Abstract
Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg-Gly-Asp(145) sequence and were generated by cleavage of the Leu(151)-Arg(152), Arg(152)-Ser(153), Ser(153)-Lys(154), Lys(154)-Ser(155), Ser(155)-Lys(156), Lys(156)-Lys(157), or Phe(158)-Arg(159) peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg(152)-Ser(153) matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys(154)-Ser(155). Another endogenous milk protease, cathepsin D, cleaved the Leu(151)-Arg(152) bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the alpha(V)beta(3)- or alpha(5)beta(1)-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.
Highlights
Cell functions, and tumor growth [1,2,3]
A significant proportion of the milk OPN is expected to pass through the gut and into the intestines largely intact upon milk consumption, as the protein is relatively resistant to proteolysis by neonatal gastric juice [6]
Thrombin and matrix metalloproteinase (MMP) cleavage of OPN can modulate its functionality, as e.g. adhesion of tumor cells to OPN via the ␣V3-integrin is significantly increased after thrombin and MMP cleavage [15]
Summary
Materials—Percoll, phorbol 12-myristate 13-acetate, plasmin, thrombin, cathepsin D, bovine serum albumin, 97% 18Owater, endoproteinase Asp-N, fibronectin, toluidine blue, and mouse IgG1 isotype control monoclonal antibody (MOPC-21). The fractions containing N-terminal OPN fragments of interest were lyophilized and further analyzed by Western blotting, and their C termini were determined by cleavage with endoproteinase Asp-N as described. The digest of the N-terminal OPN fragments with Asp-N in 50% H218O was separated by RP-HPLC (Fig. 2A), and all fractions were analyzed by MALDI-TOF-MS in reflector-ion mode. Peptides in six fractions (F2–F7 in Fig. 2) had a natural isotopic distribution, indicating that they represent C-terminal peptides The masses of these peptides all corresponded to cleavage of OPN a few residues to the C-terminal side of the integrin binding RGD145 sequence (Fig. 2C). The full-length protein was digested with the wise, full-length OPN and the mixture of N-terminal fragments three proteases, and the resulting fragments and peptides were were purified, and equimolar concentrations of all forms were separated by RP-HPLC or gel filtration (data not shown). Characterization of OPN fragments generated by thrombin, plasmin and cathepsin D
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