procathepsin B autoactivation is pH-dependent with a pH optimum at 4.5 and with no processing observed at pH s 6.0. However, in the presence of 0.5 W Wg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t 1a2 V V90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.z 1999 Federation of European Biochemical Societies.

No abstract

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/1 E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three‐amino‐acid extension at its N‐terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin BA E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.Keywords. Cathepsin B ; renaturation ; refolding ; processing ; expression.Among the lysosomal cysteine proteases, cathepsin B is the most abundant and the most thoroughly studied. This enzyme takes part in many normal cellular processes, including intracellular protein degradation (Barrett et al., 1988), bone resorption (DelaissC and Vaes, 1992) and antigen processing (Guagliardi et al., 1990). Several pathophysiological states have also been attributed to its action, e.g. Alzheimer's disease (Cataldo and Nixon, 1990), arthritic processes (Van Norden et al., 1988), cancer invasion and metastasis (Sloane et al., 1994), and others. In vivo, the protein is synthesized as a preproenzyme. After being glycosylated, procathepsin B is targeted to the lysosomes by the mannose 6-phosphate signal. Presumably in endosomallprelysosomal compartments it is converted by limited proteolysis into the single-chain form of the mature enzyme, which can be further processed in the lysosomes in a cell-type-specific manner into two-chain cathepsin B consisting of a heavy and light chain linked together by a disulphide bond (Hanewinkel et al., 1987;Mach et al., 1992).In vitro proteolytic processing of recombinant rat procathepsin B, expressed in yeast, has been studied by Rowan et al. (1992). These authors showed that cathepsins D, L, mature cathepsin B itself, and pepsin can produce a processed single-chain form of cathepsin B from its precursor. N-terminal sequencing revealed that these proteins are all elongated by a few residues with respect to the mature form found in vivo. Mach et al. (1994) showed that human procathepsin B can be autocatalytically acti- vated and processed, the mechanism of this process probably being unimolecular.The X-ray crystal structure of human liver cathepsin B has already been determined (Musil et al., 1991); this protein is the first mammalian cysteine protease whose tertiary structure has been determined.In addition to its well characterized endopeptidase activity, cathepsin B also exhibits exopeptidase (dipeptidyl carboxypeptidase) activity (Takahashi et al., ...