Identification and Characterization of the Carboxyl-terminal Region of Rat Dentin Sialoprotein

Abstract

Two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), are present in the extracellular matrix of dentin but not in bone. These two proteins are expressed in odontoblasts and preameloblasts as a single cDNA transcript coding a large precursor protein termed dentin sialophosphoprotein (DSPP). DSPP is specifically cleaved into two unique proteins, DSP and DPP. However, the cleavage site(s) of DSPP and the mechanisms for regulating the cleavages are unknown. To identify the specific site(s) of DSPP that are cleaved when the initial translation product is converted to DSP and DPP, we performed a detailed analysis (Edman degradation and mass spectrometry) on selected tryptic peptides of a size originating from the COOH-terminal region of rat DSP. After cleavage with trypsin, the DSP fragments were separated by a twodimensional method (size-exclusion chromatography followed by reversed phase high performance liquid chromatography). We characterized 13 peptides from various regions of DSP. The analyses showed that peptide Ile 409 -Tyr 421 was the major COOH-terminal fragment, ending at Tyr 421 only 9 residues from the NH 2 terminus of DPP. Peptide Gln 385 -His 406 represented a second, minor COOH-terminal peptide that terminated at His 406 . Both of these residues are well beyond the COOH terminus predicted previously by two independent studies estimating that rat DSP contained 360 -370 amino acids. Careful studies on two peptides showed that, among 9 potential casein kinase II phosphorylation sites, 2 serines were phosphorylated. We found that rat DSP was heterogeneous with respect to phosphorylation, because this same peptide sequence eluted in two discrete peaks, one with 2 phosphoserines and the other having 1. The finding that 3 lysines just preceding the COOH termini were modified by a 43-Da substituent (possibly a carbamoyl substituent) suggests that the lysines in this region were particularly susceptible to attachment of this substituent.The dentin extracellular matrix is formed by highly specialized, postmitotic cells termed odontoblasts. These cells secrete a unique set of gene products similar to those expressed by osteoblasts in the formation of bone. The noncollagenous proteins include osteonectin, osteocalcin, osteopontin (OPN), 1 bone sialoprotein, and dentin matrix protein 1 (Dmp-1), found in bone and dentin, and dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), occurring in dentin but not in bone (1-5). Because these noncollagenous proteins are very acidic and are secreted into the extracellular matrix during the formation and mineralization of these tissues, it is generally accepted that they play key biological roles in the formation of dentin and bone (6); however, details concerning their precise functions are unknown.Type I collagen is the most abundant organic constituent of dentin extracellular matrix, forming a fibrillar lattice for mineral deposition. DPP is the second most plentiful protein, accounting for as much as 50% of dentin noncollagenous proteins. Th...

In Vitro Effects of Dentin Matrix Protein-1 on Hydroxyapatite Formation Provide Insights into in Vivo Functions

“…Similarly, the gene for dentin sialophosphoprotein codes for both dentin sialoprotein and dentin phosphoprotein (phosphophoryn). Two X-Asp bonds are also cleaved in the posttranslational processing of dentin sialophosphoprotein (34). Dentin sialoprotein is a relatively weak HA nucleator (35), whereas dentin phosphoprotein is an extremely effective nucleator (24).…”

Bone Morphogenetic Protein-1/Tolloid-like Proteinases Process Dentin Matrix Protein-1

“…The carboxyl-terminal domain of DSPP, designated the dentin phosphoprotein, and the carboxyl-terminal domain of DMP1, have both been shown to stimulate hydroxyapatite crystal formation in vitro (24,25). Thus, it has been proposed that in vivo proteolytic processing of full-length DSPP and DMP1 occurs, to generate functional carboxyl-terminal cleavage fragments that can be found in the extracts of mineralized tissue (26,27). Whereas the proteinase(s) responsible for the specific cleavage of these proteins has not been identified, several predicted proteolytic sites have been mapped based on sequencing of DSPP and DMP1 fragments extracted from dentin and bone, respectively.…”

Porcine Dentin Sialoprotein Is a Proteoglycan with Glycosaminoglycan Chains Containing Chondroitin 6-Sulfate

“…Rat DSP is the N-terminal portion of DSPP and is generated by proteolytic cleavages, primarily after Tyr 421 but also following His 406 (12). The molecular mass of the major rat DSP component, which contains 29.6% carbohydrate, was determined by sedimentation equilibrium analysis to be 52.57 kDa (13), although the glycoprotein has an apparent molecular mass of 95 kDa on SDS-PAGE (14).…”