Red Cell Membrane Glucose Transporter Protein Characterization And Its Interacting Proteins

The facilitative glucose transporter 1 (GLUT1) is perhaps the most extensively studied membrane transporter. GLUT1 is the only type glucose transporter present in human erythrocyte. cDNAs encoding the GLUT1 protein have been isolated. All encode proteins of 492 amino acids and all exhibit an extraordinarily high level of amino acid identity (-97%). Using hydropathy analysis, GLUT1 was predicted to consist of 12 transmembrane-spanning α-helices with the N and C termini and a large loop between transmembrane helices 6 and 7 located on the cytoplasmic side of the membrane. The availability of GLUT1 purified in large quantity from human erythrocytes has facilitated investigation of its secondary structure by biophysical techniques. Circular dichroism (CD) spectroscopy of the reconstituted protein in lipid vesicles has shown that it contains approx. 82% α-helix, 10% /3-turns and 8% random coil structure . GLUT1 admits dehydroascorbic acid as a substrate, and also exhibits a modest water conductance, suggesting the possible presence of a pore through the protein. GLUT1 facilitates the passive diffusion of D-glucose across the cell membranes for providing the energy resource in glycolysis.GLUT1 mutations can result in many diseases, such as diabetes, glucose transporter deficiency syndrome (GTDS), infantile haemangioma, liver vascular tumor, stomach cancer and pancreas cancer. So it is very important to find the mechanism of interaction between GLUT1 and other membrane proteins, which will be helpful for clinic diagnosis and therapy.Band 3 is another kind of important proteins in the human erythrocyte membranes. The most important function of Band 3 is to mediate rapid exchange of CO₂ in the tissues with the outside O₂ through Cl^-/HCO₃^- exchange across the membranes. Firstly, we investigated the interaction between Band 3 and GLUT1 on erythrocyte by using bis(sulfosuccinimidyl)suberate (BS³) which is a membrane-impermeable Band 3 cross-linking reagent. The covalent dimer formed by BS³ is between the same two subunits that are rightly associated in a native dimer, i.e. BS³ does not cross-link two subunits of neighbour dimers.The measurement of NO₂^- transport across the erythrocyte membranes shows that BS³ inhibits the rate of anion transport. The ID₅₀ of BS³ for the inhibition of anion transport was 0.63±0.15 mmol/L. We also observed an inhibitory effect of BS³ on the transport rate of glucose across the erythrocyte membranes. For glucose entryof six experiments, ID₅₀= 0.77±0.21 mmol/L. However in the case of glucose exit, the inhibitory effect of BS³ was rather smaller. The relative rate of glucose exit was (84.0 ± 5.6) % after 6 mmol/L BS³ treatment for six blood samples. This means that the outside domain of GLUT1 was affected by BS³ more seriously. The SDS-PAGE patterns shows clearly that the quantity of Band 3 decreased after treatment with 3 mmol/L BS³ and the quantity of the proteins located around the bands of spectrin increased because of overlap with the cross-linked Band 3 dimers. Meanwhile, there was no significant difference between the quantities of GLUT1 which located at Band 4.5 in the SDS-PAGE for the samples of control and BS³ treatment. To consider all these results we can make a proposal that the information of either conformation change can be somehow transferred to the other side and induces the change in its transport function. Thus, the effect of BS³ on the glucose transport would be performed through the linkage between in our experiment. However, the direct structural interaction between them is still unclear.Next, we tried to setup the feasible in vitro research system to study the Band 3 and GLUT1 complex. We succeeded in cloning full length recombinant GLUT1 and some of its domain in E. coli. After optimizing the induction and expression condition, we successfully got recombinant carboxyl-terminal cytoplasmic domain of GLUT1 (GLUTl-Ct), the amino-terminal cytoplasmic domain of GLUT1 (GLUT1-Nt) and a large loop between transmembrane helices 6 and 7 located on the cytoplasmic side of the membrane (GLUT1-loop) with different tags: Trx-GLUT1-Nt, Trx-GLUT1-Ct, GST-GLUT1-Nt, GST-GLUT1-Ct and GST-GLUT1-loop. After optimizing the expression condition, the recombinant proteins were purified by anion exchanger column, affinity column and size exclusive column in series.We used GLUT1-Ct and the amino-terminal cytoplasmic domain of Band 3 (cdb3) to study the structural interaction between Band 3 and GLUT1. Coimmunoprecipitate (Co-IP) and pull-down assay were used to verify their interaction in vitro. Meanwhile in the experiments the interaction between recombinant cdb3 and ankyrin D34 fragment was measured as positive control. The result of Co-IP showed that using anti-GLUTl antibody (N/C ) His₆-cdb3 coimmunoprecipitated with Trx-GLUT1-ct, and using anti-band3 antibody Trx-GLUT1-ct coimmunoprecipitated with (N/C) His₆-cdb3. In the experiments, Trx and (N/C)His₆-cdb3 were used as negative control. By pull-down assay, we found that GST-GLUT1-ct in Glutathione resin can pull down (N/C)His₆-cdb3. Conversely,GST-cdb3 in glutathione resin can pull down Trx-GLUT1-ct, and the interaction between GST and Trx-GLUT1-ct was not observed.These experiments from both functional and structural aspects proved that there is direct interaction between the carboxyl-terminal cytoplasmic domain of GLUT1 and amino-terminal cytoplasmic domain of Band 3. In combination with the previous result, it is deduced that Band 3 and GLUT1 in erythrocyte membranes would be directly interacted, and perhaps they are assembled into a transport complex to facilitate the membrane transport.For the erythrocytes in vivo in the place where the activity of anion transport is higher the bigger ability of cell deformation is needed, so more amount of glucose must entered in to the erythrocytes. This would be a kind of physiological significance for the interaction between Band 3 and GLUT1.