Erythroid anion transport, nitric oxide, and blood pressure.

Glycophorin A and glycophorin B are structural membrane glycoproteins bound in the band 3 multiprotein complexes on human red blood cells (RBCs). Band 3 is an erythroid-specific anion exchanger (AE1). AE1-mediated HCO3 - transport provides the substrate for the enzyme-catalyzed conversion HCO3 - (aq) ⇌ CO2(g), which takes place inside the RBCs. Bicarbonate transport via AE1 supports intravascular acid-base homeostasis and respiratory excretion of CO2. In the past decade, we conducted several comparative physiology studies on Taiwanese people having the glycophorin variant GPMur RBC type (which accompanies greater AE1 expression). We found that increased anion transport across the erythrocyte membrane not only enhances gas exchange and lung functions but also elevates blood pressure (BP) and reduces nitric oxide (NO)-dependent vasodilation and exhaled NO fraction (FeNO) in healthy individuals with GP.Mur. Notably, in people carrying the GPMur blood type, the BP and NO-dependent, flow-mediated vasodilation (FMD) are both more strongly correlated with individual hemoglobin (Hb) levels. As blood NO and nitrite (NO2 -) are predominantly scavenged by intraerythrocytic Hb, and NO2 - primarily enters RBCs via AE1, could a more monoanion-permeable RBC membrane (i.e., GPMur/increased AE1) enhance NO2 -/NO3 - permeability and Hb scavenging of NO2 - and NO to affect blood pressure? In this perspective, a working model is proposed for the potential role of AE1 in intravascular NO availability, blood pressure, and clinical relevance.

Prevalence of GP. Mur variant phenotype among Malaysian blood donors.

BACKGROUND AND OBJECTIVE: A number of glycophorin variant phenotypes or hybrid glycophorin variants of the MNS blood group system bear multiple immunogenic antigens such as Mia, Mur, and MUT. In the East and Southeast Asian populations, glycoprotein (GP.) Mur is the most common glycophorin variant phenotype expressing those three immunogens. The aim of this study was to detect MNS system glycophorin variant phenotypes (GP. Mur, GP. Hop, GP. Bun, GP. HF, and GP. Hut) among Malaysian blood donors. MATERIALS AND METHODS: In this cross-sectional study, 144 blood donors were selected under stratified random sampling. The deoxyribonucleic acid was extracted from whole blood samples, followed by a polymerase chain reaction assay. Sanger sequencing was used to identify the specific MNS variants and then validated by a serological crossmatch with known anti-Mur and anti-MUT. RESULTS: GP. Mur was identified among Malaysian blood donors with a prevalence of 6.94%, and no other variants of the MNS system were found. CONCLUSION: The present study substantiates that GP. Mur is the main variant of the MNS system glycophorin (B-A-B) hybrid in Malaysian blood donors. GP. Mur-negative red blood cells must therefore be considered in the current transfusion policy in order to prevent alloimmunization and immune-mediated transfusion reactions, particularly in transfusion-dependent patients.

A Balance between Transmembrane-Mediated ER/Golgi Retention and Forward Trafficking Signals in Glycophorin-Anion Exchanger-1 Interaction.

Anion exchanger-1 (AE1) is the main erythroid Cl-/HCO3- transporter that supports CO2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants-GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

What Decides Your Athletic Career?-Reflection from Our Study of GP.Mur-Associated Sports Talents during the COVID-19 Pandemic Era.

This opinion article discusses the factors that attract children and teens to athletic careers. The most important attribute for the making of athletes is polished sports talent, followed by psychological, environmental, and incentive factors. Our laboratory studies a red blood cell (RBC) type called GP.Mur, which is rare in most parts of the world besides Southeast Asia. Intriguingly, the prevalence of the GP.Mur blood type is relatively high among Taiwanese elite athletes. The highest frequency of the GP.Mur blood type worldwide is found among Taiwan's Ami people (88-95% from hospital blood bank surveys in the 1980s). Though the Ami constitute only 0.6-0.8% of the Taiwanese population, from records of national track-and-field games in the past century, 10-60% of the medalists were Ami. Biologically, GP.Mur expression supports blood CO2 metabolism, which may have implications for athleticism. As many of our study subjects are elite college athletes with the GP.Mur blood type, we contemplated their upbringings and career dilemmas, especially during the difficult COVID-19 pandemic. Beyond individual sports talent, the pandemic particularly tests personal characteristics and socioeconomic support for becoming an athlete.

Study on the antigens and antibodies of Mur and Mia blood groups in southern China.

BACKGROUND: The frequency of Mur and Mia blood group antigens in Asian population is much higher than that in Caucasian population. However, due to the scarcity and high price of commercial detection reagents, there are few studies on antigen and antibody detection and comparative analysis in large samples. OBJECTIVE: To study the occurrence frequency, antigen correlation and antibody properties of Mur and Mia antigens and their corresponding antibodies in southern China. METHODS: Mur and Mia antigens and antibodies in local blood donors and patients were detected by routine serological microplate method. Statistical methods were used to calculate the incidence of two antigens and antibodies and analyze their correlation. RESULTS: Among blood donors, the positive rates of Mur and Mia antigens were 6.4 % and 6.5 % respectively, with no significant difference (P > 0.05). In this region, the incidence of anti-"Mur" and anti-"Mia" was 0.65 % and 0.45 % respectively. But significant difference existed between blood donors and patients (P < 0.05). Among the anti-"Mur" and anti-"Mia" positive patients, most of the antibodies were IgM or IgM + IgG mixed type and had saline activity. CONCLUSION: Mur and Mia antigens and their corresponding antibodies have a high frequency in the population of southern China. In the routine clinical detection of irregular antibodies, Mur or Mia positive (GP.Mur) should be added to screen erythrocytes. Moreover, given the high correlation between Mur and Mia antigens expression on red blood cells, using monoclonal antibodies against Mia could predict the presence of Mur antigen.

Influence of hemoglobin on blood pressure among people with GP.Mur blood type☆.

BACKGROUND/PURPOSE: GP.Mur is a clinically important red blood cell (RBC) type. GP.Mur and band 3 interact on the RBCs. We previously observed that healthy adults with GP.Mur type present slightly higher blood pressure (BP). Because band 3 and Hb comodulate nitric oxide (NO)-dependent vasodilation and hemoglobin (Hb) is positively associated with BP, we aimed to test whether these could contribute to higher BP in GP.Mur+ people. METHODS: We recruited 989 non-elderly adults (21% GP.Mur) free of catastrophic illness and not on cardiovascular or anti-hypertensive medication. Their body indices, blood lab data and lifestyle data were collected for analyses of potential BP-related factors (BMI, age, smoking, Hb, and GP.Mur). RESULTS: BMI and age remained the most significant contributors to BP. GP.Mur slightly increased systolic BP (SBP). The direct correlation between Hb and BP was only found in Taiwanese non-anemic men, not women. After age and BMI adjusted, we estimated an increase of 1.8 mmHg and 2.6 mmHg of SBP by 1 g/dL Hb among men without and with GP.Mur type, respectively. Hb was generally lower among people expressing GP.Mur, which likely limited their larger impact on BP. CONCLUSION: GP.Mur contributed to BP in both Hb-dependent and Hb-independent fashion. A pronounced impact of hemoglobin on BP likely requires sufficient Hb, as GP.Mur increased the sensitivity of SBP to Hb only in non-anemic Taiwanese men, and not in Taiwanese women or anemic men. The mechanism through which GP.Mur affected BP independent of Hb is unknown.

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.

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.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

BACKGROUND: The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed. STUDY DESIGN AND METHODS: DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast. RESULTS: The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals. CONCLUSION: The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Identification and Quantification of Anti-Gp.Mur Antibodies in Human Serum Using an Insect-Cell-Based System.

Gp.Mur is a clinically relevant antigen of the MNS blood group system that is highly prevalent in several Asian populations. Its corresponding antibody, anti-Gp.Mur, has been implicated in hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Currently, identifying and confirming anti-Gp.Mur antibody presence in sera via agglutination of a panel of red blood cells (RBCs) is inefficient and difficult to quantify. Using a baculovirus expression system to express Gp.Mur antigen on insect cell surfaces, we have developed a quantitative cell-based system to confirm the presence of anti-Gp.Mur antibody in human serum. We obtained 10 serum samples preidentified as having anti-Gp.Mur antibody and another 4 samples containing noncorresponding antibodies from hospital patients. Insect cells displaying Gp.Mur antigen successfully adsorbed anti-Gp.Mur antibody in the sera and inhibited the RBC agglutination mediated by this antibody. By varying the concentration of Gp.Mur-displaying cells, we could grade levels of RBC agglutination by anti-Gp.Mur antibody. Densitometric analysis further enabled quantitative determinations of hemagglutination inhibition by Gp.Mur-displaying cells. We believe that this cell-based hemagglutination inhibition system greatly improves or supplements existing technology and is a convenient means for accurately identifying and quantifying anti-Gp.Mur antibody.

Characterization of GYP*Mur and novel GYP*Bun-like hybrids in Thai blood donors reveals a qualitatively altered s antigen.

BACKGROUND AND OBJECTIVES: The Mi(a+) GP(B-A-B) hybrid phenotypes occur with a prevalence of 2%-23% across Southeast Asia. While the s antigen is alleged to be altered, no evidence for specific variants is known. Screening using a monoclonal IgM anti-s mistyped six S-s+ RBC units as S-s-. Further, alloanti-s was identified in an S+s+ patient. Our objective was to investigate the s antigen further. MATERIALS AND METHODS: DNA from 63 Thai blood donor samples PCR-positive for a GYP(B-A-B) hybrid was sequenced with primers spanning GYPB exons 3-4. Flow cytometry was used for semiquantitative analysis of s expression and correlated with the glycophorin genotype. RESULTS: DNA sequencing showed that GYP*Mur was carried by 56/63 samples (88·9%) of which 5/56 lacked normal GYPB: three of these were GYP*Mur homozygotes, one was a compound heterozygote carrying GYP*Mur and a GYP*Bun-like allele (designated GYP*Thai), and the fifth sample carried GYP*Mur and another GYP*Bun-like allele. Seven samples (7/63) were GYP*Thai heterozygotes. IgM monoclonal anti-s (P3BER) did not react with the s antigen carried by GP.Mur or GP.Bun, whereas two IgG anti-s showed enhanced reactivity. CONCLUSIONS: We confirmed that GYP*Mur is the most frequent variant in Thai blood donors and also identified GYP*Thai with a frequency of 1·1%. We showed that s antigen on Mi(a+) GP(B-A-B) hybrids is qualitatively altered and should be considered when selecting reagents for phenotyping where such hybrids are prevalent, endemically and in blood centres worldwide.

Exploring the Potential Roles of Band 3 and Aquaporin-1 in Blood CO2 Transport-Inspired by Comparative Studies of Glycophorin B-A-B Hybrid Protein GP.Mur.

The Cl-/HCO3- exchanger band 3 is functionally relevant to blood CO2 transport. Band 3 is the most abundant membrane protein in human red blood cells (RBCs). Our understanding of its physiological functions mainly came from clinical cases associated with band 3 mutations. Severe reduction in band 3 expression affects blood HCO3-/CO2 metabolism. What could happen physiologically if band 3 expression is elevated instead? In some areas of Southeast Asia, about 1-10% of the populations express GP.Mur, a glycophorin B-A-B hybrid membrane protein important in the field of transfusion medicine. GP.Mur functions to promote band 3 expression, and GP.Mur red cells can be deemed as a naturally occurred model for higher band 3 expression. This review first compares the functional consequences of band 3 at different levels, and suggests a critical role of band 3 in postnatal CO2 respiration. The second part of the review explores the transport of water, which is the other substrate for intra-erythrocytic CO2/HCO3- conversion (an essential step in blood CO2 transport). Despite that water is considered unlimited physiologically, it is unclear whether water channel aquaporin-1 (AQP1) abundantly expressed in RBCs is functionally involved in CO2 transport. Research in this area is complicated by the fact that the H2O/CO2-transporting function of AQP1 is replaceable by other erythrocyte channels/transporters (e.g., UT-B/GLUT1 for H2O; RhAG for CO2). Recently, using carbonic anhydrase II (CAII)-filled erythrocyte vesicles, AQP1 has been demonstrated to transport water for the CAII-mediated reaction, CO2(g) + H2O ⇌ HCO3-(aq) + H+(aq). AQP1 is structurally associated with some population of band 3 complexes on the erythrocyte membrane in an osmotically responsive fashion. The current findings reveal transient interaction among components within the band 3-central, CO2-transport metabolon (AQP1, band 3, CAII and deoxygenated hemoglobin). Their dynamic interaction is envisioned to facilitate blood CO2 respiration, in the presence of constantly changing osmotic and hemodynamic stresses during circulation.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

BACKGROUND: The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. MATERIALS AND METHODS: GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. RESULTS: Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. CONCLUSION: Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation.

Selection of GP. Mur antigen-negative RBC for blood recipients with anti-'Mia ' records decreases transfusion reaction rates in Taiwan.

OBJECTIVES: To evaluate the clinical significance of GP. Mur antigen-negative blood selection for transfusion in patients with anti-'Mia ' records. BACKGROUND: The GP. Mur RBC phenotype is prevalent (7·3%) in Taiwan. Antibodies against GP. Mur (anti-'Mia ') are identified in 1·24% of our population, and anti-'Mia ' screening using GP. Mur RBC has been routine for Taiwan's blood banks. However, due to the lack of commercial antibodies, only cross-matching was used to prevent transfusion of GP. Mur-positive blood to patients with anti-'Mia ' in most hospitals. There is still a risk of GP. Mur-positive RBC exposure and subsequent anti-'Mia '-related transfusion reactions. METHODS: Since February 2014, GP. Mur antigen-negative RBCs identified by reaction with anti-'Mia '-positive serum were selected for blood recipients with anti-'Mia ' records. The transfusion reactions between January 2013 and January 2014 were compared with those that occurred between February 2014 and July 2015. RESULTS: The transfusion reaction rate was significantly higher in anti-'Mia '-positive blood recipients compared to total subjects receiving an RBC transfusion before GP. Mur-negative donor RBC selection. After antigen-negative RBC selection, the transfusion reaction frequency in subjects with anti-'Mia ' became similar to total blood recipients. IgG form anti-'Mia ' antibodies were present in all cases of probable anti-'Mia '-related transfusion reactions. The time required for anti-'Mia ' boosting after transfusion was around 4-21 days. CONCLUSION: Selection of GP. Mur-negative RBC for transfusion to patients with anti-'Mia ' records could decrease the rate of transfusion reaction and antibody boosting. This procedure should be incorporated into blood bank routines in areas where anti-'Mia ' is prevalent.

Mur (MNS 10) screening with a novel loop-mediated isothermal amplification assay in Zhongshan, China.

BACKGROUND: The distribution of the Mur blood group antigen is 5-7% in the south of China, and a much higher prevalence is observed in some areas of the region. Anti-Mur can cause hemolytic disease of the newborn and severe transfusion reactions. OBJECTIVES: Genetic testing is more ideal than conventional serological tests because antibodies for detection are usually not available. METHODS: In this study, a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Mur blood group antigen was established. RESULTS: Fifteen of 275 (5·5%) samples were confirmed by LAMP as Mur antigen positive. All the Mur antigen-positive samples were GP.Mur subtype which was confirmed with sequencing. CONCLUSION: The LAMP method has identical results with conventional serology method but more suitable for large-scale screening.