CHLORATE-INDUCED INHIBITION OF TYROSINE SULFATION ON BONE SIALOPROTEIN SYNTHESIZED BY A RAT OSTEOBLAST-LIKE CELL-LINE (UMR 106-01 BSP)

Bone sialoprotein (BSP) is a major noncollagenous, RGD-containing glyc oprotein found in the extracellular matrix of bone. The RGD sequence i s flanked by two tyrosine-rich regions, which fit the established cons ensus requirements for tyrosine sulfation. Tyrosine sulfation is sugge sted to be important in the regulation of protein secretion and functi on. The role of this posttranslational modification on the cell attach ment activity and secretion of a highly sulfated form of BSP isolated from a rat osteoblast-like cell line (UMR 106-01 BSP) was investigated by inhibiting sulfation with chlorate. [S-35]Sulfate, [H-3]glucosamin e, and [H-3]tyrosine were used as metabolic precursors to monitor bios ynthetic products. Chlorate was effective in inhibiting to tal [S-35]s ulfate incorporation by 90% without altering overall protein synthesis and secretion in cultures up to 72 h under serum-free conditions. Iso lated proteoglycans and purified BSP were analyzed for sulfate incorpo ration. Proteoglycans isolated from the medium of cells treated with c hlorate displayed a difference in the hydrodynamic properties of the m olecules as compared with control cultures. An increase in the specifi c activity of proteoglycans labeled with [H-3]glucosamine isolated fro m chlorate-treated cells was also observed suggesting a change in hexo samine metabolism induced by chlorate. BSP purified from the medium of chlorate-treated cells contained similar to 7% of the S-35 incorporat ion as compared with nontreated control cultures. Quantification of su lfate incorporation into glycoconjugates versus tyrosine sulfate of BS P indicates that the amount of sulfate associated with N- and O-linked oligosaccharides was reduced by similar to 97%, while that on tyrosin e residues was reduced by similar to 90%. Using normal human bone cell s, the cell attachment activity of the reduced sulfate form of BSP was nearly equivalent to that of the fully sulfated product.