Osteopontin: Its Transglutaminase-Catalyzed Posttranslational Modifications and Cross-Linking to Fibronectin1

Abstract

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells

“…Furthermore, the predominant form of secreted OPN present in the conditioned media of the 21T series cell lines was of high molecular weight (*97 kDa), whereas MDA-MB-435 cells in addition show signi®cant accumulation of lower molecular weight forms (including a major 66 kDa band). The high molecular weight species may represent either a very heavily post-translationally modi®ed, or conjugated (by transglutaminase) form (Prince et al, 1991;Beninati et al, 1994;Sorensen et al, 1994;Sorensen and Petersen, 1995;Aeschlimann et al, 1996). In keeping with this interpretation is our ®nding that the major intracellular form of OPN present in cell lysates (of both 21T series cells and MDA-MB-435 cells) is the low molecular weight, 66 kDa form.…”

Osteopontin promotes pathologic mineralization in articular cartilage

“…3). Osteopontin is an important substrate of the transglutaminase enzymes in mineralizing tissues (Beninati et al 1994;Kaartinen et al 1999).Osteopontin is almost ubiquitous at sites of both normal and pathologic mineralization. However, its role in mineral formation remains poorly understood and somewhat controversial.…”

“…3). Osteopontin is an important substrate of the transglutaminase enzymes in mineralizing tissues (Beninati et al 1994;Kaartinen et al 1999).…”

A Locus on Chromosome 7 Determines Dramatic Up-Regulation of Osteopontin in Dystrophic Cardiac Calcification in Mice

“…One of two putative heparinbinding sites (D 276 -I 283 ) exhibited an R277H substitution. Additional V123F, N143D, D172Y, R225S, and Q233H amino acid substitutions did not occur in any known relevant protein motif including the signal peptide (M 1 -S 16 ), the aspartic acid-rich hydroxyapatite-binding site (D 86 -D 95 ), the RGD-motif (R 145 -D 147 ) and additional integrin binding sites (S 148 -L 153 ), the thrombin cleavage site (R 154 -S 155 ), and the remaining phosphorylation and glycosylation sites. 19,36 -38 The D172Y substitution may yet be functionally important, because it created a shift from an acidic to a basic amino acid.…”

“…14 The biological function of Opn may be influenced by extensive posttranslational modification including glycosylation 15 and transglutamination. 16 Phosphorylation of Opn appeared to be required for inhibition of mineralization. 17 Secondly, Opn has an Arg-Gly-Asp (RGD) amino acid sequence motif 18 serving as a ligand for integrins, a family of cell surface receptors promoting cell adhesion and migration.…”