title: Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVβ₃-integrin receptor.
abstract: Site-specific and partial proteolysis of milk proteins can both alter and increase their biological activity. The milk protein osteopontin (OPN) is a highly phosphorylated integrin-binding molecule present in most tissues and body fluids. Osteopontin is a biological substrate for matrix metalloproteinases, thrombin, plasmin, and cathepsin D. These proteases cleave OPN at several positions near the integrin-binding sequence Arg-Gly-Asp(138). This cleavage can either increase or reduce the ability of OPN to bind integrins and illustrates that small differences in the cleavage pattern can have a substantial effect on the functionality of OPN. Bovine milk OPN (bOPN) exists in both intact full-length and cleaved forms, and in this study, 6 N-terminal bOPN fragments originating from proteolytic cleavage were purified and characterized by mass spectrometry. These fragments were generated by cleavage at the Lys(145)-Ser(146), Arg(147)-Ser(148), Lys(149)-Lys(150), Phe(151)-Arg(152), Arg(152)-Arg(153), and Arg(153)-Ser(154) peptide bonds. The principal protease in milk, plasmin, appeared to cleave 3 of these sites. However, the major cleavage site was observed to be at the Phe(151)-Arg(152) bond, which does not match the specificity of plasmin. The bOPN fragments were shown to interact with the integrin receptor αVβ₃ as efficiently as the well-characterized and highly active OPN fragment Ile(1)-Arg(152), generated by thrombin cleavage of human milk OPN. These data show that OPN in milk is highly susceptible to proteolytic cleavage in the region containing the integrin-binding motifs, and the generated fragments are highly capable of binding cells via the αVβ₃-integrin.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Christensen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark. Electronic address: ess@mb.au.dk.
authors.forename: E S
authors.initials: ES
authors.lastname: Sørensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000235:genetics / Q000378:metabolism*; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D013917:Thrombin / Q000378:metabolism
chemical_list: D039302:Integrin alphaVbeta3; D008894:Milk Proteins; D009842:Oligopeptides; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin; D005341:Fibrinolysin
keywords: cell binding; integrin; osteopontin; proteolytic cleavage