Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.
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.
Beninati
S
S
Section on Developmental Genetics, Human Genetics Branch, NICHD, NIH, Bethesda, MD 20892.
Senger
D R
DR
Cordella-Miele
E
E
Mukherjee
A B
AB
Chackalaparampil
I
I
Shanmugam
V
V
Singh
K
K
Mukherjee
B B
BB
D000595:Amino Acid Sequence; D000818:Animals; D015317:Biogenic Polyamines / Q000378:metabolism; D001704:Biopolymers; D002384:Catalysis; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D005353:Fibronectins / Q000737:chemistry*; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D053495:Osteopontin; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011869:Radioligand Assay; D012795:Sialoglycoproteins / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D015317:Biogenic Polyamines; D001704:Biopolymers; D005353:Fibronectins; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D053495:Osteopontin; D011503:Transglutaminases