Dentin Sialophosphoprotein (DSPP) in Dentin Extracellular Matrix (ECM)

Objectives: Sequences for two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are coded by a single mRNA transcript. Thus, the putative translation product DSPP would be a large precursor protein that must be proteolytically converted to DSP and DPP. Although the DSPP cDNA has been cloned and completely sequenced, the presence of DSPP protein in odontoblasts or dentin ECM has never been demonstrated. Recently, we unexpectedly detected DSPP in rat dentin ECM extracts. Methods: The Gdn/EDTA extracts of rat dentin ECM were separated by DEAE-Sephacel and Bio-Gel A-50m chromatography; fractions were analyzed with 5-15% SDS-PAGE and Western immunoblots. Tryptic peptides were separated by HPLC and sequenced by Edman degradation. Results: On DEAE-Sephacel, DSPP eluted later than DSP and DPP, consistent with its larger size and increased net charge. Bio-Gel A-50m fractions showed DSPP as a series of bands migrating at 160 to 210 kDa that reacted with anti-DSP antibodies on Western immunoblots. A DSPP fraction from Bio-Gel A-50m chromatography yielded 3 peptides with the sequences, GQVGIAENAE, GQGSVSTEDDD and NSPKQGESDKP, identical to residues 119-128, 270-280 and 381-391, respectively, in the DSP portion of rat DSPP. Conclusions: These data indicate that DSPP is present in dentin ECM and that it may be processed into DSP and DPP after its secretion from odontoblasts. Supported by NIH grant DE05092 (to WTB)