Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.     

Microheterogeneity of the carbohydrate moiety of the human erythrocyte glucose transporter

The carbohydrate moiety of the human erythrocyte glucose transporter was isolated using two independent methods: hydrazinolysis andN-glycanase treatment. The major structure observed was constituted of complex-type carbohydrate chains carrying repetitive units ofN-acetyllactosamine. This structure exhibited microheterogeneity: a broad variability in the number of repetitive units, presence of branched structures and substitution by fucosyl residues. Moreover, significant amounts of bi-antennary and hybrid structures were present.     

Proteolytic and chemical dissection of the human erythrocyte glucose transporter.

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.     

Structural basis of human erythrocyte glucose transporter function in reconstituted vesicles

The transmembrane orientation of the human erythrocyte glucose transporter was assessed based on polarized Fourier transform infrared and ultraviolet circular dichroism spectroscopic data obtained from oriented multilamellar films of the reconstituted transporter vesicles. Infrared spectra revealed that there are distinct vibrations for alpha-helical structure while the vibrational frequencies specific to beta-structure are characteristically absent. Analysis of linear dichroism of the infrared spectra further indicated that these alpha-helices in the transporter are preferentially oriented perpendicular to the lipid bilayer plane forming an effective tilt of less than 38 degrees from the membrane normal. Such a preferential orientation was further supported by ultraviolet circular dichroism spectra which reveal that the 208 nm Moffit band found in the detergent-solubilized preparation is absent in the film preparation. Linear dichroism data further indicated that D-glucose, a typical substrate, further reduces this effective tilt angle slightly.     

Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.     

Endoglycosidase f cleaves the oligosaccharides from the glucose transporter of the human erythrocyte

The glucose transporter from human erythrocytes is a heterogeneously glycosylated protein that runs as a very broad band of average apparent Mr 55 000 upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the purified preparation of transporter, solubilized in Triton X-100, was treated with endoglycosidase F, much of it ran as a sharp band of Mr 46 000 upon electrophoresis. Moreover, endoglycosidase F released 80% of the radioactivity in a preparation of the transporter labeled in its oligosaccharides with galactose oxidase and tritiated borohydride, and almost none of the remaining radioactivity was located in the Mr 46 000 band. These results suggest that endoglycosidase F can release virtually all of the carbohydrate linked to the transporter polypeptide. A quantitative analysis of the gels was complicated by partial aggregation of polypeptides that occurs due to prolonged incubation in Triton X-100, but at least 65% of the protein in the preparation of purified transporter is the 46 kDa polypeptide. The extracellular domain of the transporter is very resistant to proteolysis; no cleavage occurred upon treatment of intact erythrocytes with seven different proteases at high concentration.     

Location of the maltosyl isothiocyanate binding site on the human erythrocyte glucose transporter

The covalent affinity probe maltosyl isothiocyanate (MITC) has been used previously to identify the glucose transporter of human erythrocytes as a component of band 3. By use of limited proteolysis, the site on the Mr 100 000 protein to which MITC attaches has been localized to a 17 000-dalton region near the center of the polypeptide chain which is intimately associated with the membrane. The erythrocyte anion transporter, which is probably homologous to the glucose carrier, has a corresponding segment which is known to bind the covalent affinity label 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid [Ramjeesingh, M., Gaarn, A., & Rothstein, A. (1980) Biochim. Biophys. Acta 559, 127-139]. These results suggest that, in addition to having structural features in common, the two carrier proteins may be quite similar with regard to functional organization.     

Glycation of the human erythrocyte glucose transporter in vitro and its functional consequences.

Stimulation of NIH-3T3 cells with prostaglandin F2 alpha (PGF2 alpha) caused a dose- and time-dependent generation of inositol phosphates. The first detectable changes were in the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Increases in Ins(1,3,4)P3, InsP2 and InsP were detected later, and only minor changes were observed in putative InsP5 or InsP6. The accumulation of inositol phosphates was synergistically increased by the addition of calf serum, whereas PGF2 alpha had no effects on cell proliferation in either the presence or the absence of calf serum. Stimulation of a different clone of NIH-3T3 cells (AmNIH-3T3) or Swiss 3T3 cells with PGF2 alpha resulted in both inositol phospholipid breakdown and cell proliferation. No differences were found in the characteristics of PGF2 alpha-stimulated inositol phosphate generation between the two clones of NIH-3T3 cells, nor was there any difference in receptor number of Kd. These results question the role of inositol phospholipid breakdown in mitogenesis and demonstrate significant differences in the biochemical properties of apparently the 'same' cells.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.     

Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.     

Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.     

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.     

Structural basis of human erythrocyte glucose transporter function: pH effects on intrinsic fluorescence.

The effects of pH on the intrinsic fluorescence of purified human erythrocyte glucose transporter (HEGT) were studied to deduce the structure and the ligand-induced dynamics of this protein. D-Glucose increases tryptophan fluorescence of HEGT at a 320-nm peak with a concomitant reduction in a 350-nm peak, suggesting that glucose shifts a tryptophan residue from a polar to a nonpolar environment. Cytochalasin B or forskolin, on the other hand, only produces a reduction at the 350-nm peak. The pH titration of the intrinsic fluorescence of HEGT revealed that at least two tryptophan residues are quenched, one with a pKa of 5.5, the other with a pKa of 8.2, indicating involvement of histidine and cysteine protonation, respectively. D-Glucose abolishes both of these quenchings. Cytochalasin B or forskolin, on the other hand, abolishes the histidine quenching but not the cysteine quenching and induces a new pH quenching with a pKa of about 4, implicating involvement of a carboxyl group. These results, together with the known primary structure and the transmembrane disposition of this protein, predict the dynamic interactions between Trp388 and His337, Trp412 and Cys347, and Trp412 and Glu380, depending on liganded state of HEGT, and suggest the importance of the transmembrane helices 9, 10, and 11 in transport function.