Procathepsin B is precursor of Cathepsin B, which is a lysosomal cysteine proteinase of the papain superfamily.
The nucleotide sequences predict that the primary structure of preprocathepsin B contains 339 amino acids organized as follows: a 17-residue NH2-terminal prepeptide sequence followed by a 62-residue propeptide region, 254 residues in mature (single chain) cathepsin B, and a 6-residue extension at the COOH terminus. A comparison of procathepsin B sequences from three species (human, mouse, and rat) reveals that the homology between the propeptides is relatively conserved with a minimum of 68% sequence identity. In particular, two conserved sequences in the propeptide that may be functionally significant include a potential glycosylation site and the presence of a single cysteine at position 59. Comparative analysis of the three sequences also suggests that processing of procathepsin B is a multistep process, during which enzymatically active intermediate forms may be generated1.
Mode of Action
In a study, the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, was investigated, thus recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. Further, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself 2.
Human Procathepsin B Interacts with the Annexin II Tetramer on the Surface of Tumor Cells: To study potential roles of plasma membrane-associated extracellular cathepsin B in tumor cell invasion and metastasis, the yeast two-hybrid system was used to screen for proteins that interact with human procathepsin B. The annexin II light chain (p11), one of the two subunits of the annexin II tetramer, was one of the proteins identified. Furthermore, procathepsin B could interact with the annexin II tetramer in vivo as demonstrated by coimmunoprecipitation. Cathepsin B and the annexin II tetramer were shown by immunofluorescent staining to colocalize on the surface of human breast carcinoma and glioma cells. These results suggest that the annexin II tetramer can serve as a binding protein for procathepsin B on the surface of tumor cells, an interaction that may facilitate tumor invasion and metastasis3.
Interaction of Human Breast Fibroblasts with Collagen I Increases Secretion of Procathepsin B: Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. A study determined that growth of breast fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against a1, a2, and ß1 integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. Furthermore, procathepsin B secretion was induced when cells were plated on ß1 integrin antibodies. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer4.
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