Porcine DSP is a Proteoglycan with Chondroitin-6-Sulfate Chains
Dentin Sialoprotein (DSP) is a glycoprotein that is critical for proper tooth dentin formation. Objectives: Our objectives were to isolate DSP from developing porcine molars and to characterize its carbohydrate attachments and other posttranslational modifications. Methods: Polyclonal antibodies were raised in chicken against recombinant pig DSP, and used to identify native DSP in fractions of tooth dentin proteins. Unerupted molars were extracted from 6-month-old pigs, and their pulp-sides were sequentially extracted with water, guanidine and guanidine/EDTA. DSP was isolated from the guanidine/EDTA fraction by anion exchange, hydroxyapatite affinity, size exclusion and reverse phase-high performance liquid chromatography (RP-HPLC). Purified DSP was sequentially digested with glycopeptidase A, O-glycanase and chondroitinase ABC and the digestion products were characterized. Results: Amino acid analyses and characterization of lysylendopeptidase cleavage products confirmed that purified protein was DSP, and that Arg391 is at the DSP C-terminus. On SDS-PAGE, DSP appeared as two smear bands extending from 280 to 100 kDa, but in the presence of β-mercaptoethanol the top of the DSP smear disappeared. The high molecular weight DSP was likely comprised of covalent DSP dimers connected by a disulfide bridge at Cys205. Both N- and O-linked oligosaccharides were released from DSP following glycopeptidase A and O-glycanase treatments, but these digestions had little effect on the apparent molecular weight of DSP on SDS-PAGE, when compared to the significant reduction following the chondroitinase ABC digestion. Glycosaminoglycanases with assorted glycosaminoglycan (GAG) cleavage specificities coupled with Western analyses of the cleaved GAG stubs demonstrated that DSP GAG attachments contain chondroitin-6-sulfate, but not keratan sulfate, heparan sulfate, dermatan sulfate, chondroitin or chondroitin-4-sulfate. Conclusions: DSP is a proteoglycan and that GAG attachments are the predominant structural feature of porcine DSP. This study was funded by USPHS Research Grants DE12769, DE15846, and NIDCR grant DE11301.
Division: Japanese Division Meeting
Meeting:2004 Japanese Division Meeting (Tokyo, Japan) Location: Tokyo, Japan
Year: 2004 Final Presentation ID:8 Abstract Category|Abstract Category(s):Scientific Groups
Authors
Yamakoshi, Yasuo
( The University of Michigan, Ann Arbor, MI, USA
)
Hu, Jan C-c
( The University of Michigan, Ann Arbor, MI, USA
)
Fukae, Makoto
( Tsurumi University, Yokohama, N/A, Japan
)
Iwata, Takanori
( Tokyo Medical & Dental University, Tokyo, N/A, Japan
)
Simmer, James P
( The University of Michigan, Ann Arbor, MI, USA
)