Distribution of SIBLING proteins in the organic and inorganic phases of rat dentin and bone

Abstract

The SIBLING protein family is a group of non-collagenous proteins (NCPs) that includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). In the present study, we compared these four proteins in different phases of rat dentin and bone. First, we extracted NCPs in the unmineralized matrices and cellular compartments using guanidium-HCl (G1). Second, we extracted NCPs closely associated with hydroxyapatite using an EDTA solution (E). Last, we extracted the remaining NCPs again with guanidium-HCl (G2). Each fraction of Q-Sepharose ion-exchange chromatography was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Stains-All stain, and with western immunoblotting. In dentin, the NH(2)-terminal fragment of DSPP and its proteoglycan form were primarily present in the G1 extract, whereas the COOH-terminal fragment of DSPP was present exclusively in the E extract. The processed NH(2)-terminal fragment of DMP1 was present in G1 and E extracts, whereas the COOH-terminal fragment of DMP1 existed mainly in the E extract. Bone sialoprotein was present in all three extracts of dentin and bone, whereas OPN was present only in the G1 and E extracts of bone. The difference in the distribution of the SIBLING proteins between organic and inorganic phases supports the belief that these molecular species play different roles in dentinogenesis and osteogenesis.

Biomimetic Engineering of Nanofibrous Gelatin Scaffolds with Noncollagenous Proteins for Enhanced Bone Regeneration

“…19 Since these NCPs are acidic and are secreted into the ECM during bone formation and mineralization, they are believed to play key biological roles in initiating and modulating the mineralization of collagen fibers when osteoids are converted into bone. 24 A number of studies have shown that mutation in or knocked out of genes coding for certain NCPs is associated with phenotypic abnormalities in bone mineralization. 22,[33][34][35] However, the way these NPCs in 3D scaffolds affect osteoblast response during bone tissue regeneration is largely unknown.…”

“…The presence of OPN, DMP1, and BSP was confirmed by western blot analyses using specific antibodies. 24 …”

“…24 Briefly, the long bones of the rats were dissected and frozen in liquid nitrogen to be crushed into small pieces, which were further ground into powders. The non-mineral-associated proteins and cells inside the bone powder were removed using 200 mL guanidine hydrochloride (Gdm-HCl, 4 M) in a sodium acetate (50 mM) solution.…”

FAM20C Plays an Essential Role in the Formation of Murine Teeth

“…The monoclonal anti-FAM20C antibodies (clone 3 or clone 5) were used at a dilution of 25 g of IgG/ml. A polyclonal biglycan antibody (LF-159) was used as described previously (13) as well as antibodies against bone sialoprotein (BSP), dentin sialoprotein (DSP; the N-terminal fragment of DSPP), and DMP1 (14,15). A polyclonal antibody against ameloblastin (Santa Cruz Biotechnology, Inc.) was employed following the manufacturer's instructions.…”

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

“…The NCPs were extracted from the long bones of 4-wk-old mice (8 WT vs. 12 KO) with 4 M guanidine-hydrochloride/0.5 M EDTA containing protease and phosphatase inhibitors, followed by buffer exchange into 6 M urea (29). After they were dialyzed against water, the NCP samples (0.06 mg of each) were hydrolyzed in 0.2 M NaOH, and the concentration of inorganic phosphate (P i ) liberated from the phosphoproteins was determined (6,(30)(31)(32).…”