Identification and characterization of the carboxyl-terminal region of rat dentin sialoprotein
Identification and characterization of the carboxyl-terminal region of rat dentin sialoprotein
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DOI：
10.1074/jbc.m006271200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Butler, WT
中科院分区：
文献类型：
--
作者：
Qin, CL;Cook, RG;Butler, WT
牙本质唾液蛋白（DSP）和牙本质磷蛋白（DPP）这两种酸性蛋白存在于牙本质细胞外基质中，但不存在于骨中。这两种蛋白在成牙本质细胞和前成釉细胞中表达为单个 cDNA 转录物，编码称为牙本质唾液酸磷蛋白 (DSPP) 的大前体蛋白。 DSPP被特异性切割成两种独特的蛋白质：DSP和DPP。然而，DSPP的切割位点和调节切割的机制尚不清楚。为了确定当初始翻译产物转化为 DSP 和 DPP 时 DSPP 被切割的特定位点，我们对源自大鼠 DSP COOH 末端区域的选定胰蛋白酶肽进行了详细分析（Edman 降解和质谱法）。用胰蛋白酶切割后，通过二维方法（尺寸排阻色谱法，然后 反相高效液相色谱法）。我们表征了来自 DSP 不同区域的 13 种肽，分析表明肽 II(409)-Tyr(421) 是主要的 COOH 末端片段，以 Tyr(421) 结尾，距 DPP 末端 NH 仅 9 个残基，肽 Gln(385)-His(406) 代表第二个次要 COOH 末端肽，其终止于 His(406)，这两种肽都 残基远远超出了之前两项独立研究预测的 COOH 末端，估计大鼠 DSP 含有 360-370 个氨基酸。对两种肽的仔细研究表明，在 9 个潜在的酪蛋白激酶 II 磷酸化位点中，有 2 个丝氨酸被磷酸化。我们发现大鼠 DSP 在磷酸化方面具有异质性，因为相同的肽序列在两个离散峰中洗脱，一个具有 2 个磷酸丝氨酸，另一个具有 1 个。发现 COOH 末端之前的 3 个赖氨酸被 43-Da 修饰 取代基（可能是氨基甲酰基取代基）表明该区域的赖氨酸特别容易连接该取代基。
Two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), are present in the extracellular matrix of dentin but not in bone. These two proteins are expressed in odontoblasts and preameloblasts as a single cDNA transcript coding a large precursor protein termed dentin sialophosphoprotein (DSPP). DSPP is specifically cleaved into two unique proteins, DSP and DPP, However, the cleavage site(s) of DSPP and the mechanisms for regulating the cleavages are unknown. To identify the specific site(s) of DSPP that are cleaved when the initial translation product is converted to DSP and DPP, we performed a detailed analysis (Edman degradation and mass spectrometry) on selected tryptic peptides of a size originating from the COOH-terminal region of rat DSP, After cleavage with trypsin, the DSP fragments were separated by a two-dimensional method (size-exclusion chromatography followed by reversed phase high performance liquid chromatography). We characterized 13 peptides from various regions of DSP, The analyses showed that peptide Il(409)-Tyr(421) was the major COOH-terminal fragment, ending at Tyr(421) only 9 residues from the NH, terminus of DPP, Peptide Gln(385)-His(406) represented a second, minor COOH-terminal peptide that terminated at His(406), Both of these residues are well beyond the COOH terminus predicted previously by two independent studies estimating that rat DSP contained 360-370 amino acids. Careful studies on two peptides showed that, among 9 potential casein kinase II phosphorylation sites, 2 serines were phosphorylated, We found that rat DSP was heterogeneous with respect to phosphorylation, because this same peptide sequence eluted in two discrete peaks, one with 2 phosphoserines and the other having 1. The finding that 3 lysines just preceding the COOH termini were modified by a 43-Da substituent (possibly a carbamoyl substituent) suggests that the lysines in this region were particularly susceptible to attachment of this substituent.