Recombinant GST-human osteopontin fusion protein is functional in RGD-dependent cell adhesion.
Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione-S-transferase (GST). We report that the GST-OPN fusion protein has functional activity. PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin-mediated mechanisms. Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein. Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein. Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post-translational modifications that occur in native OPN for adhesive function as measured here.
Xuan
J W
JW
London Regional Cancer Centre, Ontario, Canada.
Hota
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Chambers
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D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002461:Cell Line, Transformed; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000037:antagonists & inhibitors*; D053495:Osteopontin; D010750:Phosphoproteins / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D009842:Oligopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid; D005982:Glutathione Transferase