Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site.
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.
Hefta
S A
SA
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Hefta
L J
LJ
Lee
T D
TD
Paxton
R J
RJ
Shively
J E
JE
D000230:Adenocarcinoma / Q000032:analysis / Q000556:secondary; D002272:Carcinoembryonic Antigen / Q000302:isolation & purification / Q000378:metabolism*; D003110:Colonic Neoplasms; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D006801:Humans; D008113:Liver Neoplasms / Q000032:analysis / Q000556:secondary; D008565:Membrane Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010716:Phosphatidylinositols / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*
D002272:Carcinoembryonic Antigen; D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010716:Phosphatidylinositols; D011134:Polysaccharides; D019856:Ethanolamine