Man Erythrocyte Membrane Band 3 Protein As The Core Of The Protein Complex

The erythrocyte anion transporter, Band 3, is the most abundant protein of the erythrocyte membrane, comprising approximately 25% of the total membrane protein. The gene that encodes Band 3 (AEl) is located on chromosome 17q21-q22, and it is primarily expressed in the erythrocytes and kidney.Band 3 consists of two structurally and functionally independent domains. The C-terminal transmembrane domain (transmembrane domain of Band 3, tdb3), about 50 kDa, mediates the exchange of Cl- for HCO3- across the plasma membrane. The N-terminal cytoplasmic domain (cytoplasmic domain of erythrocyte Band 3, cdb3), about 43 kDa, serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins:1. associated with ankyrin, protein 4.1 and protein 4.2, linking cytoskeleton with cell membrane, helping to maintain the mechanical properties and integrity of the erythrocytes;2. associated with integral erythrocyte membranes, including Rh complex, glycophorin complex, glucose transporter 1, etc, and recent research found that Band 3 was also in erythrocyte lipid rafts;3. associated with glycolytic enzymes, including aldolase, glyceraldehyde-3-phosphate dehydrogenase and 6-phosphofructokinase, also regulating their activity;4. associated with reduced form hemoglobin, perhaps this interaction regulated the NO level of erythrocyte, also interact with hemichrome to form large Band 3 oligermers complex, served as autosome antigen;5. associated with carbon anhydrase II, functional as an integrated CO2/O2 gas exchange unit in the erythrocyte.Band 3 mutations can result in significant changes in the shape and deformablility of the erythrocytes. Southeast Asian ovalocytosis (SAO) with abnormal rigid, stomatocytic erythrocytes results from the heterozygous presence of a deletion in Band 3 protein. Some Band 3 mutations in the erythrocytes, especially in cdb3, destabilize erythrocyte membranes, leading to hereditary spherocytosis (HS). A truncated form of Band 3 is expressed in kidney cells and certain Band 3 mutations are associated with distal renal tubular acidosis (dRTA).Biomembranes were formed mainly by 3 kinds of lipids: glycerophospholipids,sphingolipid and cholesterol. Though sphingolipid and cholesterol formed lipid rafts microdomain of biomembrane, they were not well characterized. The proteins in lipid rafts were always correlative with signal transduction, and in lipid rafts there were 3 kinds of specific intrinsic proteins: caveolin, flotillin and stomatin. Flotillin was found important in the second signalling pathway required for insulin-stimulated glucose transport which was independent of PI(3)K. Our previous research also discovered that flotillin-1 in erythrocyte appeared to be upregulated in type 2 diabetes. Band 3 was the predominant protein of erythrocyte membrane, and flotillin was the most important protein of erythrocyte lipid rafts, were there any interactions between them?We first tried to setup the feasible in vitro research system to study the Band 3-based macrocomplex. We tried to express full length recombinant Band 3 in E. coli, but the expression level of tdb3 was poor, since tdb3's main function was transport anions, and this domain rarely interact with other proteins, so we tried to cloning and expressing cdb3 instead. We sucessfully got recombiant cdb3 with 5 different tags: (N)His6-cdb3, (C)His6-cdb3, CBD-cdb3, GST-cdb3 and Trx-cdb3. After optimizing the expression condition, the recombinant proteins were purified by anion exchanger column, affinity column and size exclusive column in series.We verified the purification procedure by SDS-PAGE and Western blot, and then measured the molecular weight of the recombinant proteins by mass spectrometry, they all accord with the theoretical calculated value. The intrinsic fluorescence of recombinant cdb3 of different pH was same as cdb3 purified from erythrocyte membrane, and when they were denatured, the pH dependent intrinsic fluorescence was eliminated. By pull-down assay, we found that recombinant cdb3's ability to interact with protein 4.2 C3 fragment was similar to natural cdb3. We also performed pull-down assay using recombinant cdb3 and recombinant ankyrin D34 fragment, found that their interaction was regulated by pH but not ion concentration, confirmed Michaely et al. 's assumption by experiment.Then we get the 2D-PAGE pattern of the erythrocyte membrane proteins that can react with recombinant cdb3 by pull-down assay and 2D electrophoresis. We tried to maximally solubilize the erythrocyte membrane proteins with non-ionic detergents, and finally chose the detergent mixture of TX100, OG and SB310 to treat the erythrocyte membrane. We performed the pull-down system of His6-cdb3 in Ni-NTA resin and GST-cdb3 in Glutathione resin, then separated the proteins interact with cdb3 by 2D electrophoresis, after silver stain, the 2D-page pattern was compared with...