Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes.
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell-cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x-specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal alpha(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.
Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Biochemie und Molekularbiologie, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
Lothar
L
Lucka
Malkanthi
M
Fernando
Detlef
D
Grunow
Christoph
C
Kannicht
Andrea K
AK
Horst
Peter
P
Nollau
Christoph
C
Wagener
D000906:Antibodies / Q000276:immunology; D015703:Antigens, CD / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000943:Antigens, Differentiation / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules; D002460:Cell Line; D006026:Glycoside Hydrolases / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D009005:Monosaccharides / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D015703:Antigens, CD; D000943:Antigens, Differentiation; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D009005:Monosaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D006026:Glycoside Hydrolases