OSTEOPONTIN - ITS TRANSGLUTAMINASE-CATALYZED POSTTRANSLATIONAL MODIFICATIONS AND CROSS-LINKING TO FIBRONECTIN

Osteopontin (OP) is a component of extracellular, bone, and urinary st one matrices, but the mechanism by which it is stably incorporated int o such matrices remains unknown. By SDS-PAGE analysis of [I-125]OP, tr eated 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 OF, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of. TG-catalyzed epsilon-(gamma-g lutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Si milar 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 I-125-labeled NRK cell surf ace proteins with anti-OF and anti-Fn antibodies, SDS-PAGE analysis, a nd autoradiography provides critical evidence for nonreducible OP-Fn c ross-linking in vivo. These results clearly suggest that TG-mediated c ross-linking between OP and Fn represents one of the most likely mecha nisms by which OP becomes covalently linked to bone matrix, urinary st one matrix, and to ECM.