The human erythrocyte nucleoside and glucose transporters are both band 4.5 membrane polypeptides.
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ABSTRACT:
SUBMITTER: Wu JS
PROVIDER: S-EPMC1152343 | biostudies-other | 1983 Sep
REPOSITORIES: biostudies-other
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Project description:Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.
| S-EPMC1148059 | biostudies-other
Project description:Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ?0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.
| S-EPMC3914982 | biostudies-literature
Project description:The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.
| S-EPMC3448790 | biostudies-literature
Project description:The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.
| S-EPMC3790964 | biostudies-literature
Project description:The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.
| S-EPMC3112043 | biostudies-literature
Project description:The altered metabolism observed in cancer cells generally consists in increased glucose uptake and glycolytic activity. This is associated with an overexpression of glucose transporter proteins (GLUTs), which facilitate glucose uptake across the plasma membrane and play a crucial role in the survival of cancer cells. Therefore GLUTs are considered as suitable targets for the treatment of cancer. Herein we review some of the most relevant GLUT inhibitors that have been recently developed as prospective anticancer agents.
| S-EPMC5198910 | biostudies-literature
Project description:The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by tyrosine kinase. To study the regulation of band 3 phosphorylation, we examined phosphotyrisine phosphatase (PTP) activity in the human erythrocyte. We show that the human erythrocyte membrane contains a band 3-associated neutral PTP which is activated by Mg2+ and inhibited by Mn2+ and vanadate. The PTP is active in the intact cell and in the isolated membrane. A major fraction of the PTP is tightly bound to the membrane and can be extracted from it by Triton X-100; a minor part is associated with Triton X-100-insoluble cytoskeleton. The behaviour of the PTP parallels that of band 3, the major fraction of which is extractable by detergents with a minor fraction being anchored to the cytoskeleton. Moreover, band 3 is co-precipitated when the PTP is immunoprecipitated from solubilized membranes, and PTP is co-precipitated when band 3 is immunoprecipitated. The PTP appears to be related to PTP1B (identified using an antibody to an epitope in its catalytic domain and by molecular mass). The system described here has a unique advantage for PTP research, since it allows the study of the interaction of a PTP with an endogenous physiological substrate that is present in substantial amounts in the cell membrane. The membrane-bound, band 3-associated, PTP may play a role in band 3 function in the erythrocyte and in other cells which have proteins analogous to band 3.
| S-EPMC1217139 | biostudies-other
Project description:Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 μM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 μM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.
| S-EPMC7318791 | biostudies-literature
Project description:The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector lambda gt11. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2(+)-dependent cross-linking proteins, guinea pig liver transglutaminase (protein-glutamine gamma-glutamyltransferase; protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular gamma-glutamyl-epsilon-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit of factor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly, within the five contiguous consensus residues of the transglutaminase active site, Gly-Gln-Cys-Trp-Val, band 4.2 has an alanine substituted for cysteine (which is apparently essential for activity). Consistent with this active site substitution, erythrocyte membranes or inside-out vesicles, which contain band 4.2, show no evidence of transglutaminase activity by two types of in vitro assay.
| S-EPMC53315 | biostudies-other
Project description:Evidence accumulated over the years suggests that human erythrocyte membrane protein 4.2 is one of the proteins involved in strengthening the cytoskeleton-membrane interactions in the red blood cell. Deficiency of protein 4.2 is linked with a variety of hereditary haemolytic anaemia. However, the interactions of protein 4.2 with other proteins of the erythrocyte membrane remain poorly understood. The major membrane-binding site for protein 4.2 resides on the cytoplasmic domain of band 3 (CDB3). In order to carry out an initial characterization of its interaction with the CDB3, protein 4. 2 was subjected to proteolytic cleavage and gel renaturation assay, and the 23-kDa N-terminal domain was found to interact with band 3. This domain contained two putative palmitoylatable cysteine residues, of which cysteine 203 was identified as the palmitoylatable cysteine. Recombinant glutathione S-transferase-fusion peptides derived from this domain were characterized with respect to their ability to interact with the CDB3. Whereas these studies do not rule out the involvement of other subsites on protein 4.2 in interaction with the CDB3, the evidence suggests that the region encompassing amino acid residues 187-211 is one of the domains critical for the protein 4.2-CDB3 interaction. This is also the first demonstration that palmitoylation serves as a positive modulator of this interaction.
| S-EPMC1220278 | biostudies-other