The anatomy of bone sialoprotein immunoreactive sites in bone as revealed by combined ultrastructural histochemistry and immunohistochemistry.

Bone sialoprotein was immunolocalized at the EM level in thin Lowicryl K4M sections of rat bone. Because of the unconventional EM morphology of the bone matrix seen in thin demineralized acrylate sections, the pattern of immunolabeling was compared with detailed structural images of demineralized bone obtained using an en bloc treatment of tissue samples with the cationic electron 'dye,' Malachite Green (MG), which provides stabilization and retention of anionic material throughout specimen processing. A system of structures corresponding to the sites of bone sialoprotein (BSP) immunoreactivity, as seen in Lowicryl K4M this sections, could be readily identified in the MG-treated, epoxy thing sections. This system includes the cement lines, and aggregates of similar material within mineralized bone and mineralizing osteoid. The virtual identity of BSP distribution with the arrangement of the MG-visualized material indicates that a BSP-enriched, noncollagenous phase can be demonstrated using different, unrelated tissue preparation and imaging protocols for EM. Besides improving our understanding of the distribution of bone sialoprotein in bone, these data assign a previously unrecognized structural dimension to noncollagenous material in the bone matrix.

Cloning and sequence analysis of bovine bone sialoprotein cDNA: conservation of acidic domains, tyrosine sulfation consensus repeats, and RGD cell attachment domain.

We isolated and sequenced a cDNA encoding bovine bone sialoprotein (BSP) using a bovine cDNA library made from mRNA isolated from bone-derived cell cultures and ligated to a phage lambda gt11. One of the cDNA clones isolated from this library had a 1800 base pair long insert and was found to contain the entire protein-encoding region. The deduced protein sequence revealed a 310 amino acid protein containing a signal peptide sequence of 16 hydrophobic amino acids. The protein sequence shows remarkable conservation with previously published human and rat sequences (more than 80% similarity for both species). The potential functional domains of BSP, including three acid amino acid-rich sequences, tyrosine sulfation consensus repeats, and the RGD cell binding sequence, are all present in the bovine sequence. Northern analysis of RNA from different bovine tissues indicated the presence of BSP message in bone but not in other nonmineralized tissues, confirming that bone is the major site of BSP message production.