Physiological Implications of Miltenberger blood group antigen subtype III (Mi.III)

Authors


  • This work was supported by a grant from Taiwan National Science Council (NSC 99-2320-B195-001-MY3) and by Mackay Memorial Hospital to K.H.

  • 3A-S1-02

Kate Hsu, PhD, 45 Min-Sheng Rd, Research building 616, Tamsui, Taiwan 251
E-mail: khsu1@ms1.mmh.org.tw

Abstract

In Taiwan, Miltenberger blood group antigen subtype III (Mi.III) is the second most important blood group antigen following ABO. Mi.III presumably evolved from homologous recombination between GYPA and GYPB, and expresses a hybrid configuration of glycophorin B-A-B and the Mur antigen in the crossover region.

GPA and GPB oligomerize on the erythrocyte cell membrane. GPA also forms protein complexes with band 3 and facilitates band 3 expression. GPB is believed not to involve in the GPA-band 3 interaction. In Mi.III+ blood, half or all GPB is replaced with the glycophorin BAB hybrid, Gp.Mur. We previously showed that upon heterologous expression in HEK-293 cells, Gp.Mur, like GPA, facilitates the protein expression of band 3. Quantitative proteomics by iTRAQ revealed 25–67% more band 3 on the Mi.III+ erythrocyte membrane, as compared to the non-Mi.III. Band 3 is a Cl/HCO3 exchanger. With more band 3 protein expression, Mi.III+ RBCs exhibit larger capacities for Cl/HCO3 exchange across the cell membrane. Conceivably, those with the Mi.III phenotype might have larger capacities for CO2 respiration.

On the other hand, GPB is a component of the Rh protein complex, and has been shown to assist the surface expression of RhAG. Because GPB is half or completely replaced by Gp.Mur in Mi.III+ RBCs, we also examined if the expression of the Rh complexes could be affected in Mi.III. We found that homologous Mi.III RBCs exhibit reduced levels of RhAG and Rh antigens. The functions of Rh polypeptides and RhAG in erythrocytes are unclear, but RhAG in other species might function as gas channels for physiologically important molecules such as CO2, NH3, NO, and/or O2. Thus, substitution of GPB with Gp.Mur in Mi.III+ RBCs affects the structure of the band 3/Rh-associated macrocomplex, and is expected to affect functional coordination within the metabolon of O2/CO2 gas exchange as well.

Among the 11 Miltenberger blood group antigen subtypes (Mi.I → Mi.XI) of the MNS system identified up to date (Fig. 1), Mi.III has the highest prevalence among Southeast Asians: 2–7% in Taiwanese [1], 6·3% in Hong Kong Chinese [2], 6·5% in Vietnamese [3], 9·7% in Thais [1,2,4], and 7·6% in Filipinos [5]. But Mi.III is considered a rare phenotype among Caucasian (0·0098%), northern Han Chinese, and Japanese (0·006%) [6]. In the west, the Rh system is the most clinically important blood group system following ABO. In Taiwan, Mi.III is more important than Rh in the field of transfusion medicine, because only 0·3% of the Taiwanese population is RhD negative and only 0·13% of the RhD-negative Taiwanese carry anti-D [7,8]. In contrast, from the statistics of Mackay Memorial Hospital blood bank in Taiwan, ∼1% of the Mi.III-negative patients have alloantibodies against Mia, a major Miltenberger antigen that is present in multiple subtypes including Mi.III [8,9].

Figure 1.

 Miltenberger blood group antigens are products of homologous gene recombination between glycophorin A and glycophorin B, and can be categorized based on their structural arrangement (configurations): A-B, B-A, A-B-A and B-A-B [26]. Mi.III and Mi.VI, two phenotypes found in southeast Asian countries, exhibit the configuration of glycophorin B-A-B. (Bottom) Protein sequence alignment for GPA, GPB, Mi.III (Gp.Mur), and Mi.VI (Gp.Bun). The unique Mur antigen of Mi.III and the unique Hop antigen of Mi.VI are marked.

Miltenberger antigens are hybrid proteins that primarily evolved from homologous gene recombination of glycophorin A (GYPA) and glycophorin B (GYPB) (Fig. 1) [10]. Some of the glycophorin hybrid structures could be highly antigenic and elicit alloimmune Ig responses following pregnancy or transfusion. As early as in 1975, there was already a report in Philippines about a haemoytic disease of the fetus and newborn (HDFN) due to the Mur antigen, which is present in both Mi.III and Mi.VI antigens [11]. Alloantibodies elicited by various Miltenberger antigens (e.g. Mia) are associated with hydrops fetalis [12], HDFN [13], as well as acute intravascular haemolytic transfusion reactions (HTRs) [9,14]. Not surprisingly, most of these clinically relevant cases were reported from Southeast Asia countries.

Both Mi.III and Mi.VI antigens share a similar glycophorin B-A-B hybrid configuration (Fig. 1); that is, glycophorin B is inserted with a piece of glycophorin A sequence in the middle [15]. This unique glycophorin B-A-B gives rise to the antigenic Mur peptide in the crossover junction between glycophorin B and glycophorin A. So the Mi.III-specific glycophorin hybrid protein is also known as Gp.Mur (Fig. 1). From immunoblot studies of the red cell membrane, Gp.Mur is abundantly expressed with an equivalent level to glycophorin B (GPB) in Mi.III+/− RBCs [16]. In homozygous Mi.III RBCs, GPB is completely replaced by Gp.Mur. It is estimated that there are ∼200,000 Gp.Mur molecules in a Mi.III+/+ red cell and ∼100,000 Gp.Mur in a Mi.III+/− cell.

The impacts of Gp.Mur on the protein expression of the band 3/Rh complexes

Glycophorin A (GPA) functions as a chaperone for band 3 [17,18], presumably through a one-to-one interaction (each with about a million molecules per red cell). GPB exhibits chaperone-like activities for Rh-associated glycoprotein (RhAG) instead: GPB has been shown to facilitate intracellular trafficking and protein maturation of RhAG [19]. Since Gp.Mur is a hybrid protein of GPB and GPA, it might bear similar or modified chaperone activities. We previously compared the chaperone activities of GPA versus Gp.Mur for band 3 in heterologous expression experiments, and found that Gp.Mur is as potent as GPA in enhancing the protein production and surface expression of band 3 [16]. On the other hand, GPB has relatively small effects on the surface expression of band 3. It is thus conceivable that expression of Gp.Mur instead of GPB in Mi.III erythrocytes shall enhance the overall expression of band 3. Indeed, from an iTRAQ proteomic experiment that quantitatively compared protein components of the band 3-associated macrocomplexes in Mi.III versus non-Mi.III erythrocytes, there is 25% or more band 3 on the surface of Mi.III erythrocytes [16].

Band 3 and Rh complex proteins are structurally linked. The expression levels of band 3 and the Rh complex are also quantitatively correlated, according to a biochemical study on the red cell membrane of band 3 knockout mice and of a human patient with homozygous band 3 Coimbra mutation [20]. From this study, expression of the Rh complex proteins decreases significantly in the absence of band 3 (Fig. 2) [20]. However, in homozygous Mi.III red cells that lack GPB completely, there are more band 3 molecules and fewer Rh antigens and RhAG (Fig. 2) [21]. Therefore, Mi.III+/+ RBCs might express fewer band 3/Rh-associated complexes on the cell membrane. The shift in the proportion of the band 3/Rh-associated macrocomplexes in Mi.III+/+ suggests that GPB/Gp.Mur probably plays a modulatory role in complex association between band 3 and Rh/RhAG.

Figure 2.

 The relative expression of band 3 and Rh/RhAG in the RBCs of band 3−/−, band 3 Coimbra, wild-type (WT), and heterozygous and homozygous Mi.III. Drawn not to scale. In the absence of band 3 (band 3−/− or band 3 Coimbra), erythrocyte Rh antigens and RhAG are much reduced. Mi.III RBCs exhibit higher levels of band 3. Rh antigens and RhAG are only reduced in Mi.III+/+ erythrocytes but not in Mi.III+/−.

The expression levels of Rh antigens and RhAG in heterozygous Mi.III RBCs are not significantly different from that in the non-Mi.III cells, but there are structural variations in the interface between RhAG and GPB/Gp.Mur on the Mi.III+/− membrane (Fig. 2), as probed by anti-U [21]. This alteration on the RhAG-GPB/Gp.Mur interface of Mi.III+/− is reminiscent of our earlier finding about the higher Wrb expression in Mi.III erythrocytes [22]. The Wrb antigen is situated on a specific interfacial region between band 3 and GPA [23]. The increase of Wrb in Mi.III RBCs suggests that the interface between band 3 and Gp.Mur could also be recognized by anti-Wrb, and/or that higher band 3 expression in Mi.III could drive oligomerization between band 3 and GPA/Gp.Mur and increase formation of the Wrb antigen [21]. As Mi.III expression affects the interface between RhAG and GPB/Gp.Mur and the interface between band 3 and GPA/Gp.Mur, conceivably band 3 and the Rh complex are structurally linked by oligomerization of GPA and GPB/Gp.Mur. The protein–protein interaction between GPA and its homologous counterparts – GPB/Gp.Mur is substantial and stable, since GPA-GPB/Gp.Mur heterodimers could be readily observed on SDS-PAGE, with similarly strong band intensities as that for GPA homodimers or GPB homodimers [16]. We also recently found that GPA and GPB/Gp.Mur partially colocalize in transfected mammalian cultured cells (unpublished data). The new finding suggests that the dynamics of GPA-GPB/Gp.Mur interaction during erythropoiesis may play an important role in driving the formation of band 3/Rh-associated macrocomplexes.

The impacts of Gp.Mur on erythrocyte band 3 activities

Band 3, also known as anion exchanger-1 (AE1), is a Cl/HCO3 exchanger abundantly expressed on the red cell membrane. This Cl/HCO3 antiport activity of band 3 is essential for CO2 respiration, since during respiration, 60–90% of blood CO2 is converted to HCO3 by an enzyme inside the red cell. CO2 freely diffuses through the erythrocyte membrane, but HCO3 needs to pass through the lipid bilayer of a red cell via AE1. In tissues with high CO2, CO2 diffuses into circulating RBCs where CO2 is rapidly catalysed by carbonic anhydrase II (CAII) and converted into HCO3. HCO3 is then exported to the plasma via band 3-mediated anion exchange. While in the lungs with low CO2 pressure, CO2 diffuses out of circulating RBCs, and plasma HCO3 rushes into these RBCs via band 3 to be converted to CO2. Notably, the enzyme activity of CAII is one order faster than band 3, so band 3-mediated anion exchange is the rate-limiting step for CO2 respiration [24]. As Mi.III RBCs express 20% or more band 3 on the cell membrane, the bicarbonate transport capacity of these cells is expected to be higher. Interestingly, we found that the Cl/HCO3 exchange capacity of Mi.III erythrocytes only became significantly higher when these cells were in an environment of high HCO3 [16], indicating that the anion exchange capacity of Mi.III RBCs is expandable when the demand for CO2 metabolism rises.

Blood pH is primarily determined by the ratio of HCO3 to CO2. Indeed, the superior Cl/HCO3 exchange of Mi.III erythrocytes also facilitates acid-base homoeostasis. Upon acidosis challenge, the intracellular pH (pHi) of Mi.III erythrocytes appears to be less affected as compared to that of the non-Mi.III cells [16]. The greater pHi–buffering capacity of Mi.III is expected to be an asset for one’s health or even survival under certain pathophysiological stress that disturbs pH homoeostasis.

Our recent findings about the reduction of Rh/RhAG protein expression in Mi.III RBCs probably will complicate functional or mechanistic studies in the future. This is because RhAG probably functions as a gas channel for several physiologically important molecules, such as CO2, NH3, NO and/or O2 [25], though there is no direct evidence yet that demonstrates the gas transport activities of RhAG in erythrocytes or related tissue types. It has been proposed that the band 3/Rh-associated macrocomplex may serve as a hub or metabolon for CO2/O2 gas exchange [20]. If this is indeed the case, the coexistence of more band 3 and less Rh/RhAG on the Mi.III+ erythrocyte membrane may imply a shift in the proportion of the band 3/Rh macrocomplex to other band 3 complexes, as well as differences in the structural arrangement and functional coordination within the metabolon.

Disclosures

No potential conflict of interests to declare.

Ancillary