Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal beta 1-3GlcNAc.
The monoclonal antibody LU-BCRU-G7, that was generated by in vitro immunization, shows clinical value as a prognostic marker in early stage breast carcinoma. It has now been characterized with regard to its binding epitope. Using a recently described method based on the construction of N-substituted polyacrylamide (PAA) derivatives of carbohydrates (pseudopolysaccharides), the structure of the epitope for the monoclonal antibody LU-BCRU-G7 has been determined. Competitive binding assays and inhibitory enzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharides have shown the LU-BCRU-G7 epitope to be a disaccharide Gal beta 1-3GlcNAc (Lec; where Gal is D-galactose, Glc is D-glucose and GlcNAc is N-acetyl-D-glucosamine). Both galactose and N-acetyl glucosamine moieties are essential for binding. Substitution on C-2 or C-3 of the terminal galactose abolished binding, as did galactose-alpha terminated oligosaccharides. The galactose moiety alone, as expressed by the Gal beta-PAA conjugate, appeared to be a more important feature of the epitope than the GlcNAc-PAA conjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody. In the N-acetyl glucosamine moiety, binding was decreased but not eliminated by fucose substitution, as in Lea, or change in configuration of C-4, as in Gal beta 1-3GlcNAc. Omission of the NAc group resulted in complete loss of activity. The tetrasaccharide lacto-N-tetraose, although containing the terminal Lec disaccharide, does not react with the antibody, suggesting conformational interference of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Rye
P D
PD
Department of Tumour Biology, Norwegian Radium Hospital, Oslo.
Bovin
N V
NV
Vlasova
E V
EV
Walker
R A
RA
D000117:Acetylglucosamine / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry* / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D005690:Galactose / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D004187:Disaccharides; D000939:Epitopes; D006023:Glycoproteins; D000117:Acetylglucosamine; D005690:Galactose