Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter.
Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.
Nishimura
H
H
Second Division, Internal Medicine, Kyoto University School of Medicine, Japan.
Kuzuya
H
H
Kosaki
A
A
Okamoto
M
M
Okamoto
M
M
Kono
S
S
Inoue
G
G
Maeda
I
I
Imura
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D003571:Cytochalasin B / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D010446:Peptide Fragments / Q000494:pharmacology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D003571:Cytochalasin B; D005947:Glucose