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The use of antibodies in a study of the structure, function and distribution of glucose transport proteins

Davies, Anthony; (1990) The use of antibodies in a study of the structure, function and distribution of glucose transport proteins. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Antibodies were raised against synthetic peptides corresponding to most of the regions of the human erythrocyte glucose transporter predicted to be extramembranous in a model proposed by Mueckler et al. (1985) Science 229, 941-945 for the arrangement of the membrane-bound protein. The antibodies were used as probes to investigate the topology and to identify functionally important regions of this glucose transport protein. In addition, the antibodies were used in a study of the distribution of homologous glucose transporters in other tissues both of mammalian and non-mammalian origin. Of the 21 antibodies produced all but two recognized the denatured glucose transporter, whilst only 7 recognized the native, functionally-active glycoprotein, even after its extensive deglycosylation. Use of these 7 antibodies as structural probes demonstrated that a large, central hydrophilic region of the sequence (residues 217-272) and the C-terminal region (residues 450-492) were both exposed at the cytoplasmic face of the membrane. The anti-peptide antibodies were used to identify the sequence location of fragments of the protein produced by partial proteolytic digestion. This led to the identification of the exofacial site of glycosylation as being located in the N-terminal half of the protein, and the site of photolabelling by cytochalasin B as lying in the C- terminal half of the protein. The location of the sites of glycosylation and of the C-terminus of the protein were in agreement with the model of Mueckler et al. (1985), and disproved an alternative model proposed by Shanahan and D'Artel-Ellis (1984). Furthermore, identification of the sequence locations of proteolytic fragments of the protein enabled the epitopes of eight monoclonal antibodies against the transporter to be located. All proved to be within the central or C-terminal hydrophilic regions of the sequence, on the cytoplasmic surface of the membrane. Antibodies against the central hydrophilic region of the sequence, but not against the C-terminal region, were found to inhibit cytochalasin B binding to the protein. Furthermore, the binding of antibodies to the central region was affected by the presence of bound substrate. These findings suggested that the central region of the sequence is important for transporter function, and is involved in the conformational changes that accompany substrate binding. Proteins homologous to the human erythrocyte glucose transporter were found to be widely distributed in mammalian tissues. An apparently homologous transporter was even detected in the erythrocytes of the hagfish, the most primitive living vertebrate. The anti-peptide antibodies were also used to begin to probe the subcellular distribution of glucose transporters in mammalian tissues. Evidence was obtained that glucose transporters are present in the Golgi membranes of lactating rat mammary gland epithelial cells. They probably function here to transport glucose across the membranes to the intralumenal site of lactose synthesis. This finding is one of the first demonstrations that intracellular glucose transporters may be functionally important in some mammalian cells.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The use of antibodies in a study of the structure, function and distribution of glucose transport proteins
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10120015
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