CARCINOEMBRYONIC ANTIGEN IS ANCHORED TO MEMBRANES BY COVALENT ATTACHMENT TO A GLYCOSYLPHOSPHATIDYLINOSITOL MOIETY - IDENTIFICATION OF THE ETHANOLAMINE LINKAGE SITE
CARCINOEMBRYONIC ANTIGEN IS ANCHORED TO MEMBRANES BY COVALENT ATTACHMENT TO A GLYCOSYLPHOSPHATIDYLINOSITOL MOIETY - IDENTIFICATION OF THE ETHANOLAMINE LINKAGE SITE
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DOI：
10.1073/pnas.85.13.4648
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发表时间：
1988-07-01
影响因子：
11.1
通讯作者：
SHIVELY, JE
中科院分区：
文献类型：
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作者：
HEFTA, SA;HEFTA, LJF;SHIVELY, JE
癌胚抗原（CEA）的COOH-末端氨基酸与乙醇胺共价连接，这与CEA通过磷脂酰肌醇-聚糖尾锚定到质膜的证据一致。用胰蛋白酶消化纯化的CEA，并通过反相HPLC分离所得肽。胰蛋白酶消化的六肽T12以丙氨酸终止，在序列（Ser-Ile-Thr-Val-Ser-Ala）上与成熟CEA蛋白中第三重复结构域的最后六个残基（637-642）相对应。通过快原子轰击质谱法测定六肽的质量表明存在额外的乙醇胺部分。这一发现和随后的26个疏水残基的cDNA序列预测的情况下是证据，六肽T12是成熟CEA的COOH-末端肽。制备与六肽T12相同的合成肽，并将乙醇胺偶联至其COOH-末端丙氨酸;该合成乙醇胺偶联肽与肽T12的色谱性质相同。乙醇胺偶联的合成肽和肽T12的B/E-连接-扫描质谱分析揭示了与COOH-末端乙醇胺的存在一致的碎片离子系列。用磷脂酰肌醇特异性磷脂酶C处理后，通过间接免疫荧光和流式细胞术显示CEA表达细胞系LS 174 T中膜结合CEA的释放。我们的结论是，CEA的处理后，以消除疏水性的COOH-末端残基（643-668），随后加入乙醇胺-糖基磷脂酰肌醇部分和治疗的结肠细胞系与磷脂酰肌醇特异性磷脂酶C释放膜结合的CEA。
The COOH-terminal amino acid of carcinoembryonic antigen (CEA) is shown to covalently link with ethanolamine, evidence consistent with the anchorage of CEA to the plasma membrane through a phosphatidylinositol-glycan tail. Purified CEA was digested with trypsin, and the resulting peptides were isolated by reverse-phase HPLC. Tryptic hexapeptide T12, terminating atypically with alanine, corresponded in sequence (Ser-Ile-Thr-Val-Ser-Ala) with the last six residues (637-642) of the third repeating domain in the mature CEA protein. Mass determination of the hexapeptide by fast atom bombardment mass spectrometry suggested the presence of an additional ethanolamine moiety. This finding and the absence of the subsequent 26 hydrophobic residues predicted by cDNA sequence is evidence that hexapeptide T12 is the COOH-terminal peptide of mature CEA. A synthetic peptide identical to hexapeptide T12 was prepared, and ethanolamine was coupled to its COOH-terminal alanine; chromatographic properties of this synthetic ethanolamine-coupled peptide and peptide T12 were the same. B/E-linked-scan mass spectral analysis of the ethanolamine-coupled synthetic peptide and peptide T12 revealed a fragment ion series consistent with the presence of a COOH-terminal ethanolamine. Release of membrane-bound CEA from the CEA-expressing cell line LS 174T was shown by indirect immunofluorescence and flow cytometry after treatment with phosphatidylinositol-specific phospholipase C. We conclude that CEA is processed posttranslationally to remove the hydrophobic COOH-terminal residues (643-668) with subsequent addition of an ethanolamine-glycosylphosphatidylinositol moiety and that treatment of a colonic cell line with phosphatidylinositol-specific phospholipase C releases membrane-bound CEA.