RESUMO
Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-ß activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-ß receptor inhibitor. We then purified TGF-ß-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-ß isoform bound to DPP and DSP was identified as being TGF-ß1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-ß1 (CF-hTGF-ß1) with TGF-ß-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-ß1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-ß1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-ß to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-ß1. We conclude that both DPP and DSP help retain TGF-ß1 activity in porcine dentin.
