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.

Fig. 1

Stains-All staining of fractions 25–84 of dentin G1 (A), E (B), and G2 (C) extracts. The digits at the top of a figure represent the fraction number after Q-Sepharose chromatography. Note the presence of a blue band at ≈ 100 kDa (representing dentin sialoprotein) in fractions 29–33 of the G1 extract (A) and the absence of this protein band in the E extract (B) and the G2 extract (C).

Fig. 2

Western immunoblotting of dentin extracts using monoclonal anti-DSP IgG2b 2G7.3 as a probe. Dentin sialoprotein (DSP) (≈ 100 kDa in fractions 26–34) and the proteoglycan form of DSP (DSP-PG) (≈ 110 to ≈ 200 kDa in fractions 42–76) were detected in G1 extract (A), but not in E extract (B). Note the small amount of DSP in fractions 26–32 in G2 extract (C). Cont, 2 μg of DSP purified from rat dentin was used as a positive control.

Fig. 3

Western immunoblotting of dentin extracts using anti-DMP1 IgG2b. (A) The 37 kDa fragment of dentin matrix protein 1 (DMP1) was detected in fractions 41–45 of the dentin G1 extract by monoclonal anti-DMP1 IgG2b 9B6.3 that specifically recognizes the NH₂-terminal region of DMP1. (B) The 37 kDa fragment of DMP1 was also detected in similar fractions of dentin E extract by antibody 9B6.3. (C) The 57 kDa fragment of DMP1 was detected in fractions 45–49 of dentin E extract by polyclonal anti-DMP1-C that specifically recognizes the COOH-terminal region of DMP1. Note that more than one immunoreactive band for anti-DMP1 was present. We believe that the double or triple bands of DMP1 represent the processed products of DMP1 resulting from cleavage at several sites (20). The positive control (Cont) in panels A and B was 4 μg of 37 kDa fragment purified from rat bone. The positive control (Cont) in panel C was 4 μg of 57 kDa fragment purified from rat bone.

Fig. 4

Western immunoblotting of dentin extracts using monoclonal anti-BSP IgG1 10D9.2 as a probe. Bone sialoprotein (BSP) was detected in G1 (A), E (B), and G2 (C) extracts of dentin. BSP was least abundant in G1 extract and most abundant in G2 extract. The positive control (Cont) was 2 μg of BSP purified from rat bone.

Fig. 5

Stains-All staining of fractions 30–92 of bone G1 (A), E (B), and G2 (C) extracts. Bone sialoprotein (BSP), a blue band just below the 100 kDa molecular weight marker, was present in all three extracts; this blue band was recognized by anti-BSP IgG1 10D9.2 (see Fig. 7). Note the presence of osteopontin (OPN) migrating between the 54- and 100-kDa molecular weight markers in fractions 40–45 of G1 extract and E extract, but not in G2 extract; these bands were immunoreactive to anti-OPN serum (see Fig 8).

Fig. 6

Western immunoblotting for E extract of bone using anti-DMP1 IgG2b. (A) The 37 kDa fragment of dentin matrix protein 1 (DMP1) was mainly detected in fractions 45–47 of bone E extract by monoclonal anti-DMP1 IgG2b 9B6.3. (B) The proteoglycan form of DMP1 (DMP1-PG) was detected in fractions 68–78 of bone E extract by antibody 9B6.3. (C) The 57 kDa fragment of DMP1 was primarily detected in fractions 46–49 of bone E extract by polyclonal anti-DMP1-C serum. The positive control (Cont) in panel A was 4 μg of 37 kDa fragment purified from rat bone. The positive control (Cont) in panel C was 4 μg of 57 kDa fragment purified from rat bone.

Fig. 7

Western immunoblotting for bone extracts using monoclonal anti-BSP IgG1 10D9.2. Bone sialoprotein (BSP) was detected in G1 (A), E (B), and G2 (C) extracts of bone. The positive control (Cont) was 2 μg of BSP purified from rat bone.

Fig. 8

Western immunoblotting for bone extracts using polyclonal anti-OPN. Osteopontin (OPN) was detected in G1 (A) and E (B) extracts of bone. OPN eluted mainly in fractions 40–45. The positive control (Cont) was 2 μg of OPN purified from rat bone.