PURIFICATION AND FRAGMENTATION OF NONDENATURED BONE SIALOPROTEIN - EVIDENCE FOR A CRYPTIC, RGD-RESISTANT CELL ATTACHMENT DOMAIN

Bone sialoprotein (BSP), a small (approximately 80,000 M(r)) integrin binding, RGD-containing bone matrix glycoprotein, has been purified in milligram quantities from the serum-free medium of the rat osteosarco ma cell line UMR-106-BSP using nondenaturing conditions. Routine prote in purification without serine protease inhibitors or reducing agents consistently resulted in three major fragments. The largest fragment ( El) started at amino add 117 and did not bind to antibodies made to th e RGD region of the protein. Furthermore, the smallest fragment (E3), was shown by sequencing to contain the RGD region of the protein. Dige stion of intact BSP with highly purified chymotrypsin also resulted in a large fragment (Cl) with properties nearly identical to those of El . The large, non-RGD-containing fragments, El and Cl, as well as the i ntact BSP, supported attachment by normal human bone cells and human s kin fibroblasts in vitro. Attachment to the intact BSP was totally blo cked by 0.4 mM GRGDS peptide. Both preparations of skin fibroblasts an d approximately half of the preparations of normal human bone cells, h owever, also would not attach to the El and Cl fragments in the presen ce of 0.4 mM GRGDS peptide. In contrast, half of the bone cell prepara tions had significant attachment activity to El (> 50%) and Cl (> 25%) in the presence of 0.4 mM GRGDS peptide. These data suggest that clea vage of the BSP results in either (1) the exposure of a previously una vailable or cryptic cell attachment site or (2) a conformational chang e that increase the affinity of the complex between a non-RGD-encoded binding region of the El and Cl fragments and at least one receptor. T he possible homology of the second, non-RGD-suppressible site of BSP w ith the second cell attachment site on the gamma chain of fibrinogen i s discussed.