Microsequence and mass spectral analysis of nonspecific cross-reacting antigen 160, a CD15-positive neutrophil membrane glycoprotein. Demonstration of identity with biliary glycoprotein.
Sequence information was obtained from low picomole amounts of nonspecific cross-reacting antigen (NCA) 160 (M(r) 160,000), a granulocyte membrane glycoprotein. Following affinity purification and SDS-polyacrylamide gel electrophoresis, the protein was electrotransferred to nitrocellulose, digested with trypsin, and the peptides were isolated using capillary reversed-phase liquid chromatography. Analysis of these peptides by Edman microsequencing and mass spectrometry established that NCA-160 was identical to biliary glycoprotein I, a protein that we previously cloned from a human colon library (1). NCA-160 from human granulocytes is a CD15-positive glycoprotein belonging to the carcinoembryonic antigen family and possesses putative transmembrane and cytoplasmic domains. Previous efforts to characterize this antigen at the protein level were hampered by a blocked NH2 terminus. In this study, we confirmed 20% of the deduced amino acid sequence starting with approximately 50 pmol of sample. Carbohydrate structural data is also presented on a single N-linked oligosaccharide moiety located in the A' domain. The capillary high performance liquid chromatography techniques used here, as well as mass spectrometry, were essential for high sensitivity analysis of the blotted, digested glycoprotein.
Mahrenholz
A M
AM
Beckman Research Institute of City of Hope, Duarte, California 91010.
Yeh
C H
CH
Shively
J E
JE
Hefta
S A
SA
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000032:analysis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules; D003429:Cross Reactions; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D016256:Lewis X Antigen; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry*
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins