Hefta S A, Hefta L J, Lee T D, Paxton R J, Shively J E
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4648-52. doi: 10.1073/pnas.85.13.4648.
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.
癌胚抗原（CEA）的羧基末端氨基酸被证明可与乙醇胺共价连接，这一证据与CEA通过磷脂酰肌醇聚糖尾锚定在质膜上相符。用胰蛋白酶消化纯化的CEA，所得肽段通过反相高效液相色谱法分离。胰蛋白酶消化产生的六肽T12以丙氨酸非典型结尾，其序列（Ser-Ile-Thr-Val-Ser-Ala）与成熟CEA蛋白第三个重复结构域的最后六个残基（637 - 642）一致。通过快原子轰击质谱法对该六肽进行质量测定表明存在一个额外的乙醇胺部分。这一发现以及cDNA序列预测的后续26个疏水残基的缺失证明六肽T12是成熟CEA的羧基末端肽。制备了与六肽T12相同的合成肽，并将乙醇胺与它的羧基末端丙氨酸偶联；这种合成的乙醇胺偶联肽和肽T12的色谱性质相同。对乙醇胺偶联的合成肽和肽T12进行B/E连接扫描质谱分析，发现了与羧基末端乙醇胺存在相符的碎片离子系列。在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从表达CEA的细胞系LS 174T释放出膜结合的CEA。我们得出结论，CEA在翻译后进行加工，去除疏水的羧基末端残基（643 - 668），随后添加乙醇胺 - 糖基磷脂酰肌醇部分，并且用磷脂酰肌醇特异性磷脂酶C处理结肠细胞系会释放膜结合的CEA。
