title: Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments.
abstract: 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 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A 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 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.
authors.affiliation: Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive Scarborough, ME 04074, USA.
authors.forename: Yu Alice
authors.initials: YA
authors.lastname: Gao
authors.forename: Renu
authors.initials: R
authors.lastname: Agnihotri
authors.forename: Calvin P H
authors.initials: CP
authors.lastname: Vary
authors.forename: Lucy
authors.initials: L
authors.lastname: Liaw
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002448:Cell Adhesion; D002460:Cell Line; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019169:Jurkat Cells; D020782:Matrix Metalloproteinases / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D053495:Osteopontin; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D013997:Time Factors
chemical_list: D010455:Peptides; D011506:Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D020782:Matrix Metalloproteinases