Sequence and glycosylation site identity of two distinct glycoforms of nonspecific cross-reacting antigen as demonstrated by sequence analysis and fast atom bombardment mass spectrometry.
Nonspecific cross-reacting antigen (NCA) is a highly glycosylated membrane protein which is immunologically and structurally related to carcinoembryonic antigen, an important tumor-associated antigen. Two glycoforms of NCA were purified from a single liver metastasis of a colonic carcinoma and characterized with respect to their primary sequence and position of glycosylation sites. The two glycoforms (designated TEX (tumor-extracted antigen), Mr 75,000, and NCA, Mr 45,000) each showed a deglycosylated Mr of 35,000 and yielded identical peptide maps. The structural characterization of TEX and NCA and the assignment of glycosylation sites was performed by fast atom bombardment mass spectrometry and microsequence analysis of the resulting peptides. This approach showed that TEX and NCA were identical with respect to primary sequence and provided direct evidence that 11 of the 12 predicted asparagine-linked glycosylation sites were glycosylated in both TEX and NCA. Indirect evidence was obtained for glycosylation at the other site. Both glycoforms also contain ethanolamine linked to Gly-286, a finding consistent with the conclusion that these proteins are anchored to the plasma membrane through a glycosyl-phosphatidylinositol tail. The large difference in the molecular weights of glycosylated TEX and NCA suggests significant variations in their oligosaccharide structures.
Hefta
S A
SA
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Paxton
R J
RJ
Shively
J E
JE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D015815:Cell Adhesion Molecules; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008113:Liver Neoplasms / Q000276:immunology / Q000556:secondary; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid
D000596:Amino Acids; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D006023:Glycoproteins