DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin
DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin
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DOI：
10.1177/0022034514534690
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发表时间：
2014-07-01
影响因子：
7.6
通讯作者：
Oida, S.
中科院分区：
文献类型：
--
作者：
Yamakoshi, Y.;Kinoshita, S.;Oida, S.
猪牙本质涎磷蛋白（DSPP）是牙本质中含量最丰富的非胶原蛋白。它被蛋白酶加工成3种独立的蛋白质：牙本质唾液蛋白（DSP）、牙本质糖蛋白（DGP）和牙本质磷蛋白（DPP）。我们通过离子交换（IE）色谱法从发育中的猪磨牙中分离DPP和DSP沿着TGF-β活性，并在有或没有TGF-β受体抑制剂的情况下测量它们在人牙周（HPDL）细胞中的碱性磷酸酶（ALP）刺激活性。然后，我们使用ALP-HPDL系统通过反相高效液相色谱法（RP-HPLC）纯化TGF-β未结合或结合的DPP和DSP。通过ELISA和LC-MS/MS分析，将与DPP和DSP结合的TGF-β同种型鉴定为TGF-β 1。我们将无载体的人重组TGF-β 1（CF-hTGF-β 1）与未结合TGF-β的DPP或DSP孵育，并使用ALP-HPDL系统在IE-HPLC上表征结合。当仅孵育CF-hTGF-β 1时，保留约3.6%的ALP刺激活性。DPP和DSP挽救了TGF-β 1活性的丧失。约19%和10%的ALP刺激活性分别通过TGF-β与DPP和DSP的结合而保留。I型胶原蛋白很少与CF-hTGF-β 1结合。我们得出结论，DPP和DSP有助于保持猪牙本质中的TGF-β 1活性。
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.