Characterization of porcine bone sialoprotein: primary structure and cellular expression

Bone sialoprotein (BSP) is a highly glycosylated and sulphated phosphoprotein that is a major non-collagenous protein of bone. To further characterize the porcine protein and to study its expression during bone formation BSP cDNA clones were isolated from a porcine bone cDNA library. The primary sequence of the protein was derived from the nucleotide sequence of the largest cDNA insert and from the amino-terminal amino acid sequence determined by the automated Edman degradation procedure. When compared with sequences obtained from the human and rat BSPs 74% and 64% of the amino acids, respectively, were identical and a further 11% and 17%, respectively, were conservative replacements. Moreover, 60% of the amino acids in a concensus sequence derived from the primary sequences of mammalian BSPs were conserved with 16% conservative replacements. The two stretches of polyglutamic acid, through which the protein is capable of binding to hydroxyapatite, and an RGD motif that mediates cell attachment are retained in conserved sequences as are a number of potential sites of serine, threonine and tyrosine phosphorylation, glycosylation and tyrosine sulphation. Secondary structure prediction and hydrophilicity analysis indicate that the nascent BSP has an open flexible structure with the potential to form significant amounts of alpha-helix and some beta-sheet. In situ hybridization of fetal porcine bone with cRNA probes to porcine BSP mRNA shows that BSP is specifically expressed in differentiated osteoblasts on the surface of newly-forming bone trabeculae with especially high levels of hybridization at sites of de novo bone formation. The highly conserved features of BSP and its restricted distribution indicate an important role for this sialoprotein in the formation of bone.