Our laboratory has previously demonstrated that increased malignancy of several histological types of human and animal tumours is associated with increases in their cathepsin B activity, particularly cathepsin B activity associated with plasma-membrane/endosomal vesicles or shed vesicles. Here we report that cathepsin B from normal or tumour tissues degrades purified extracellular-matrix components, type IV collagen, laminin and fibronectin, at both acid pH and neutral pH. The number and sizes of degradation products were analysed by SDS/PAGE. Cathepsin B from both sources exhibited similar activities towards, and similar patterns of cleavage of, the extracellular-matrix proteins. At neutral pH, cathepsin B from both sources appeared to undergo autodegradation, a process that was decreased in the presence of alternative substrates such as the extracellular-matrix proteins. Cathepsin B readily degraded type IV collagen at 25 degrees C, indicating activity towards native type IV collagen. Fibronectin degradation products of 100-200 kDa and of 18 and 22 kDa were observed. A single 70 kDa fragment was released from laminin under non-reducing conditions and multiple fragments ranging from 45 to 200 kDa under reducing conditions. These results suggest that cathepsin B at or near the surface of malignant tumour cells may play a functional role in the focal dissolution of extracellular matrices.
Cathepsin B activity, including that of a plasma membrane-associated cathepsin B, has been linked to tumor malignancy. As cathepsin B at the tumor cell surface has been hypothesized to play a role in the focal degradation of basement membrane during the metastatic cascade, we have examined the ability of human tumor cathepsin B to degrade laminin, an adhesive glycoprotein found exclusively in the basement membrane. We report that at pH 6.5 and 7.0 tumor cathepsin B degraded by specific, limited proteolysis both subunits of native laminin. The disappearance of both subunits and the appearance of lower Mr protein bands could be observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Accumulation of degradation products was also observed using gel filtration chromatography and a fluorescamine assay. The proteolysis of laminin by tumor cathepsin B could be inhibited by an active site titrant for cysteine proteinases or stefin B, an endogenous low-Mr cysteine proteinase inhibitor.
IGF in milk possibly promote maturation of the gastrointestinal tract in newborns. We studied the composition of milk samples derived from 99 healthy women at regular intervals during a period beginning 3 d and ending 6 mo after birth. The concentrations measured by RIA on d 3 were 10.7+/-0.4 ng/mL for IGF-II, 1.9+/-0.1 ng/mL for IGF-I, 100+/-5 ng/mL for IGF binding protein-3 (IGFBP-3), and 2163+/-108 ng/mL for IGFBP-2. All factor concentrations decreased by up to 70% in the course of the 6 mo. The most striking finding was an IGFBP-2-specific protease activity. Protease assays performed by incubation of 125I-IGFBP-2 with milk yielded fragments of 14, 16, 23, and 25 kD. 125I-IGFBP-3 was not cleaved. Proteolysis occurred only in milk from mothers until 4 wk postpartum and could be visualized by immunoblots. Since the affinity of the fragments to 125I-IGF-II was low, they were not demonstrable by ligand blot. Inhibitor studies and pH optimizing classified the IGFBP-2 protease as an Me2(+)-dependent serine protease with a pH optimum of 7 to 8. The proteolytic activity of further milk proteins, known as IGFBP proteases, was analyzed. Epidermal growth factor receptor peptide and prostate-specific antigen did not cleave IGFBP-2, although the protease activity correlated (r = 0.84, p < 0.00003) with the prostate-specific antigen concentration in milk. The y-nerve growth factor cleaved 125I-IGFBP-2, but in a completely different manner than the milk protease. In conclusion, the IGFBP-2 protease in milk is most probably a kallikrein. The specific proteolysis of IGF/IGFBP-2 complexes may increase the biologic availability of IGF in early milk. This mechanism may promote growth of the maternal breast epithelium and maturation of the gastrointestinal tract of newborns.
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