title: DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin.
abstract: 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-β 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-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan yamakoshi-y@tsurumi-u.ac.jp.
authors.forename: Y
authors.initials: Y
authors.lastname: Yamakoshi
authors.affiliation: Department of Pediatric Dentistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Division of Regenerative Medicine, The Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-0003, Japan.
authors.forename: L
authors.initials: L
authors.lastname: Izuhara
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Karakida
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Fukae
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Oida
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects; D000818:Animals; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D003804:Dentin / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000494:pharmacology*; D006801:Humans; D010513:Periodontal Ligament / Q000166:cytology / Q000201:enzymology; D010750:Phosphoproteins / Q000032:analysis / Q000494:pharmacology*; D011485:Protein Binding; D020033:Protein Isoforms / Q000032:analysis; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins; D012795:Sialoglycoproteins / Q000032:analysis / Q000494:pharmacology*; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000032:analysis / Q000187:drug effects*; D000099311:Dentin Sialophosphoprotein
chemical_list: D000469:Alkaline Phosphatase; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D053773:Transforming Growth Factor beta1; D000099311:Dentin Sialophosphoprotein
keywords: HPLC; cell culture; extracellular matrix (ECM); isolation and purification; phosphophoryn; tooth