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 osteosarcoma cell line UMR-106-BSP using nondenaturing conditions. Routine protein purification without serine protease inhibitors or reducing agents consistently resulted in three major fragments. The largest fragment (E1) started at amino acid 117 and did not bind to antibodies made to the RGD region of the protein. Furthermore, the smallest fragment (E3), was shown by sequencing to contain the RGD region of the protein. Digestion of intact BSP with highly purified chymotrypsin also resulted in a large fragment (C1) with properties nearly identical to those of E1. The large, non-RGD-containing fragments, E1 and C1, as well as the intact BSP, supported attachment by normal human bone cells and human skin fibroblasts in vitro. Attachment to the intact BSP was totally blocked by 0.4 mM GRGDS peptide. Both preparations of skin fibroblasts and approximately half of the preparations of normal human bone cells, however, also would not attach to the E1 and C1 fragments in the presence of 0.4 mM GRGDS peptide. In contrast, half of the bone cell preparations had significant attachment activity to E1 (> 50%) and C1 (> 25%) in the presence of 0.4 mM GRGDS peptide. These data suggest that cleavage of the BSP results in either (1) the exposure of a previously unavailable or cryptic cell attachment site or (2) a conformational change that increases the affinity of the complex between a non-RGD-encoded binding region of the E1 and C1 fragments and at least one receptor. The possible homology of the second, non-RGD-suppressible site of BSP with the second cell attachment site on the gamma chain of fibrinogen is discussed.
Mintz
K P
KP
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland.
Grzesik
W J
WJ
Midura
R J
RJ
Robey
P G
PG
Termine
J D
JD
Fisher
L W
LW
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001842:Bone and Bones / Q000166:cytology* / Q000378:metabolism; D002448:Cell Adhesion; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012867:Skin / Q000166:cytology; D014407:Tumor Cells, Cultured
C546871:IBSP protein, human; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D009842:Oligopeptides; D012795:Sialoglycoproteins; C047981:arginyl-glycyl-aspartic acid; C049554:glycyl-arginyl-glycyl-aspartyl-serine