Complementary DNA cloning for galactosyltransferase associated with tumor and determination of antigenic epitopes recognized by specific monoclonal antibodies.
The galactosyltransferase associated with tumor (GAT) was the name given to the isoenzyme that tends to polymerize resulting in slower moving in a nondenaturing polyacrylamide gel electrophoresis than normal (beta 1-4)galactosyltransferase (normal GalT). A complementary DNA (cDNA) library was constructed from a human ovarian cancer cell line, RMG-I, which secreted an amount of GAT into the culture supernatant and screened with monoclonal antibodies (MAbs) against GAT and normal GalT. One of six cDNA clones, UG86-1, encoded an epitope recognized by a GAT-specific MAb, 8513. Recombinant proteins expressed by UG86-1 in Escherichia coli also had antigenic epitopes recognized by the other MAbs against normal GalT. The 229-base pair nucleotide sequence encoded by UG86-1 was identical to the stem region sequence of HGT832 which encodes a full-length cDNA of human GalT. Using recombinant proteins directed by deletion mutant cDNAs, the antigenic epitopes recognized by each MAb were determined. The epitope of MAb8628, which reacts to both the GAT and normal GalT, was localized to the COOH-terminal side of proteolytic cleavage site where the membrane-bound form enzyme is cleaved to be converted to soluble forms, while MAb8513 epitope was at the NH2-terminal side from this cleavage site between the COOH-terminal end of the membrane-binding domain and the cleavage site. These results demonstrate that GAT is produced by aberrant proteolytic cleavage at the different site, closer to the membrane-binding domain, from the normal GalT.
Uejima
T
T
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
Uemura
M
M
Nozawa
S
S
Narimatsu
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D015152:Blotting, Northern; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010051:Ovarian Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D017385:Sequence Homology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D000939:Epitopes; D007527:Isoenzymes; D012333:RNA, Messenger; D005700:Galactosyltransferases