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.

Milk osteopontin retains integrin-binding activity after in vitro gastrointestinal transit

“…The MAB222p antibody recognized OPN species migrating, corresponding to masses of approximately 50 and 25 to 30 kDa in SDS-PAGE of the sample not incubated with enzymes ( Figure 2B, lane 2). These bands represented the full-length protein and N-terminal OPN fragments naturally present in bovine milk (Christensen and Sørensen, 2014). Fragments migrating at 25 to 30 kDa also reacted with the MAB222p antibody in both pepsin digests of OPN ( Figure 2B, lane 3-4).…”

“…These threonines are well conserved among mammalian OPN sequences, but no clear functional role for the glycosylations has been described. In human and bovine milk, OPN is present as both an intact full-length protein and as several proteolytically generated N-terminal fragments Christensen and Sørensen, 2014). The N-terminal fragments are generated by proteolytic cleavage in the sequence to the Cterminal side of the RGD and SVVYGLR sequences.…”

Immunological blockade of adipocyte inflammation caused by increased matrix metalloproteinase‐cleaved osteopontin in obesity

“…The fact that OPN action in adipocytes could be blocked with CMIP 9‐3, a monoclonal antibody specifically directed against the integrin‐binding region of OPN, underlines the impact of OPN cleavage in obesity. Cleavage of OPN can lead to enforcement of integrin binding by two mechanisms, namely binding of RGD‐domain to αvβ3 and αvβ5 integrins and interaction with SVVYG(LR)‐site via α4 (and α9) integrins . Our data show that adipocytes mainly express RGD‐binding integrins and MMP‐cOPN effects are primarily reduced by αvβ3 and αvβ5 blockade.…”

Osteopontin is proteolytically processed by matrix metalloproteinase 9

“…OPN contains an Arg-Gly-Asp (RGD) amino acid sequence that facilitates OPN-cell interactions via the α v β 3 integrin that promotes cell adhesion (McFarland et al 1995). Proteolytic cleavage can either enhance or reduce the ability of OPN to bind to integrins (Christensen and Sorensen 2014). This illustrates that slight differences in the cleavage pattern, i.e., different cleavage sites, can have substantial effects on the biological function of OPN.…”

“…OPN is a biological substrate for MMPs, thrombin, plasmin, and cathepsin D (Christensen et al 2010; Christensen and Sorensen 2014). Of these proteases, MMPs are robustly expressed post-MI and play a major role in LV remodeling (Iyer et al 2014).…”