Erythrocyte GLUT1 Research Articles

Genetic Modulation of the GLUT1 Transporter Expression-Potential Relevance in Complex Diseases.

The human GLUT1 (SLC2A1) membrane protein is the key glucose transporter in numerous cell types, including red cells, kidney, and blood-brain barrier cells. The expression level of this protein has a role in several diseases, including cancer and Alzheimer's disease. In this work, to investigate a potential genetic modulation of the GLUT1 expression level, the protein level was measured in red cell membranes by flow cytometry, and the genetic background was analyzed by qPCR and luciferase assays. We found significant associations between red cell GLUT1 levels and four single nucleotide polymorphisms (SNP) in the coding SLC2A1 gene, that in individuals with the minor alleles of rs841848, rs1385129, and rs11537641 had increased, while those having the variant rs841847 had decreased erythrocyte GLUT1 levels. In the luciferase reporter studies performed in HEK-293T and HepG2 cells, a similar SNP-dependent modulation was observed, and lower glucose, serum, and hypoxic condition had variable, cell- and SNP-specific effects on luciferase expression. These results should contribute to a more detailed understanding of the genetic background of membrane GLUT1 expression and its potential role in associated diseases.

Higher lactate and purine metabolite levels in erythrocyte-rich fresh venous thrombus: Potential markers for early deep vein thrombosis

BackgroundThrombolytic therapy is effective in fresh deep vein thrombosis (DVT) although the benefit may fall below the risk of bleeding in non-fresh thrombosis. Markers reflecting fresh DVT have not been established. The present study aims to identify metabolites reflecting fresh venous thrombus and their role in thrombus formation. MethodsMetabolites of rabbit venous blood and jugular venous thrombus 4 h after thrombus induction were analysed using electrophoresis-time of flight mass spectrometry. The effects of the altered metabolites on blood coagulation and platelet aggregation were assessed by using rotation thromboelastometry and platelet aggregometer. Cellular contents and glucose transporter (Glut)-1 expression in aspirated human DVT samples were pathologically analysed. ResultsMetabolome analysis identified 226 metabolites (133 cationic and 93 anionic metabolites). Largely altered 18 metabolites (thrombus/blood ratio: >5 or <0.5) included glycolytic metabolites, redox-related metabolites, purine nucleotides and tryptophan metabolites. Among the metabolites with >5-fold increase, lactic acid was most abundant and guanine modestly enhanced whole blood clotting with thromboelastometry. Lactic acid and adenosine monophosphate inhibited collagen-induced platelet aggregation. Human DVTs were rich in erythrocytes expressing Glut-1. The erythrocyte content and Glut-1 expression were negatively correlated with the time after onset of DVT. ConclusionsGlycolysis-, purine-, and redox-related metabolites may reflect fresh erythrocyte-rich venous thrombus, and altered metabolites may affect venous thrombus formation. An increased level of lactate may reflect active glycolysis of thrombus cellular components, predominantly erythrocytes.

Akita dogs possess GLUT1 in erythrocytes, and Na,K-ATPase activity enables more efficient ascorbic acid recycling.

We investigated hematologic characteristics of healthy Akita dogs. All were found to contain glucose transporters, GLUT1 and GLUT4, in erythrocyte membrane, whereas Beagle and any other Western dogs have only GLUT4. Of 47 Akitas, ten showed high K and low Na concentrations with elevated glutathione (GSH) in erythrocytes due to Na,K-ATPase activity in the membrane (HK). Akitas showed increased capacity for recycling vitamin C or ascorbic acid (AA) from oxidized ascorbic acid (DHA) compared to Beagle dogs. Particularly, HK Akitas performed even greater AA recycling and ferricyanide reduction than normal Akitas which have normal GSH, low K and high Na concentrations (LK). All HK Akitas also had stomatin in erythrocyte membrane, while half of LK Akitas had it at lower levels than HK Akitas. Stomatin did not have any influence on AA recycling. GLUT1, Na,K-ATPase and stomatin in erythrocytes are characteristics of Akita dogs, and the high prevalence of these proteins suggests their positive roles in biological efficiency.

Inheritance of erythrocyte GLUT1 and age-dependent change in Japanese Shiba dogs

Inheritance of erythrocyte GLUT1 and age-dependent change in Japanese Shiba dogs

Inheritance of erythrocyte GLUT1 and age-dependent change in Japanese Shiba dogs

Three families of Shiba dogs were investigated for GLUT1 erythrocyte inheritance. Of 22 adult dogs, 14 contained both GLUT1 and GLUT4 (GLUT1 type) while others had only GLUT4 (normal). Erythrocytes with GLUT1 showed greater capacity for ascorbic acid (AA) recycling than those with only GLUT4. Puppies at 1 month of age presented uniformly high levels of GLUT1 and AA recycling capacity. Eight puppies were examined for postnatal changes until differences between types were apparent. Two puppies completely lost GLUT1 after 6 months of age and had only GLUT4, indicating normal type. Six others including an offspring of a normal pair showed persistent GLUT1, and were eventually GLUT1 type. GLUT4 was invariably present in all dogs. Hence, GLUT1 in erythrocytes of Shiba dogs is inherited in an autosomal recessive mode. Expression and function of GLUT1 are not associated with Na,K-ATPase or stomatin in Shiba dog erythrocytes.

More on vitamin C deficiency in mice and humans

I read with great interest the article by Park etal.,1 who found that a vitamin C deficiency ameliorated liver fibrosis via the up-regulated expression of peroxisome proliferator-activated receptor gamma in senescence marker protein 30 (SMP30) knockout mice. The results are interesting and shed light on the possible mechanisms underlying the attenuated liver fibrosis of SMP30 knockout mice. Here I propose that if these results in SMP30 knockout mice are to be extended to humans, we will need more comprehensive studies based on the new and significant finding that humans employ a unique strategy for adapting to vitamin C deficiency. As we know, humans and some other mammals such as guinea pigs and fruit bats have an inborn metabolic error, that is, an inability to synthesize vitamin C from glucose due to a defective form of the gene for the L-gulonolactone oxidase enzyme required in the last step of vitamin C synthesis.2 Montel-Hagen etal.3 recently demonstrated that human erythrocytes from the vitamin C auxotrophs can dramatically enhance their vitamin C–transporting ability by switching the preference of glucose transporter 1 (Glut1) from glucose to the oxidized form of ascorbic acid [L-dehydroascorbic acid (DHA)]. The membrane protein stomatin has been shown to regulate the substrate preference switch. More interestingly, erythrocyte Glut1 and its associated DHA uptake are specific to species unable to produce vitamin C from glucose. Mice can synthesize vitamin C, and further experimentation has indicated that their mature erythrocytes do not harbor Glut1 or transport DHA. Therefore, in comparison with mice, humans possess a compensatory mechanism to adapt to a vitamin C deficiency, and this may lead us to reevaluate the human vitamin C deficiency status and potentially result in more complicated reflections on vitamin C deficiency in human liver tissues. I do not mean to question the rationality and significance of the findings of Park etal.,1 but Isubmit that this unique compensatory mechanism should be considered in future studies on the relationships between vitamin C deficiency and liver fibrosis in humans. Liang Shen Ph.D.*, * Shandong Provincial Research Center for Bioinformatic Engineering and Technique, Shandong University of Technology, Zibo, People's Republic of China.

Japanese Shiba dogs possessing erythrocytes with high Glut-1 activity and high ascorbic acid recycling capacity

Glut-1 was discovered in erythrocytes of some Japanese Shiba dogs. They have Glut-1 as well as Glut-4 in erythrocytes, while other Shiba dogs and other breeds have only Glut-4. Dog erythrocytes with Glut-1 showed much higher affinity for entry of glucose and oxidized ascorbic acid and greater capacity for ascorbic acid (AA) recycling than those with only Glut-4. Dog is among numerous AA-synthesizing species that normally do not have Glut-1 in erythrocytes. However, this variant of dog is the first example of an AA-synthesizing animal which maintains Glut-1 in erythrocytes throughout life. Shiba dogs can be a new animal model for further understanding the mechanism of regulation and functions of AA in the organism.

Relationship between erythrocyte GLUT1 function and membrane glycation in type 2 diabetes

This paper investigates the effect of glycation on glucose transport in erythrocytes. Glucose transporter function, numbers and erythrocyte phosphorylation rates are simultaneously studied using 30 Caucasian patients with diabetes and 30 Caucasian control volunteers (mean±SD where P≤0.05; age 48±8 vs. 45±8 years [ns]; body mass index [BMI] 31±7 vs. 27±5 [P=0.035]; blood glucose 12±7 vs. 5±0.6 mmol/L [P=0.001]; HbA1c 8±2 vs. 5±0.3% [P=0.0001]; fructosamine 336±64 vs. 237±16 µmol/L [P=0.0001]; disease duration 13±11 years, respectively). Significant differences were found for glucose transporter function, with 3-O-methylglucose uptake rates (108±49 vs. 146±55 µmol/L/sec/1012 cells [P=0.010]); D-glucose influx (64±30 vs. 117±45 µmol/L/sec/1012 cells [P=0.0001]); and D-glucose net transport (31±22 vs. 74±55 µmol/L/sec/ 1012 cells [P = 0.0001]). No differences were found for phosphorylation rates using 2-deoxyglucose (33±17 vs. 38±12 µmol/L/sec/1012 cells [P=0.194]). The number of functional transporters using cytochalasin B studies measured via Bmax, was not found to be significantly different between the groups (195±139 vs. 264±174 [P=0.206]). However, Kd was lower for those with diabetes, suggesting higher binding affinity (12±11 vs. 32±25 nmol/L [P=0.006]). A negative correlation between HbA1c and D-glucose influx involving both groups was found (r=–0.670, P=0.0001). Glucose transport is shown to be decreased in people who have diabetes compared to normoglycaemic volunteers, whereas the number of glucose transporters is apparently unchanged; however, affinity for binding is increased.

The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis

AbstractGlucose is a major source of energy for living organisms, and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with more than 200 000 molecules per cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C–deficient mammalian species, comprising only higher primates, guinea pigs, and fruit bats. Here, we show that in all other tested mammalian species, Glut1 was transiently expressed in erythrocytes during the neonatal period. Glut1 was up-regulated during the erythroblast stage of erythroid differentiation and was present on the vast majority of murine red blood cells (RBCs) at birth. Notably though, Glut1 was not induced in adult mice undergoing anemia-induced erythropoiesis, and under these conditions, the up-regulation of a distinct transporter, Glut4, was responsible for an increased glucose transport. Sp3 and Sp1 transcriptions factors have been proposed to regulate Glut1 transcription, and we find that the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented Sp3/Sp1 ratio. Glucose transporter expression patterns in mice and human erythrocytes are therefore distinct. In mice, there is a postnatal switch from Glut1 to Glut4, with Glut4 further up-regulated under anemic conditions.

Analysis of Glucose Transporter Topology and Structural Dynamics

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.

Differential Glucose Transporter Expression during Perinatal and Postnatal Erythropoiesis

Glucose is a major source of energy for living organisms and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with >200,000 molecules/cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C-deficient mammalian species, comprising only higher primates, guinea pigs and fruit bats (Montel-Hagen et al., Cell, 2008). We now show that in all other tested mammals, including mice, rats, dogs and cows, Glut1 is in fact transiently expressed in erythrocytes during the neonatal period. This is in marked contrast with humans, where Glut1 is present at equivalently high levels on both neonatal and adult RBC. In mice, we found that Glut1 expression was not associated with primitive erythropoiesis but was highly expressed during definitive fetal erythropoiesis. Indeed, this transporter was present at significantly earlier stages of erythropoiesis in fetal spleen and liver than immediately following birth. It was therefore important to determine whether erythrocyte Glut1 expression in mice is specifically associated with fetal erythropoiesis or alternatively, is common to any physiological state where an extensive erythropoiesis is provoked. Induction of a hemolytic anemia in adult mice resulted in a massive erythropoiesis with significant increase in glucose uptake but notably, Glut1 was not detected. Rather, in these conditions as well as following birth, Glut4, an insulin-sensitive transporter previously thought to be responsible for glucose uptake in muscle and adipose tissue, was highly expressed. Following birth, the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented ratio of the Sp3 to Sp1 zinc-finger transcription factors. Thus, in contrast to humans, murine Glut1 is highly expressed during definitive erythropoiesis and is then downregulated at birth. Further erythroid development is characterized by the upregulation of a distinct glucose transporter, Glut4. Expression of distinct glucose transporters in nonhuman erythrocytes, regulated at the transcriptional level, therefore characterizes different states of erythroid development and differentiation.

Erythrocyte Glut1 Triggers Dehydroascorbic Acid Uptake in Mammals Unable to Synthesize Vitamin C

Of all cells, human erythrocytes express the highest level of the Glut1 glucose transporter. However, the regulation and function of Glut1 during erythropoiesis are not known. Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut1 transcripts. In contrast, Glut1-mediated transport of L-dehydroascorbic acid (DHA), an oxidized form of ascorbic acid (AA), is dramatically enhanced. We identified stomatin, an integral erythrocyte membrane protein, as regulating the switch from glucose to DHA transport. Notably though, we found that erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose. Accordingly, we show that mice, a species capable of synthesizing AA, express Glut4 but not Glut1 in mature erythrocytes. Thus, erythrocyte-specific coexpression of Glut1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.

Induction of GLUT1 during Erythropoiesis Triggers a Switch from Glucose to DHA Uptake in Vitamin C-Defective Mammals.

Glucose provides a key supply of energy and carbon and its transport is achieved via multimembrane-spanning glucose transporters (GLUTs). The human erythrocyte is the cell type expressing the highest level of the GLUT1 glucose transporter, harboring greater than 200,000 molecules per cell. We now demonstrate that GLUT1 transcripts increase by 3-logs during erythropoiesis and high GLUT1 surface expression is observed following passage through the basophilic erythroblast stage. Paradoxically though, glucose transport significantly decreases. As GLUT1 also transports L-dehydroascorbic acid (DHA), the oxidized form of ascorbic acid (AA), transport of this molecule was assessed and indeed, it increases dramatically during erythropoiesis. The switch from glucose to DHA transport is coupled to the physical association of GLUT1 with stomatin, an integral erythrocyte membrane protein. We find that stomatin inversely regulates the relative transports of glucose and DHA by GLUT1. Moreover, in a patient with overhydrated hereditary stomatocytosis, a rare genetic disorder of red cell membrane permeability wherein stomatin is absent, glucose uptake is significantly higher but there is a concomitant 50% reduction in DHA transport. Intriguingly, erythrocyte-specific DHA transport is not conserved amongst all mammalian species; we did not detect GLUT1 on mature murine erythrocytes where DHA uptake is minimal. Notably though, humans differ from the vast majority of the greater than 5,000 mammalian species in that they are unable to synthesize ascorbic acid from glucose. This trait is shared only with other higher primates, guinea pigs and and fruit bats. We have determined that erythrocyte GLUT1 expression and associated DHA transport are specific features of these diverse ascorbic acid-deficient mammals. Within the primate order, defective ascorbic acid synthesis is due to an inactivation of the L-gulonolactone oxidase (GLO) enzyme at the Haplorrhini-Strespsirrhini split. In accord with the data presented above, we find that erythrocyte GLUT1 expression and associated DHA transport are characteristic of erythrocytes from primates within the Haplorrhini suborder but not Strepsirrhini lemurs. Thus, the erythrocyte-specific co-expression of GLUT1 and stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize the essential ascorbic acid metabolite.

Ultrastructure of Human Erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.

The effects of phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin on cellular glucose transport

Glucose is the principal fuel for brain metabolism and its movement across the blood-brain barrier depends on Glut1. Impaired glucose transport to the brain may have deleterious consequences. For example, Glut1 deficiency syndrome (Glut1DS) is the result of heterozygous loss of function Glut1 mutation leading to energy failure of the brain and subsequently, epileptic encephalopathy. To preserve the integrity of the energy supply to the brain in patients with compromised glucose transport function, consumption of compounds with glucose transport inhibiting properties should be avoided. Phenytoin is a widely used anticonvulsant that affects carbohydrate metabolism. In this study, the hypothesis that phenytoin and its metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) affect cellular glucose transport was tested. With a focus on Glut1, the effects of phenytoin and HPPH on cellular glucose transport were studied. Glucose uptake assay measuring the zero-trans influx of radioactive-labeled glucose analogues showed that phenytoin and HPPH did not exert immediate effects on erythrocyte Glut1 activity or glucose transport in Hs68 control fibroblasts, Glut1DS primary fibroblasts isolated from two patients, or in rat primary astrocytes. Prolonged exposure to the two compounds could stimulate glucose transport by up to 30–60% over the control level (p < 0.05) in Hs68 and Glut1DS fibroblasts as well as in rat astrocytes. The stimulation of glucose transport by HPPH was dose-dependent and accompanied by an up-regulation of GLUT1 mRNA expression (p < 0.05). In conclusion, phenytoin and HPPH do not compromise cellular glucose transport. Prolonged exposure to these compounds can modify carbohydrate homeostasis by up-regulating glucose transport in both normal and Glut1DS conditions in vitro.

Membranous expression of glucose transporter‐1 protein (GLUT‐1) in embryonal neoplasms of the central nervous system

The human erythrocyte GLUT-1 is a transmembrane protein which facilitates transport of glucose in the cell in an energy-independent fashion. Neuroectodermal stem cells show strong membrane immunoreactivitry with this marker at early developmental stages in rodents. Membranous expression by undifferentiated neuroectodermal cells gradually decreases while GLUT-1 becomes confined to the endothelial cells, when these acquire blood-brain barrier function. We thus sought to determine whether GLUT-1 expression was limited to embryonal neoplasms of the central nervous system (CNS) which are presumably derived from developmentally arrested neuroectodermal stem cells. Archival material of 40 primary CNS neoplasms were examined for immunoreactivity with anti-GLUT-1. This included both non-embryonal neoplasms (18 astrocytic tumours, one ependymoma and three oligodendroglioma) and embryonal neoplasms (12 cerebellar medulloblastomas, four supratentorial PNETs and two atypical teratoid/rhabdoid tumours (AT/RhT)). In addition, cell lines and nude mice xenografts derived from both undifferentiated and differentiated tumours were assessed for GLUT-1 immunoreactivity by both immunohistochemistry and Western blotting. All embryonal tumours, MBs and PNET xenografts consistently showed GLUT-1 membrane staining. Non-embryonal neoplasms were negative except for vascular staining. Membrane protein fraction of embryonal tumours cell lines immunoreacted by immunoblot with GLUT-1, whereas the glioblastoma cell line was negative. Expression of GLUT-1 supports the stem cell nature of the cells of origin of MBs, supratentorial PNET and AT/RhTs. As a result, GLUT-1 is a useful marker to define the embryonal nature of CNS neoplasms.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.Key words: anion exchanger, band 3, erythrocyte, glycoprotein, HEMPAS.

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport.

Net sugar import by human erythrocytes consists of ATP-modulated rapid and slow phases while sugar export consists of a single slow phase. We have proposed that this behaviour results from obligate substrate tunnelling from transporter to bulk cytosol through a complex containing high-affinity, low-capacity sugar binding sites (Cloherty, Sultzman, Zottola & Carruthers, 1995). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to compartmentalize ATP delivery to erythrocyte membrane ATPases and interact directly with the erythrocyte glucose transporter in vitro. The present study examines the possibility that GAPDH is an obligate component of the hypothesized sugar-binding complex. GAPDH remains associated with the erythrocyte membrane following cell lysis and remains associated with the cytoskeleton under conditions where more than 99% of the membrane glucose transport protein (GLUT1) is released by detergent (Triton X-100). GAPDH is released from erythrocyte membranes upon exposure to Mg.ATP or to NADH. ATP displacement of membrane-bound GAPDH is half-maximal at 200 microM ATP and appears to involve ATP interaction with multiple, co-operative sites. GAPDH interaction with purified tetrameric GLUT1 is saturable, co-operative and also inhibited by ATP. ATP inhibition of GAPDH binding to purified tetrameric GLUT1 is less effective than ATP inhibition of GAPDH binding to intact erythrocyte membranes. Removal of cellular GAPDH by exposing erythrocyte membranes to NADH prior to membrane resealing neither affects ATP modulation of sugar transport nor reduces biphasic net sugar uptake to a single phase. We conclude that ATP-sensitive GAPDH interaction with the cytoplasmic surface of erythrocyte membranes and GLUT1 is responsible neither for ATP modulation of sugar transport nor for multiphasic net sugar import by human red cells.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins.

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.

Synthesis of O-methylsulfonyl derivatives of d-glucose as potential alkylating agents for targeted drug delivery to the brain. Evaluation of their interaction with the human erythrocyte GLUT1 hexose transporter

In order to obtain hydrophilic analogues of 1,4-dimethylsulfonyloxybutane (busulfan) with enhanced selectivity and improved brain penetration, we have synthesized 6-O-methylsulfonyl- d-glucose, 3-O-methylsulfonyl- d-glucose, 3,6-di-O-methylsulfonyl- d-glucose, 4-O-methylsulfonyl- d-glucose, and 4,6-di-O-methylsulfonyl- d-glucose, and we have studied their interactions with the human erythrocyte GLUT1 hexose transport system. Mesylation of OH-4 and OH-6 of glucose resulted in a slightly diminished affinity for the GLUT1 glucose transporter, whereas mesylation of OH-3 led to complete loss of affinity.