Bone sialoprotein (BSP-II)

The other major sialoprotein is BSP, composed of 50% carbohydrate (12% is sialic acid) and stretches of polyglutamic acid (as opposed to polyaspartic acid in OPN). The RGD sequence is located at the carboxy terminus of the molecule, whereas it is located centrally in OPN. The sequence is also characterized by multiple tyrosine sulfation consensus sequences found throughout the molecule, in particular, in regions flanking the RGD (Fisher et al., 1990). Sulfated BSP has been isolated in a number of animal species, but the levels appear to be variable.

BSP exhibits a more limited pattern of expression than OPN. In general, its expression is tightly associated with mineralization phenomena (although there are exceptions). In the skeleton, it is found at low levels in chondrocytes, in hypertrophic cartilage, in a subset of osteoblasts at the onset of matrix mineralization, and in osteoclasts (Bianco et al., 1991). Consequently, BSP expression marks a late stage of osteoblastic differentiation and an early stage of matrix mineralization. Outside of the skeleton, BSP is found in trophoblasts in placental membranes, which in late stages of gestation fuse and form mineralized foci.

Ethylene Diamine Tetraacetate Acid Tetrasodium Salt (EDTA-4Na)

BSP may be multifunctional in osteoblastic metabolism. It is very clear that it plays a role in matrix mineralization as supported by the timing of its appearance in relationship to the appearance of mineral and its Ca²⁺-binding properties. BSP has a high capacity for binding calcium ions. The polyglutamyl stretches were thought to be solely responsible for this property; however, studies using recombinant peptides suggest that although the polyglutamyl stretches are required, they are not the sole determinants (Stubbs et al., 1997). Unlike OPN, BSP does nucleate hydroxyapatite deposition in a variety of in vitro assays.

It is also clear from in vitro assays that BSP is capable of mediating cell attachment, most likely through interaction with the somewhat ubiquitous αVβ3 (vitronectin) receptor. Bone cells attach to the intact molecule in an RGD-dependent fashion. However, when BSP is fragmented, either endogenously by cells or using commercially available enzymes, the fragment most active in cell attachment does not contain the RGD sequence (Mintz et al., 1993). Studies indicate that the sequence upstream from the RGD mediates attachment (in an RGD-independent fashion) and suggest that the integrin-binding site is more extended than had been envisioned previously. Sequences flanking the RGD site are often tyrosine sulfated. However, it is not known how sulfation influences BSP activity, as in vitro, unsulfated BSP appears to be equivalent in its activity. Once again, it is not clear if currently available in vitro assays are sufficiently sophisticated to determine what influence post-translational modifications, such as sulfation, have on the biological activity. In addition to sulfation, conformation of the RGD site may also influence the activity of the protein. Although the RGD region in fibronectin is found in a looped-out region that is stabilized by disulfide bonding, there are no disulfide bonds in BSP. However, the flanking sequences most likely influence the conformation of the region. The cyclic conformations also appear to have a higher affinity for cell surface receptors than linear sequences (van der Pluijm et al., 1996).

BSP-knockout mice have been extensively studied by the Malaval laboratory (Bouleftour et al., 2016). Initial studies of the knockout mouse clearly implicate BSP in playing an important role in mineralization and cortical or primary bone formation, but not endochondral bone formation (Bouleftour et al., 2014). More recent studies have demonstrated that BSP can also influence hematopoiesis and vascularization (Granito et al., 2015). Interestingly, in the absence of BSP, OPN is upregulated and it is speculated that some aspects of the BSP-deficient phenotype may be secondary to upregulation of OPN (Granito et al., 2015; Bouleftour et al., 2016)