Abstract
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.
Highlights
Membrane Glycoprotein have not yet been fully elucidated
Sequence information was obtained from lowpico- are three N-linked glycosylation sites in the NH2-terminaldomole amounts of nonspecific cross-reacting antigen main and nine other N-linked sites in the other domains [6]
Polyacrylamide gel electrophoresis, the protein was Whereaspast work has been focused on establishingthe electrotransferred to nitrocellulose, digested with trypsin, and the peptides were isolated using capillary reversed-phaseliquid chromatography. Analysis of these peptides byEdman microsequencingand massspectrometry established that Nonspecific cross-reacting antigens (NCAs)-160 was identical to biliary glycoprotein I, a protein that we identities of the NCAs, recent studies aredirected at establishing a function.The expression on phagocytic cells and the structural similarity with immunoglobulins suggest a function for NCAs in immunerecognition
Summary
Sequencing studies on the isolated clones [7,8,9] have revealed that NCA-50 ( M , 50,000) and NCA-90 (Mr90,000) are identical with respect to sequence and placement of glycosylation sites (Received for publication,April 2, 1993) despite differingby 40,000 in apparent mass (6N).CA-95 has a. Polyacrylamide gel electrophoresis, the protein was Whereaspast work has been focused on establishingthe electrotransferred to nitrocellulose, digested with trypsin, and the peptides were isolated using capillary reversed-phaseliquid chromatography Analysis of these peptides byEdman microsequencingand massspectrometry established that NCA-160 was identical to biliary glycoprotein I, a protein that we identities of the NCAs, recent studies aredirected at establishing a function.The expression on phagocytic cells and the structural similarity with immunoglobulins suggest a function for NCAs in immunerecognition. In addition to the structural similarity shwariethd CEA, the NCA glycoproteins share sequence homology with BGP I, a Nonspecific cross-reacting antigens (NCAs)’ are a polymorphic family of highly glycosylated cell-surface proteins immunologically related to carcinoembryonic antigen (CEA). The abbreviations used are: NCA, nonspecific cross-reacting antigen; CEA,carcinoembryonic antigen; BGP I, biliary glycoprotein I; PVP-40, polyvinylpyrrolidone-40;SIMS, secondary ion mass spectrometry; HPLC, high performance liquid chromatography;PBS, phosphatebufferd saline; PAGE, polyacrylamide gel electrophoresis. Termining the structures of intact oligosaccharide chains (reviewed in Ref. 21)
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