Complete Topographical Distribution of Both the in Vivo and in Vitro Phosphorylation Sites of Bone Sialoprotein and Their Biological Implications

Abstract

Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of stateof-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solidphase N-terminal peptide sequencing that were within peptides 12-22 (LEDS(P)EENGVFK), 42-62 (FAVQSSSD- SS(P)EENGNGDS(P)S(P)EE), 80 -91 (EDS(P)DENEDEE-S(P)E), and 135-145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[14 C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250 -264 (DNGYEIYES(P)ENGDPR), and 282-289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-offlight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125-130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site.Evidence supporting many facets of the observed biological functions of bone sialoprotein (BSP) 1 and osteopontin (OPN) has been accumulating, with a wide range of implications in both mineralizing and non-mineralizing tissues. BSP and OPN are the major non-collagenous extracellular matrix (ECM) phosphoproteins of calcified tissues such as bone and cartilage. Their intimate relationship with biomineralization suggests that they play key roles during the development and maintenance of these tissues (1-8). In vitro studies using BSP and OPN indicate that, whereas BSP induces calcium phosphate apatite formation (4 -6, 9), OPN lacks this property or is inhibitory (3, 4, 10). The biological functions of BSP and OPN are not limited to mineral deposition, but impact cell behavior such as cell motility, cell adhesion, and bone resorption (10 -16). Despite extensive studies, however, the precise roles of these phosphoproteins remain to be clearly defined. Although some in vitro studies indicate that BSP promotes bone resorption and hence participates in bone degradation (17), other studies such as those using glucocorticoids, which increase expression of this protein, suggest its involvement in the anabolic phase of bone remodeling (18). In this laboratory, in vivo implants of BSP-collagen composites in the calvarial critical defect bone repair model (19) and during reparative dentinogenesis (7, 8, 20 -22) highlighted the impact of BSP during biomineralization and new bone/dentin formation.As interest continues and evid...

Family with sequence similarity member 20C is the primary but not the only kinase for the small‐integrin‐binding ligand N‐linked glycoproteins in bone

“…Other studies have identified several phospho-Thrs in the SIBLINGs, with phosphorylation that is not governed by FAM20C (3,28,39). We confirmed the existence of some of these phospho-Thrs in the Fam20C-KO mice.…”

“…Recent in vitro analyses identified FAM20C as a Golgi-enriched kinase that phosphorylates Ser in the Ser-X-Glu/pSer motifs in SIBLINGs and many other proteins on the secretory pathway (26,27), implying an association of the assumed phosphorylation failure in SIBLINGs with the severe bone defects in the FAM20C-inactivated subjects. However, the lack of in vivo evidence for the loss of phosphorylation in SIBLINGs, the fewer Ser-X-Glu motifs than the actual number of phosphates in the SIBLINGs (1), and the presence of phospho-Thrs in the SIBLINGs that do not conform to a substrate for FAM20C kinase (3,28) led us to argue that FAM20C may not be the only kinase phosphorylating the SIBLINGs. This evidence requires further investigation to gain more insight into the consequences of Fam20C inactivation.…”

“…A prominent category of NCPs in bone ECM is termed the small-integrin-binding ligand N-linked glycoprotein (SIBLING) family, which includes osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP)-1. These polyanionic proteins play pivotal roles in osteogenesis and biomineralization, and their biologic functions are closely related to posttranslational modifications, such as phosphorylation (1)(2)(3)(4)(5)(6)(7).…”

A structural and functional model for human bone sialoprotein

“…Detailed experimental studies of the bovine protein have identified a total of eleven phosphorylated serines [29,30], of which nine are conserved in human BSP. Using this information, together with predictions from the NetPhos server [31] and our sequence alignment, we chose the six phosphorylation sites included in our model ( Figure 1).…”

Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian

“…On the other hand, tetrapod IBSPs have a number of Thr residues for O-linked glycosylation sites (Zaia et al, 2001: Salih et al, 2004 in the segments encoded by exon 7 (Thr-rich region in Fig. 6).…”