Abstract:
Proteinase(s) were isolated and purified from the cytoplasmic fraction of human skin fibroblast cells. Proteins of the cytoplasmic fraction were identified to consistently exhibit a substantial amount of proteolytic activity in comparison to the proteins extracted from the cell membranes. Purification was achieved by ion-exchange and affinity column chromatography. These proteinases displayed activity against the chromogenic substrate Bnz-phe-val-arg-ρNA suggesting that the enzymes exhibited trypsin-like specificity. The purified proteins were analysed by SDS-PAGE. The identification of proteinases was determined by Western blot analysis and from 3H-DFP-labelling experiments.
Two proteins with apparent molecular weights of 45 kDa and 55 kDa were visualized following immunodetection with the leukocyte 80 kDa antiproteinase. Analysis of autoradiographs revealed that two proteins with molecular weights of 55 and 60 kDa were susceptible to labelling by DFP. A comparison of the deduced amino acid sequences of these proteins with other proteins failed to identify these proteins as known proteinases. While the former was identified as sharing extensive amino acid identity with a mouse phosphodiesterase, the latter was identified to be homologous to fetuin.
Similarly amino acid sequence analysis of a leukocyte 80 kDa component revealed the identity of this protein as either thrombospondin or lactotransferrin. Sequence alignment of three other leukocyte proteins indicated that these proteins possessed sequence identity with fibrinogen. It is clear that the antibody used for Western blotting, previously prepared against a growth-related proteinase, was not specific. Two lines of evidence have been presented that argue for and against the identity of these proteins as proteinases.
Functional studies of fibroblast growth-related proteinase were addressed from two aspects. An 80 kDa and a 14 kDa growth regulatory proteins were isolated and purified from human urine samples and medium conditioned by fibroblast cells respectively. The existence of a similar protein in medium conditioned by mouse embryo fibroblast cells was previously reported by Wells and Malluchi (1991). This protein, identified as β-galactoside binding protein, is continuously degraded during cell growth and accumulates as cells become confluent or in the presence of proteinase inhibitors, suggesting the possibility that it might be a substrate for the growth-related proteinase. The effects of the cellular protein on the growth of several cell lines was investigated. While this protein was observed to exert an inhibitory effect on three human cell lines, it was identified as having a stimulatory effect on bovine corneal endothelial cells. The growth inhibitory effects of this protein parallel the effects of inhibitors of the growth related proteinase.
The ability of urinary trypsin inhibitor to inhibit cell proliferation and cell-surface proteinase activity suggested that this inhibitor was exerting an effect by acting on a cell-surface protein. Experiments involving 125l-urinary trypsin inhibitor revealed that a fibroblast component between 45-50 kDa in size was able to form a complex with urinary trypsin inhibitor. In fact the molecular size of this protein is in close agreement with the protein purified from the cytoplasmic fraction of fibroblast cells during the present study.