title: 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 Arg391 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 beta-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 Cys205. 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.
authors.affiliation: University of Michigan Dental Research Laboratory, Ann Arbor, Michigan 48108, USA.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamakoshi
authors.forename: Jan C-C
authors.initials: JC
authors.lastname: Hu
authors.forename: Makoto
authors.initials: M
authors.lastname: Fukae
authors.forename: Takanori
authors.initials: T
authors.lastname: Iwata
authors.forename: Jung-Wook
authors.initials: JW
authors.lastname: Kim
authors.forename: Hengmin
authors.initials: H
authors.lastname: Zhang
authors.forename: James P
authors.initials: JP
authors.lastname: Simmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002809:Chondroitin Sulfates / Q000032:analysis* / Q000737:chemistry; D019281:Dimerization; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans / Q000032:analysis* / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins; D011498:Protein Precursors; D020836:Protein Structure, Quaternary; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine / Q000235:genetics; D000099311:Dentin Sialophosphoprotein
chemical_list: D002809:Chondroitin Sulfates; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D006026:Glycoside Hydrolases; D006820:Hyaluronic Acid; D010750:Phosphoproteins; D011498:Protein Precursors; D011509:Proteoglycans; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein