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

Abstract

Dentin sialoprotein (DSP) is a glycoprotein that is critical for proper tooth dentin formation, but little is known about the nature of its carbohydrate attachments and other post-translational modifications. We have isolated DSP from pig dentin and demonstrate that it is a proteoglycan. Polyclonal antibodies were raised in chicken against recombinant pig DSP, and used to identify native DSP in fractions of tooth dentin proteins extracted from developing pig molars. Amino acid analyses and characterization of lysylendopeptidase cleavage products confirmed that the purified protein was DSP, and that Arg 391 is at the DSP C terminus. On SDS-PAGE and on urea gels, DSP appeared as a smear extending from 280 to 100 kDa, but in the presence of ␤-mercaptoethanol the top of the DSP smear disappeared. The high molecular weight material was likely comprised of covalent DSP dimers connected by a disulfide bridge at Cys 205 . Oligosaccharides were released from DSP following N-and O-linked glycosidase digestions, but these digestions had little effect on the apparent molecular weight of DSP on SDS-PAGE, when compared with the significant reduction following chondroitinase ABC digestion. Glycosaminoglycanases with assorted glycosaminoglycan (GAG) cleavage specificities coupled with Western analyses of the cleaved GAG "stubs" demonstrated that the DSP GAG attachments contain chondroitin 6-sulfate, but not keratan sulfate, heparan sulfate, chondroitin, or chondroitin 4-sulfate. DSP binds biotin-labeled hyaluronic acid, and such binding is inhibited by the addition of unlabeled hyaluronic acid. We conclude that DSP is a proteoglycan and that GAG attachments are the predominant structural feature of porcine DSP.The dentin sialophosphoprotein gene (DSPP) 1 on human chromosome 4q21.3 encodes the two major noncollagenous proteins in tooth dentin: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (1, 2), and these proteins are critical for proper dentin formation. As of this writing, eight mutations have been identified in the human DSPP gene that cause hereditary dentin defects (3-7). In each case, the phenotype is inherited in an autosomal dominant pattern and is usually limited to the teeth. Occasionally the condition is associated with progressive neurosensory high frequency hearing loss (DFNA39/DGI1, MIM 605594). In addition, DSPP-null mice developed dentin defects in the absence of other symptoms (8).Whereas it is known that DSPP gene products are essential for normal dentin biomineralization, DSP structural features are only partially characterized, and its functions are unknown.Much of our current understanding of the structure and function of dentin sialoprotein comes from studies of the rat protein (9 -11). 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 t...

Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo

“…Porcine (21). These glycosylations are likely to protect regions of DSP-DGP that might otherwise be susceptible to proteolysis and preclude the use of mass spectrometry analyses to deduce the C termini of the DSPP cleavage products.…”

“…The antipeptide antibody was designated pDGP. We used the chicken polyclonal antibody raised against recombinant porcine DSP expressed in bacteria that extended from Ile 1 to Lys 368 (21), the rabbit antibody raised against the peptide Cys-QEEGVGEPRGDNPD corresponding to the Gln 173 -Asp 186 segment of porcine DMP1 (dentin matrix protein 1) (26), and the chicken polyclonal antibody (ab14311; Abcam, Cambridge, UK) raised against a peptide extending from Pro 60 to His 180 of human MMP-2, which shares 96% identity with the pig homologue. The antibody dilutions used in Western blot analyses were as follows: DSP, 1:50,000; DGP, 1:2000; DMP1, 1:10,000, and MMP-2, 1:1000.…”

“…Previously, we cloned and characterized DSPP cDNA, providing us with the deduced amino acid sequence of porcine DSPP excepting the redundant region in the DPP code, which was partially deleted during the cloning process (25). We determined that DSP is a proteoglycan that forms covalent dimers through an intermolecular disulfide bridge at Cys 190 (21) and discovered and characterized dentin glycoprotein (DGP), a phosphorylated glycoprotein derived from the middle of DSPP between DSP and DPP (3). Our objectives in the present study were to map the cleavage sites in DSPP that are used to split DSPP into its component parts during odontogenesis, isolate dentin resident matrix metalloproteinases (MMPs), and use them to cleave high molecular weight DSPP-derived proteins.…”

Dentin Sialophosphoprotein (DSPP) and Dentin

“…The C-terminus of pig DSP varies depending upon the cleavage site used. The main cleavage site is on the Cterminal side of Arg 391 , which was confirmed by demonstrating that Ser 392 is at the N-terminus of DGP 25,26) . The genetic and proteolytic cleavage data are consistent and show that DSP is the N-terminal domain of DSPP and DSP is encoded by exons 2 through 4 of the DSPP.…”

“…Another DSP-only transcript was previously reported for the rat 24) , but this cDNA is a likely cloning artifact as its generation cannot be explained by any known mechanism (the internal deletion does not obey the GT… AG rule for splicing). We expressed recombinant porcine DSP in bacteria as a GST fusion product and raised polyclonal antibodies that allowed us to isolate DSP from developing pig teeth 25) . Using this antibody we are able to track DSP and DSP-derived cleavage products throughout our purification procedures25- 27) .…”