Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in L-167 and L-211 do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a L-167 to (167)A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to (131)ELVTDFPTDLPATE(144) had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These 162 166 results suggest that a shorter motif that is found in MMP-cleaved OPN, (162)SVVYG(166), is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the betal integrins, which have been previously characterized to bind the SVVYGLR sequence. (C) 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.