Dentinogenesis and Dentin Sialophosphoprotein (DSPP)

Abstract

Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and understanding its structure and function should yield important insights into how dentin biomineralization is controlled. During the recent six years, I have focused on characterizing DSPP-derived proteins isolated from developing porcine teeth. Porcine DSPP is expressed and secreted by odontoblasts and is processed by BMP-1, MMP-20 and MMP-2 into three main parts: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We have learned that DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein that shows extensive length polymorphisms due to the genetic heterogeneity of its coding region.

Figure 1. DSPP derived proteins in porcine dentin extracts

The four primary dentin extracts are GS, GP, A, and AN extract. A and B, porcine dentin extracts analyzed by SDS-PADE stained with CBB and Stains-all. DPP is the CBB-negative, Stains-all-positive band migrating at 98 kDa in the AN extract (a, arrowheads). C and D, Western blots of SDS-PAGE using the DSP, and DGP antibodies. Lower molecular weight (LMW) DSP cleavage products from 22 to 38 kDa are observed in the A extract (b, bracket). Higher molecular weight (HMW) DSP-positive (c, bracket) and DGP-positive (d, bracket) proteins are observed in the AN extract. Most of lower molecular weight DGP-positive bands in the A extract are also DSP-positive except for the only DGP-positive proteins from 16 to 21 kDa (e, bracket).

Figure 2. Amino acid sequence and posttranslational modification sites in porcine DSP and DGP

A, potential and known carbohydrate attachment and cleavage sites by MMP-2 and MMP-20 in porcine DSP. In the primary amino acid sequence of porcine DSP there are eight predicted N-linked glycosylation (N at positions 52, 82, 92, 150, 170, 176, 191 and 330) (green), four potential O-linked glycosylations (T at positions 214, 215, 228, and 279) (pink), and four potential glycan attachment sites (S at positions 232, 235, 253, and 265) (orange). We have now demonstrated at the protein level that six of the potential N-linked sites (N52, N92, N151, N170, N176 and N191) (green squares) are glycosylated, but one (N330) is not (black cross). Two of the four predicted O-linked glycosylation sites (T214 and T228) are not glycosylated (black crosses), but one predicted site (T215) and two unpredicted sites (T231 and T331) are O-glycosylated (pink squares) ; and two of the four potential glycan attachment sites (S253 and S265) (orange squares) are used, but other two (S232 and S235) are not. The arrows indicate the locations of cleavage sites by proteinases. We have demonstrated that three sites are cleaved by MMP-2 (2) and MMP-20 (20). B, amino acid sequence and structures of oligosaccharides of DGP. DGP is consisted of 81 amino acids. We have demonstrated that one potential N-linked oligosaccaride binding site (N397) is glycosylated (green square) and four serines (S453, S455, S457 and S462) are phosphorylated (red squares). We also have identified biantennary glycan structures by analyses of oligosaccharides released from DGP and fluorescent labeled.

Figure 3. Length polymorphisms of DPP protein and coding region, and DPP allelic variations

A, SDS-PAGE showing variations in the mobility of DPP protein isolated from 22 individual pigs. Nine of the 22 pigs showed only a single band of DPP (A, B, C, D, G, H, L, O, Q). Thirteen pigs showed two DPP bands (E, F, I, J, K, M, N, P, R, S, T, U, V). Although no individual pig showed more than 2 DPP bands, six bands of non-identical size could be discerned among the 22 pigs. B, Dephosphorylation of DPP from the 22 pigs shifted the bands lower on the gel but did not alter the pattern of bands. C, DPP PCR products using genomic DNA as template generated an identical pattern of 1 or 2 bands. D, Mapping of sequence variations in the porcine DPP coding region. Point mutations are indicated by numbered arrowheads; deletions are indicated by solid rectangles. On the left is the number of basepairs in each DPP allele. The first map (1863 bp, brown) is not an allele, but a merged reference sequence that contains all of the DPP code found on the four DPP alleles characterized. The four DPP alleles had 1785 (black), 1770 (blue), 1728 (green) and 1656 (red) basepairs. E, Key to the 27 sequence variations, the number of coding basepairs (bp) in each allele, the number of amino acids (aa) in the DPP protein, and the pigs shown to host each allele. The predominant alleles in the 22 pigs were 1656 and 1728 bp. Based upon the mobility of the proteins (A), and the cloning results, these alleles were likely present in 15 or 14 of the 22 pigs, respectively (1656: C2, D2, E, F, H2, J, K, M, N, O2, Q2, R, S, T, U; 1728: A2, B2, E, F, G2, I, J, L2, N, P, S, T, U, V). Only 6 of the 22 pigs hosted other alleles (I, K, M, P, R, V). The 1770 allele in pig P also appeared to be present in pigs I and V. The 1785 allele in pigs M and R also appeared to be in pig K. The frequency of the four characterized alleles in the 22 pigs (44 alleles) were 1656: 20 alleles; 1728: 18 alleles; 1770: 3 alleles; 1785: 3 alleles.

Figure 4. Proteolysis of porcine DSPP

Numebers indicate cleavage sites discovered by characterizing DSPP-derived cleavage products from developing porcine molars or by analyzing FRET-DSPP peptide. DSPP is first processed by bone mophogenetic protein-1 (BMP-1) to generate DSP-DGP complex and DPP. Then, DSP-DGP is further cleaved in the dentin extracellular matrix by MMP-2 and MMP-20, generating DSPs and DGPs. MMP-20 performs the cleavage that separates DSP and DGP (at Ser³⁹²) and generates N-terminal DSP cleavage products. On the other hand, MMP-2 cleaves DSP-DGP primarily within the C-terminal region of the DSP (at Ser³⁴⁵ and Ile³⁷⁷), releasing DGP-containing cleavage products. The mechanism of DPP degradation in vivo is unknown.