Different Involvement of Band 3 in Red Cell Deformability and Osmotic Fragility—A Comparative GP.Mur Erythrocyte Study

GP.Mur is a clinically important red blood cell (RBC) phenotype in Southeast Asia. The molecular entity of GP.Mur is glycophorin B-A-B hybrid protein that promotes band 3 expression and band 3–AQP1 interaction, and alters the organization of band 3 complexes with Rh/RhAG complexes. GP.Mur+ RBCs are more resistant to osmotic stress. To explore whether GP.Mur+ RBCs could be structurally more resilient, we compared deformability and osmotic fragility of fresh RBCs from 145 adults without major illness (47% GP.Mur). We also evaluated potential impacts of cellular and lipid factors on RBC deformability and osmotic resistivity. Contrary to our anticipation, these two physical properties were independent from each other based on multivariate regression analyses. GP.Mur+ RBCs were less deformable than non-GP.Mur RBCs. We also unexpectedly found 25% microcytosis in GP.Mur+ female subjects (10/40). Both microcytosis and membrane cholesterol reduced deformability, but the latter was only observed in non-GP.Mur and not GP.Mur+ normocytes. The osmotic fragility of erythrocytes was not affected by microcytosis; instead, larger mean corpuscular volume (MCV) increased the chances of hypotonic burst. From comparison with GP.Mur+ RBCs, higher band 3 expression strengthened the structure of RBC membrane and submembranous cytoskeletal networks and thereby reduced cell deformability; stronger band 3–AQP1 interaction additionally supported osmotic resistance. Thus, red cell deformability and osmotic resistivity involve distinct structural–functional roles of band 3.

Comodulation of NO-Dependent Vasodilation by Erythroid Band 3 and Hemoglobin: A GP.Mur Athlete Study

GP.Mur, a red blood cell (RBC) hybrid protein encoded by glycophorin B-A-B, increases expression of erythroid band 3 (Anion Exchanger-1, SLC4A1). GP.Mur is extremely rare but has a prevalence of 1–10% in regions of Southeast Asia. We unexpectedly found slightly higher blood pressure (BP) among healthy Taiwanese adults with GP.Mur. Since band 3 has been suggested to interact with hemoglobin (Hb) to modulate nitric oxide (NO)-dependent hypoxic vasodilation during the respiratory cycle, we hypothesized that GP.Mur red cells could exert differentiable effects on vascular tone. Here we recruited GP.Mur-positive and GP.Mur-negative elite male college athletes, as well as age-matched, GP.Mur-negative non-athletes, for NO-dependent flow-mediated dilation (FMD) and NO-independent dilation (NID). The subjects were also tested for plasma nitrite and nitrate before and after arterial occlusion in FMD. GP.Mur+ and non-GP.Mur athletes exhibited similar heart rates and blood pressure, but GP.Mur+ athletes showed significantly lower FMD (4.8 ± 2.4%) than non-GP.Mur athletes (6.5 ± 2.1%). NO-independent vasodilation was not affected by GP.Mur. As Hb controls intravascular NO bioavailability, we examined the effect of Hb on limiting FMD and found it to be significantly stronger in GP.Mur+ subjects. Biochemically, plasma nitrite levels were directly proportional to individual band 3 expression on the red cell membrane. The increase of plasma nitrite triggered by arterial occlusion also showed small dependency on band 3 levels in non-GP.Mur subjects. By the GP.Mur comparative study, we unveiled comodulation of NO-dependent vasodilation by band 3 and Hb, and verified the long-pending role of erythroid band 3 in this process.

Schleswig-Holsteinische Bibliographie - Band 3, 1939-1944

Die Schleswig-Holsteinische Bibliographie ist eine Literaturdokumentation. Sie verzeichnet Bücher, Karten, Zeitschriften, Aufsätze aus Zeitschriften und (in Ausnahmefällen) aus Tageszeitungen sowie Einzelbeiträge aus Sammelwerken, die einen Bezug zu Schleswig-Holstein, einzelnen Regionen, Inseln oder Orten des Landes oder zu schleswig-holsteinischen Persönlichkeiten haben. Dabei sind ehemals schleswig-holsteinische Gebiete einbezogen, soweit die erfasste Literatur die Zeit der Zugehörigkeit zu Schleswig-Holstein oder grenzüberschreitende Fragen betrifft. Der inhaltliche Bezug wird weit ausgelegt und schließt alle Wissensgebiete und Lebensbereiche ein. Zwischen 1930 und 2006, für die Berichtsjahre 1928 bis 1998, sind 17 gedruckte Bände der Bibliographie erschienen. Nachdem im Jahre 2000 die bibliographischen Aufnahmen ab dem Berichtsjahr 1987 (Bd. 13) in eine Datenbank überführt worden waren und auch die weitere Erfassung der Titel dort stattfindet (https://bibliographie.schleswig-holstein.de), wurde die Druckausgabe eingestellt.

Mutations in the Hemoglobin Binding Domain of Band 3 Perturb Cellular Metabolism and Alter the Sickling of SS Erythrocytes

Under oxygenated conditions, 4 glycolytic enzymes that perform the terminal steps of glycolysis (phospho-fructoKinase [PFK], lactate dehydrogenase [LDH], aldolase [ALD] and glygeraldehyde 3 phosphate dehydrogenase [GAPDH]) bind to the cytoplasmic domain of band 3. Under deoxy conditions deoxy hemoglobin (Hb) is bound to band 3 and PFK, LDH, ALD and GAPDH are displaced (Campanella et al. PNAS 102, 2005; Blood 112, 2008). We generated transgenic mice in which the sequence encoding the first 35 amino acids of the wild type human band 3 cytoplasmic domain replaced the endogenous mouse band 3 sequences in the Slc4a1 gene, a mutant line in which human amino acids 12-21 were deleted removing the deoxy Hb binding site (-Hb) and a third line in which amino acids 1-11 were deleted creating a high affinity binding site for deoxyHb (++Hb). Erythrocytes from the mutant lines were insensitive to Oxygen concentration resulting in changes in oxygen dependent deformability and other physical properties compared to the wild type line (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019). We crossed our humanized band 3 mouse strains to the Townes Sickle Cell Disease (SCD) mouse model, maintaining both the human βA and βS alleles to generate human AA, AS and SS mice homozygous for each of the human band 3 cytoplasmic domain sequences. Using an assay in which SS red cells in phosphate buffer are deoxygenated to 6% oxygen over time (Dunkelberger et al., J. Phys. Chem. B 122, 2018), we observed that -Hb band 3/SS mice showed an accelerated rate of sickle cell formation and a higher percent of sickled cells than wild type band 3/SS mice (p<0.01). Conversely, ++Hb band 3/SS mice showed an inhibition of both the rate of sickling and the precent of sickled cells compared to wild type band 3/SS mice (p<0.05). We hypothesized that the inability of the glycolytic enzymes to reversibly bind to band 3 in the mutant mice were responsible for the differences in sickling. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in 12 mice (6 female, 6 male) of each genotype: wild type band 3/AA, -AS and -SS, -Hb band 3/AA, -AS, -SS and ++Hb/AA, -AS, -SS. The metabolites were quantified by LC-MS/MS using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with chromatographic resolution enabled on a polymeric amino column (apHera by Supelco) under alkaline mobile phase conditions (pH ~9.3). Stable isotope dilution and 8pt calibration curves allowed the absolute quantification of each metabolite. Consistent with the constitutive binding of the terminal glycolytic enzymes to band 3 in -Hb erythrocytes, glycolysis was inhibited after the phosphoenol pyruvate step, as evidenced by significant accumulation of the intermediates at top of the glycolysis pathway, including fructose 1,6 biphosphate (FBP; p<0.01), dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate (G3P; p<0.01), and 3-phosphoglycerate/2-phosphoglycerate (PG; p<0.01). In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from band 3, significantly lower levels of FBP, G3P and PG were observed (p<0.01). The levels of these metabolites in wild type band 3/SS erythrocytes were intermediate between the two mutant strains. We hypothesized that the accumulation of FBP, G3P and PG contributed to the increased rate of sickling in the -Hb band 3/SS mice. To test this, we incubated wild type band 3/SS cells with either FBP or PG. Both intermediates increased the rate of sickle cell formation and percentage of sickled cells in a dose dependent fashion with no alteration in any RBC indices including MCV and osmotic fragility. We next hypothesized that reduction of the levels of glycolytic intermediates would have an antisickling effect. To test this, we incubated wild type band 3/SS cells with 2,3 diphosphoglycerol (DPG), which is a potent inhibitor of glycolysis. We found that DPG treatment led to a dose dependent decrease in the rate of sickle cell formation and percentage of sickled cells, again with no alteration in any RBC indices including MCV and osmotic fragility. We conclude that the accumulation of glycolytic intermediates leads to increased sickle cell formation. We propose that reduction in the levels of glycolytic intermediates either by accelerating the terminal stages of glycolysis or by redirection to the pentose phosphate pathway may offer a means to treat SCD. Disclosures No relevant conflicts of interest to declare.

Cell Physiology and Molecular Mechanism of Anion Transport by Erythrocyte Band 3/AE1

The major transmembrane protein of the red blood cell, known as band 3, AE1, and SLC4A1, has two main functions: 1) catalysis of Cl-/HCO3- exchange, one of the steps in CO2 excretion; 2) anchoring the membrane skeleton. This review summarizes the 150 year history of research on red cell anion transport and band 3 as an experimental system for studying membrane protein structure and ion transport mechanisms. Important early findings were that red cell Cl- transport is a tightly coupled 1:1 exchange and band 3 is labeled by stilbenesulfonate derivatives that inhibit anion transport. Biochemical studies showed that the protein is dimeric or tetrameric (paired dimers) and that there is one stilbenedisulfonate binding site per subunit of the dimer. Transport kinetics and inhibitor characteristics supported the idea that the transporter acts by an alternating access mechanism with intrinsic asymmetry. The sequence of band 3 cDNA provided a framework for detailed study of protein topology and amino acid residues important for transport. The identification of genetic variants produced insights into the roles of band 3 in red cell abnormalities and distal renal tubular acidosis. The publication of the membrane domain crystal structure made it possible to propose concrete molecular models of transport. Future research directions include improving our understanding of the transport mechanism at the molecular level and of the integrative relationships among band 3, hemoglobin, carbonic anhydrase, and gradients (both transmembrane and subcellular) of HCO3-, Cl-, O2, CO2, pH, and NO metabolites during pulmonary and systemic capillary gas exchange.