Physiological properties and differential glycosylation of phosphorylated and nonphosphorylated forms of osteopontin secreted by normal rat kidney cells.
In a previous study we have shown that normal rat kidney (NRK) cells in vitro secrete a 69-kDa osteopontin in both phosphorylated (pp69) and nonphosphorylated (np69) forms. Only pp69 interacts with the cell surface and np69 forms a heat-dissociable complex with plasma fibronectin, suggesting functional modulation of osteopontin by phosphorylation. Using tunicamycin, an inhibitor of N-linked glycosylation, and peptide:N-glycosidase F, which removes N-linked oligosaccharide chains from glycoproteins, we show here that np69, but not pp69, contains N-linked carbohydrates. Our results also demonstrate that tunicamycin treatment does not inhibit the cell surface binding of pp69; however, np69 secreted by the treated cells fails to complex with plasma fibronectin, suggesting importantly, our data show that pp69 forms a heat-stable complex with cell surface fibronectin, suggesting that it is an integral component of the extracellular matrix of NRK cells. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of deglycosylated and in vitro translated osteopontin suggests that the acidic nature of osteopontin as well as its post-translational modifications play a role in the anomalous behavior of osteopontin in sodium dodecyl sulfate gels, observed in several laboratories. The data presented here provide evidence for possible functional roles of 69-kDa osteopontin and suggest that its physiological properties are regulated by post-translational modifications.
Department of Biology, McGill University, Montreal, Quebec, Canada.
K
K
Singh
M W
MW
DeVouge
B B
BB
Mukherjee
D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D066298:In Vitro Techniques; D007668:Kidney / Q000502:physiology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D053495:Osteopontin; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D012795:Sialoglycoproteins / Q000502:physiology*; D014415:Tunicamycin / Q000494:pharmacology
D005353:Fibronectins; D006023:Glycoproteins; D046911:Macromolecular Substances; D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D014415:Tunicamycin