DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin.

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-beta 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-beta receptor inhibitor. We then purified TGF-beta-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-beta isoform bound to DPP and DSP was identified as being TGF-beta 1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-beta 1 (CF-hTGF-beta 1) with TGF-beta-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-beta 1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-beta 1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-beta to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-beta 1. We conclude that both DPP and DSP help retain TGF-beta 1 activity in porcine dentin.