C-terminal-specific monoclonal antibodies against the human red cell glucose transporter. Epitope localization with synthetic peptides.
Mice were immunized with human red cell glucose transporter for production of monoclonal antibodies. Four peptides were synthesized that correspond to relatively hydrophilic segments of the human HepG2 glucose transporter (Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W. J., Lienhard, G.E., and Lodish, H.F. (1985) Science 229, 941-945), including a C-terminal segment. After identification of hybridomas that were positive for the red cell glucose transporter, enzyme-linked immunosorbent assays were done with the synthetic peptides in solution to detect peptide-binding monoclonals. The very hydrophilic C-terminal peptide 478-492 (P2), but no other peptide, gave strong and selective inhibition of antibody binding to the glucose transporter. Two C-terminal-specific monoclonal antibodies were selected. The binding of these two antibodies to immobilized inside-out vesicles of human red cell membranes could be inhibited with the peptide P2. The antibodies did not react with right-side-out vesicles. The binding of these C-terminal-specific antibodies to the glucose transporter, to immobilized vesicles, and to the peptide P2 was enhanced by the presence of the peptide 218-232 (P1), although the peptide P1 alone showed no reaction with these antibodies. This suggests that the C terminus and the segment 218-232 of the red cell glucose transporter are exposed at the cytoplasmic face of the membrane and interact in the transporter. The C-terminal-specific monoclonal antibodies reacted strongly in Western blotting with the human red cell glucose transporter.
Andersson
L
L
Department of Biochemistry, University of Uppsala, Sweden.
Lundahl
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009004:Monosaccharide Transport Proteins