Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVβ₃-integrin receptor

• Published in: Journal of dairy science Vol. 97; no. 1; p. 136
• Main Authors: Christensen, B, Sørensen, E S
• Format: Journal Article
• Language: English
• Published: United States 2014
• Subjects: Amino Acid Sequence; Animals; Cattle; Cell Adhesion; Cell Line, Tumor; Female; Fibrinolysin - metabolism; Humans; Integrin alphaVbeta3 - genetics; Integrin alphaVbeta3 - metabolism; Milk - chemistry; Milk Proteins - metabolism; Molecular Sequence Data; Oligopeptides - chemistry; Osteopontin - genetics; Osteopontin - metabolism; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Proteolysis; Thrombin - metabolism; integrin; cell binding; proteolytic cleavage; osteopontin
• ISSN: 1525-3198
• DOI: 10.3168/jds.2013-7223

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.