The functional significance of dentin sialoprotein-phosphophoryn and dentin sialoprotein

Abstract

Phosphophoryn (PP) and dentin sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.

Fig. 1

Rat DSP-PP Genomic Organization. The rat DSP-PP gene is distributed in five exons and four introns. E1, exon 1, the 5′ noncoding sequence; E2, exon 2, the 5′ noncoding region, the leader sequence and the N-terminal two amino acids for DSP; E3, exon 3, the DSP coding sequence; E4, exon 4, the DSP coding sequence; E5, exon 5, the C-terminal DSP coding sequence and the mature PP sequence (i.e., 1569 bp) as well as the 3′ noncoding sequence (i.e., 1523 bp); I₁, intron 1; I₂, intron 2; I₃, intron 3; I₄, intron 4. Two polyadenylation sites (aataaa) were represented by ; Represents DSP exons; Represents PP exon; Represents the 3′ noncoding sequence; Represents the DSP-PP promoter region; Represents the transcription start site; Represents the stop codon

Fig. 2

Multiple DSP-PP and DSP-only transcripts from the rat DSP-PP gene. The DSP-PP₂₄₀ transcript, using the first polyadenylation site, will generate a mature DSP protein and a PP₂₄₀ isoform. DSP-PP₅₂₃ and DSP-PP₁₇₁ transcripts, using the second polyadenlyation site, will give rise to a mature DSP protein, a mature PP₅₂₃, and a mature DSP protein and PP₁₇₁ isoform. The DSP-only transcript will produce a DSP₃₀₃ protein

Fig. 3

DSP-PP₂₄₀ precursor protein is generated by translating the DSP-PP₂₄₀ transcript. This precursor protein undergoes post-translational modifications, such as signal peptide cleavage, Asn-linked glycosylation, phosphorylation, and finally proteolytic processing (via tolloid-related-1 protein (TLR1) in insect cells/BMP1 in mammalian cells) to produce mature DSP₄₃₀ and PP₂₄₀

Fig. 4

Altered epithelial/mesenchymal interactions in 1- and 6-day-old DSP-PP KO mice. The sections were stained with H&E. a 1-day-old DSP-PP KO M1 (400× magnification). b 6-day-old DSP-PP KO M1 (100× magnification). c 6-d DSP-PP KO M2 (100× magnification). Note: dentin formed a circular structure within the dental pulp in 6-day-old DSP-PP KO and the enamel and dentin layers were abnormally separated. M1, molar 1; M2, molar 2; am, ameloblast; dp, dental pulp; d, dentin; E, enamel; O, odontoblast

Fig. 5

The presence of the chondrocyte marker collagen type II and acidic proteoglycan in 21-day-old DSP-PP KO mice. a–c Immunohistochemical (IHC) staining of 21-day-old wt M2 and 21-day-old DSP-PP KO M2/M3 sections using anticollagen type II antibodies at 400x magnification. a 21-day-old wt M2 section showed no antibody staining. b 21-day-old DSP-PP KO M2 section showed antibody staining in blue as indicated by the arrows. c 21-day-old DSP-PP KO M3 section showed antibody staining in blue as indicated by the arrows. d, e Sections were stained with Safranin O/fast green for the acidic proteoglycan at 1600x magnification. d 21-day-old wt M2 section showed no Safranin O staining in the dental pulp. e 21-day-old DSP-PP KO M2 showed Safranin O staining in the dental pulp