Site-specific antibodies as probes of the topology and function of the human erythrocyte glucose transporter.
Antibodies were raised against synthetic peptides corresponding to most of the regions of the human erythrocyte glucose transporter predicted to be extramembranous in the model of Mueckler, Caruso, Baldwin, Panico, Blench, Morris, Lienhard, Allard & Lodish [(1985) Science 229, 941-945]. Most of the antibodies (17 out of a total of 19) recognized the intact denatured protein on Western blots. However, only seven of the antibodies recognized the native membrane-bound protein, even after its deglycosylation. These antibodies, against peptides encompassing residues 217-272 and 450-492 in the hydrophilic central and C-terminal regions of the transporter, bound to the cytoplasmic surface of the erythrocyte membrane. This finding is in agreement with the prediction of the model that these regions of the sequence are cytoplasmic. Antibodies against peptides from the central cytoplasmic loop of the transporter were found to inhibit the binding of cytochalasin B to the membrane-bound protein, whereas antibodies against the C-terminal region had no effect. The anti-peptide antibodies were then used to map the sequence locations of fragments of the transporter arising from tryptic digestion of the membrane-bound protein. This in turn enabled the epitopes for a number of anti-transporter monoclonal antibodies to be located within either the central cytoplasmic loop or the C-terminal region of the protein. Of those monoclonal antibodies which inhibited cytochalasin B binding to the protein, all but one were found to have epitopes within the central region of the sequence. In conjunction with the results of the anti-peptide antibody studies, these findings indicate the importance of this part of the protein for transporter function.
Department of Biochemistry, Royal Free Hospital School of Medicine, University of London, U.K.
A
A
Davies
T L
TL
Ciardelli
G E
GE
Lienhard
J M
JM
Boyle
A D
AD
Whetton
S A
SA
Baldwin
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D003571:Cytochalasin B / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D004910:Erythrocyte Membrane / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000276:immunology / Q000502:physiology*; D010455:Peptides / Q000276:immunology; D011489:Protein Denaturation; D011817:Rabbits; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D003571:Cytochalasin B