Molecular identification of a human carcinoma-associated glycoprotein antigen recognized by mouse monoclonal antibody FU-MK-1.
Mouse monoclonal antibody FU-MK-1, raised against a human gastric adenocarcinoma, recognizes an antigen (termed MK-1 antigen) present on the majority of carcinomas. The present study aimed to identify the MK-1 molecule and to establish its relationship to other carcinoma antigens. Immunoprecipitation studies of human tumor cell lines revealed that FU-MK-1 recognizes a monomeric membrane glycoprotein with two forms, 40 kDa (major form) and 42 kDa (minor form), and with a molecular mass of 35 kDa following treatment with the N-glycosylation inhibitor tunicamycin. The partial amino acid sequence of a main fragment of the MK-1 molecule obtained by spontaneous cleavage under hypotonic conditions was examined, and the 17 contiguous NH2-terminal amino acids were found to be identical with residues 81-97 of the 314-residue GA733-2 protein [Szala et al.; Proc. Natl. Acad. Sci. USA, 87, 3542-3546 (1990)]. Hence, the GA733-2 cDNA was cloned and the specificity of FU-MK-1 was confirmed using four recombinant forms of the GA733-2 antigen expressed in COS-1 cells. Immunoprecipitation with FU-MK-1 of the cell lysate transfected with the full-length GA733-2 cDNA revealed two bands corresponding to those obtained from the tumor cell lines. FU-MK-1 also precipitated three other recombinant proteins consisting of amino acids 1-265, 1-201, and 1-139 of the GA733-2 protein, respectively. Furthermore, immunoblotting analysis indicated that FU-MK-1 binds to a small fragment (6 kDa) generated from a tumor cell line under hypotonic conditions, suggesting that the FU-MK-1 epitope exists on the distal 6-kDa peptide of the extracellular domain of the GA733-2 molecule. We thus conclude that the MK-1 antigen is the GA733-2 antigen, which is currently being used as a target in clinical trials with monoclonal antibodies.
Tomita
Y
Y
First Department of Biochemistry, School of Medicine, Fukuoka University.
Arakawa
F
F
Yamamoto
T
T
Kuwahara
M
M
Watanabe
R
R
Iwasaki
H
H
Kikuchi
M
M
Kuroki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D000071858:Epithelial Cell Adhesion Molecule; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule