Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

<p>Abstract</p> <p>Background</p> <p>Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in <it>DSPP </it>cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties.</p> <p>Results</p> <p>To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn³⁷, Asn⁷⁷, Asn¹³⁶, Asn¹⁵⁵, Asn¹⁶¹, and Asn¹⁷⁶. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr²⁰⁰, Thr²¹⁶and Thr³¹⁶. Porcine Dsp GAG attachments were found at Ser²³⁸and Ser²⁵⁰and were comprised of chondroitin 6-sulfate and chondroitin 4-sulfate in a ratio of 7 to 3, respectively.</p> <p>Conclusions</p> <p>The distribution of porcine Dsp posttranslational modifications indicate that porcine Dsp has an N-terminal domain with at least six N-glycosylations and a C-terminal domain with two GAG attachments and at least two O-glycosylations.</p

Characterization of porcine dentin sialoprotein (DSP) and dentin sialophosphoprotein (DSPP) cDNA clones

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74726/1/j.1600-0722.2003.00009.x.pd

Porcine Enamel Protein Fractions Contain Transforming Growth Factor‐β1

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141064/1/jper1688.pd

Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73278/1/j.1600-0722.2006.00284.x.pd

MMP-20 Is Predominately a Tooth-Specific Enzyme with a Deep Catalytic Pocket that Hydrolyzes Type V Collagen †

Matrix metalloproteinase-20 (MMP-20, enamelysin) has a highly restricted pattern of expression. In healthy tissues, MMP-20 is observed in the enamel organ and pulp organ of developing teeth and is present only as an activated enzyme. To identify other tissues that may express MMP-20, we performed a systematic mouse tissue expression screen. Among the non-tooth tissues assayed, MMP-20 transcripts were only identified in minute quantities within the large intestine. The murine Mmp20 promoter was cloned, sequenced and assessed for potential tooth-specific regulatory elements. In silico analysis identified four promoter modules that were common to Mmp20 and at least two of three co-regulated predominantly tooth-specific genes that encode ameloblastin, amelogenin, and enamelin. We asked if the highly restricted MMP-20 expression pattern was associated with a broad substrate specificity that might preclude its expression in other tissues. An iterative mixture-based random doedecamer peptide library screen with Edman sequencing of MMP-20 cleavage products revealed that, among MMPs previously screened, MMP-20 had unique substrate preferences. These preferences indicate that MMP-20 has a deep and wide catalytic pocket that can accommodate substrates with large aromatic residues in the P1′ position. Based on matrices derived from the peptide library data, we identified and then confirmed that Type V collagen is an MMP-20 substrate. Since Type V collagen is not present in dental enamel but is an otherwise widely distributed collagen, and since only active MMP-20 has been observed in teeth, our data suggests that control of MMP-20 activity is primarily regulated by transcriptional means

Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein

Summary: Dentin sialophosphoprotein (DSPP) plays an important role in the formation of dentin. Understanding its structure and function would provide important insights into the regulation of dentin mineralization. For the past 15 years, we have been studying DSPP-derived proteins isolated from pig dentin. Porcine DSPP is synthesized and secreted by odontoblasts and processed into three proteins, i.e., dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP), by bone morphogenetic protein 1 and matrix metalloproteinase-20 and -2. DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein with genetic polymorphisms. Furthermore, DPP is not detected in dental pulp. This is possibly due to the existence of two mRNA variants of the DSPP gene: one that encodes the DSP region alone and another that encodes full-length DSPP. The mRNA variant encoding DSP alone is expressed in dental pulp and odontoblasts, but the variant encoding full-length DSPP is predominantly expressed in odontoblasts and barely in dental pulp. Keywords: Dentin, Dentin sialophosphoprotein, Protease, Gene, Proteoglycan, Toot