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Originally published In Press as doi:10.1074/jbc.M309826200 on November 5, 2003

J. Biol. Chem., Vol. 279, Issue 4, 2414-2420, January 23, 2004
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Altered Structure and Anion Transport Properties of Band 3 (AE1, SLC4A1) in Human Red Cells Lacking Glycophorin A*

Lesley J. Bruce{ddagger}§, Rui-jun Pan¶, Diane L. Cope||, Makoto Uchikawa**, Robert B. Gunn{ddagger}{ddagger}, Richard J. Cherry¶, and Michael J. A. Tanner{ddagger}

From the {ddagger}Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom, the Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom, the ||Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom, the **Tokyo Blood Center, Japanese Red Cross, Tokyo 150-0012, Japan, and the {ddagger}{ddagger}Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, September 4, 2003 , and in revised form, November 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (MkMk cells) or GPA (En(a–) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to ~60% of that in normal control red cells as a result of an increased apparent Km for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the Vmax for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal Kd for iodide binding, in agreement with the unchanged Km found in transport studies. However, the apparent diffusion quenching constant (Kq) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in Vmax observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The anion exchange protein AE1 (band 3) and glycophorin A (GPA)1 are both present in the human red cell membrane at ~1 x 106 copies/cell. Evidence suggests that these two proteins associate both in internal cell membranes during their biosynthesis and in red cell membranes (1–12). Reduction of the amount of band 3 in the human red cell membrane results in membrane defects, and the almost total absence of band 3 in man compromises life and requires intervention (13, 14). However, GPA can be reduced or even absent as a result of genetic defects without known clinical consequences (15). In some individuals, GPA is completely absent in the red cell membrane (En(a–) cells), whereas other individuals have red cells that lack both GPA and glycophorin B (GPB) (MkMk cells). In other cases, only GPB is absent in the red cell membrane (S–s–U– cells). The GPA and GPB genes are located in tandem on chromosome 4, and these two highly homologous genes are prone to deletions, gene conversions, and crossovers.

The structure and function of band 3 are altered in En(a–) red cells (16, 17) and MkMk red cells (18–20). In these cells, band 3 has a higher apparent molecular weight due to an increase in the size of the N-glycan chain (16, 17, 20). In MkMk cells, the sulfate transport activity of band 3 is ~60% of that in normal red cells as a result of an increase in the apparent Km for sulfate (20). It has been argued that this altered transport activity is due to the lack of GPA in MkMk cells, as there is no evidence for an interaction between band 3 and GPB.

The transport domain of band 3 has been studied by the fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3 with I– (21). It was found that the Stern-Volmer plot of the quenching reaction between EMA-labeled band 3 and I– exhibits a downward curvature. Moreover, the quenching is inhibited by the specific inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and transportable anions. The quenching data were quantitatively analyzed by a binding-diffusion model. The model supposes that, when covalently bound to Lys430 of band 3, EMA is located in a pocket in band 3, which is probably the anion access channel (22). Based on the binding-diffusion quenching model, two parameters are obtained, viz. the anion dissociation constant (Kd) and the apparent diffusion quenching constant (Kq). These two parameters are likely to be related to the structure and dynamic features of the transport domain.

This study compares the structure and function of band 3 in red cells that entirely lack GPA (En(a–) and MkMk cells), that lack part of the GPA molecule (MiV cells), or that lack GPB (S–s–U– cells). We show that sulfate transport activity in En(a–) and MiV cells was reduced to the same extent (50–60% of that in normal red cells) as previously reported for MkMk cells (20). As expected, the sulfate transport activity of band 3 in S–s–U– red cells was normal. The reduced sulfate transport activity in MkMk, En(a–), and MiV red cells resulted from a reduction in the apparent affinity of sulfate binding to band 3 (apparent Km). We also measured monovalent anion transport and show that chloride transport activity in En(a–) cells was reduced to ~40% of that in normal red cells. The iodide transport activities of band 3 in MkMk, En(a–), and MiV red cells were reduced, but this reduction was due to a decrease in the maximum velocity of iodide transport (apparent Vmax). Fluorescence quenching experiments with EMA-labeled band 3 showed that the Kd values for I– and binding to band 3 in these variant red cells were almost the same as those in normal red cells, but the Kq values were significantly increased in En(a–), MkMk, and MiV red cells. The fluorescence polarization of band 3-bound EMA was decreased in the GPA variant cells.

The results suggest that, when GPA is absent, there is increased flexibility of the membrane domain of band 3 that is associated with the reduced anion transport activity of the molecule. Our observations indicate that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The MkMk blood sample was obtained from the Osaka Red Cross Blood Center (Osaka, Japan). The homozygous En(a–) and MiV(Japan (Jp)) blood samples were obtained from the Japanese Red Cross Central Blood Center (Tokyo, Japan). The homozygous MiV(Norway (No)) and S–s–U– blood samples were obtained from the International Blood Group Reference Laboratory (Bristol, United Kingdom). The MiV cells contain a hybrid glycophorin composed of residues 1–58 of GPA and residues 27–72 of GPB (23). Normal control blood samples were either drawn and shipped under the same conditions as for the GPA variant samples or were age-matched samples obtained from the Blood Transfusion Centre (Bristol). Monoclonal antibodies R1.3 and BRIC 170 were from Professor D. J. Anstee (Bristol Institute for Transfusion Sciences, Bristol).

Analysis of Red Cell Membrane Proteins—En(a–), MiV(Jp), and control red cells (1 µl of packed cells) were solubilized with 1 mg/ml bovine serum albumin, 125 mM Tris-HCl, pH 8, 6.25 mM EDTA, 0.0625% SDS, 1.25% C12E8 (octaethylene glycol dodecyl ether), 4 mM phenylmethanesulfonyl fluoride, 100 µg/ml leupeptin, and 50 µg/ml antipain and treated with or without 40 units of peptide N-glycanase F at 37 °C for 17 h. Untreated red cell ghosts were also prepared for each red cell type. Samples were separated by SDS-PAGE and immunoblotted according to the methods of Wainwright et al. (24).

Anion Transport Assays—A time course for sulfate transport at 30 °C was done over 60 min and found to be linear for the first 15 min. A time course for iodide transport at 8 °C was done over 15 min and found to be linear for the first 7 min. All transport measurements were performed within the linear range of the assay. The [35S]sulfate influx into red cells was measured using variant and control cells at 10% hematocrit, and the number of DIDS-binding sites was determined by titration of sulfate transport as described previously (25). The Vmax and apparent Km for [35S]sulfate influx were calculated from sulfate influx measurements in the absence of DIDS using sulfate concentrations between 0.4 and 50 mM. The Vmax and apparent Km for [125I]iodide influx were calculated from iodide influx measurements in the absence of DIDS using iodide concentrations between 0.4 and 60 mM. Red cell [36Cl]chloride efflux was measured as described by Gunn and Frohlich (26).

Labeling of Band 3 with EMA—EMA-labeled red cell membranes were prepared according to Nigg and Cherry (27). Briefly, blood samples were washed three times with phosphate-buffered saline (5 mM Na2HPO4, 150 mM NaCl, and 1 mM EDTA, pH 7.8) at 4 °C and then two times with buffer A (90 mM Na2HPO4 and 20 mM NaH2PO4, pH 7.5). The washed red cells (70% hematocrit in buffer A) were incubated with EMA (1 mg of EMA/5 ml of packed red cells) in the dark at the room temperature for 45 min. To stop the labeling reaction and to remove excess free EMA, the labeled red cells were washed with ice-cold phosphate-buffered saline several times until the supernatant was clear. EMA-labeled ghosts were prepared, and the samples were flushed with nitrogen and stored in the dark at 4 °C. The subsequent fluorescence quenching measurements were carried out within 2 days after preparation of the membranes.

Fluorescence Spectroscopic Measurements and Data Analysis—The fluorescence quenching measurements were performed as described previously (21). Fluorescence intensities were determined on a Shimadzu RF500PC spectrofluorometer. Eosin was excited at 510 nm, and the emission was recorded at 547 nm with excitation and emission slits adjusted to 2.5 nm. The eosin concentration was generally ~10–7 M. Freshly prepared KI (4 M) stock solution contained 3 mM Na2S2O3 to prevent formation of . For each quencher concentration, the ghosts were suspended in buffer B (5 mM Na2HPO4, pH 7.8) containing an amount of trisodium citrate such that, after addition of different amounts of I–, the ionic strength remained constant at 250 mM. Fluorescence intensities were corrected for the small dilution factor (<5%) arising from the addition of the quencher stock solution. The fluorescence quenching data were analyzed by the binding-diffusion model (21) (Equation 1),

(Eq. 1)
where F0 and F are the fluorescence intensities in the absence and presence of quenchers, respectively; Kq is the apparent diffusion quenching constant; and Kd is the dissociation constant for I– binding to the anion-binding site on band 3. Rearrangement of Equation 1 gives Equation 2,

(Eq. 2)
and hence, the values of Kq and Kd can be determined from the slope and y intercept of plots of F/(F0 – F) against 1/[I–].

When the quenching titration is performed in the presence of the other band 3 substrate anion (), the apparent dissociation constant (Kd') for I– binding (21) is as shown in Equation 3,

(Eq. 3)
where Kd(a) is the dissociation constant for . Thus, a plot of Kd' against [] is linear, and Kd(a) can be determined from the slope.

The fluorescence polarization (p) was determined for band 3-bound EMA by Equation 4,

(Eq. 4)
where Ivv is the fluorescence intensity when the excitation polarizer is vertical and the emission polarizer is vertical, and Ivh is the fluorescence intensity when the excitation polarizer is vertical and the emission polarizer is horizontal. The polarization factor of the emission monochromator (G) is given by G = Ihv/Ihh.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GPA and GPB in Variant and Control Red Cells—Red cell membrane proteins (separated by SDS-PAGE) were immunoblotted using monoclonal antibody R1.3 to confirm that the variant red cells contained the expected profiles of GPA and GPB (reviewed by Anstee (28)). Monoclonal antibody R1.3 reacts more strongly with GPB than with GPA (29). Both GPA and GPB were detected in control red cell membranes in their monomeric, dimeric, and heterodimeric forms (Fig. 1a). En(a–) red cell membranes lacked GPA and contained only the GPB forms (monomers, dimers, and trimers) (Fig. 1a). Immunoblotting of MiV(No) and MiV(Jp) red cell membranes with monoclonal antibody R1.3 did not show any of the normal GPA or GPB bands (Fig. 1a), but detected the monomeric and dimeric forms of the MiV GPA/GPB hybrid protein (23).



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  		FIG. 1.
SDS-PAGE and immunoblotting of proteins in En(a–), MiV, and control red cells. a, glycophorins present in En(a–), MiV, and control red cells. Red cell membranes were prepared, and the proteins were separated on an 8% SDS-polyacrylamide gel and immunoblotted with a monoclonal antibody directed against an epitope on both GPA and GPB (R1.3). Monoclonal antibody R1.3 reacts much more strongly with GPB than with GPA (29). The various monomers and dimers of GPA (A) and GPB (B) and the two bands associated with the homozygous MiV hybrid glycophorin ((A-B) and (A-B)2) are indicated. The upper band in the En(a–) sample is the GPB trimer. b, peptide N-glycanase F treatment of band 3 in En(a–), MiV, and control red cells. Red cells were treated with peptide N-glycanase F (PNGase F) or left untreated. The proteins were separated by SDS-PAGE and immunoblotted with a monoclonal antibody directed against the N terminus of band 3 (BRIC 170).

 
The Size of the N-Glycan Chain of Band 3 Is Increased in En(a–) and MiV Red Cells—It has been shown previously that band 3 in En(a–) and MiV red cells has a higher apparent molecular weight (16, 17, 23) and that, in En(a–) red cells, this is due to an increase in the size of the N-glycan chain (16, 17). We showed that the increased apparent molecular weight of band 3 in MiV red cells is also due to an increase in the size of the N-glycan chain. MiV, En(a–), and control red cells treated with or without peptide N-glycanase F and subjected to 8% SDS-PAGE were immunoblotted with a monoclonal antibody reactive with the N terminus of band 3 (BRIC 170). In the untreated samples, band 3 in both MiV and En(a–) red cells had a higher apparent molecular weight compared with band 3 in control red cells (Fig. 1b). However, band 3 had the same mobility in all the samples after treatment of the red cells with peptide N-glycanase F (Fig. 1b), showing that the increased apparent molecular weight of band 3 in both MiV and En(a–) red cells is due to an increase in the size of the associated N-glycan chain.

En(a–) and MiV Red Cells Have an Increased Apparent Km for Sulfate, Resulting in Reduced Divalent Anion Transport— The divalent anion transport activities in En(a–), MiV(Jp), MiV(No), S–s–U–, and control red cells were compared by titration of the sulfate influx with DIDS. The results show that sulfate transport activity in En(a–) and MiV red cells was ~55% of that in control cells, whereas the total number of reversible and irreversible DIDS-binding sites was the same in all three cell types (Fig. 2, a and b; and Table I). The latter observation shows that all the variant cells contained the same number of band 3 molecules/cell compared with normal control cells. The sulfate transport activity of S–s–U– red cells was unchanged compared with two control blood samples (Fig. 2c).



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  		FIG. 2.
DIDS titration of sulfate influx into GPA- and GPB-deficient red cells. The anion transport activities of En(a–), MiV, S–s–U–, and control red cells were compared by titration of the sulfate influx by DIDS at an external sulfate concentration of 4 mM (as described under "Experimental Procedures"). Each data point was derived from measurements in triplicate. The S.D. is indicated by the error bars. The lines show the results of linear regression analysis of the data. a, sulfate influx into En(a–) (•), MiV(Jp) ({blacktriangleup}), and control ({circ}) red cells; b, sulfate influx into MiV(No) ({blacksquare}) and control ({circ}) red cells; c, sulfate influx into S–s–U– ({diamondsuit}) and control ({circ}, {diamond}) red cells.

 


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  		TABLE I
Summary of the anion transport data

All data are expressed as a percent of the values obtained in parallel experiments with control red cells. The S.D. values are shown (n >= 3). NT, Not tested.

 
The Vmax and apparent Km for sulfate influx were calculated for MkMk, En(a–), MiV(No), and control red cells from sulfate influx measurements over a range of sulfate concentrations. The Vmax for sulfate influx into each type of GPA-deficient red cell was similar to that into control cells, but the apparent Km for sulfate influx into the GPA-deficient red cells was increased (Fig. 3 and Table I). The apparent Km for sulfate influx into MkMk red cells was 185%, and 170% of that into control cells (Fig. 3a and Table I). In En(a–) red cells, the apparent Km for sulfate was 160%, and 160% of that in control cells (Fig. 3b and Table I); and in MiV(No) red cells, the apparent Km for sulfate was 160% of that in control cells (Fig. 3c and Table I).



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  		FIG. 3.
Eadie-Hofstee plots of sulfate transport into GPA-deficient red cells. Sulfate influx was measured in the absence of DIDS at sulfate concentrations between 0.4 and 40 mM. a, MkMk ({blacksquare}) and control ({circ}, {diamond}) red cells; b, En(a–) (•) and control ({circ}, {diamond}) red cells; c, MiV(No) ({blacktriangleup}) and control ({circ}) red cells.

 
MkMk, En(a–), and MiV Red Cells Have a Decreased Vmax for Monovalent Anions, Resulting in Reduced Monovalent Anion Transport—The monovalent anion transport activities of En(a–), MkMk, MiV(Jp), and control red cells were compared by measuring [125I]iodide influx into and [36Cl]chloride efflux out of the cells. [125I]Iodide influx was measured in En(a–), MkMk, MiV(Jp), and control red cells, and the Vmax and apparent Km for [125I]iodide influx were calculated from these measurements. The apparent Km for iodide in the En(a–), MkMk, and MiV(Jp) red cells was similar to that in control cells; however, the Vmax for iodide influx was reduced (Fig. 4, a and b; and Table I). The Vmax for iodide influx into En(a–), MkMk, and MiV(Jp) cells was 64, 58 ± 6, and 61%, respectively, of that into control cells (Fig. 4, a and b; and Table I). It was possible to measure [36Cl]chloride efflux only for the En(a–) red cells because of the difficulty of access to the other rare blood samples. En(a–) cells were found to have only 40% of the chloride efflux transport activity of normal control red cells (Fig. 4c).



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  		FIG. 4.
Monovalent anion transport into GPA-deficient red cells. Iodide influx was measured in the absence of DIDS at iodide concentrations between 0.4 and 60 mM. Chloride efflux was measured using the method of Gunn and Frohlich (26). a, Eadie-Hofstee plot of iodide influx into MkMk ({blacksquare}) and control ({circ}, {diamond}) red cells; b, Eadie-Hofstee plots of iodide influx into En(a–) (•), MiV(Jp) ({blacktriangleup}) and control ({circ}) red cells; c, chloride efflux from En(a–) (•) and control ({circ}) red cells. The results of duplicate experiments are shown. at, the concentration of [36Cl]chloride in the medium at the time of sampling; ai, the concentration of [36Cl]chloride after isotopic equilibrium has been attained.

 
GPA-deficient Red Cells Show Enhanced Fluorescence Quenching Efficiencies for Band 3-bound EMA Due to an Increased Kq—Stern-Volmer plots of quenching of EMA-band 3 fluorescence in both normal and GPA-deficient red cell membranes exhibited a downward curvature similar to that previously reported (21). Values of Kd and Kq were obtained from linear plots of F/(F0 – F) against 1/[I–] (Equation 2). Fig. 5 shows that, compared with control red cells, En(a–), MkMk, and MiV(Jp) samples have reduced intercept values on the axis of F/(F0 – F), which indicates that the Kq for each GPA variant is increased. However, the Kd values for I– binding to band 3 were found to be the same for normal and GPA-deficient red cells. The values of Kq and Kd for normal and GPA-deficient red cells are listed in Table II.



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  		FIG. 5.
Fluorescence quenching of band 3-bound EMA in normal and variant red cell membranes. a, comparison between control ({circ}) and En(a–) (•) red cell membranes; b, comparison between control ({circ}) and MkMk ({blacksquare}) red cell membranes; c, comparison between control ({circ}) and MiV ({blacktriangleup}) red cell membranes. The data were fitted by Equation 2 as described under "Experimental Procedures." The increased Kq values are clearly shown by the reduced y axis intercept for these GPA variant samples. All measurements were carried out in buffer B with varying amounts of trisodium citrate to maintain the ionic strength constant at 240 mM. [EMA] was ~10–7 M; the temperature was 18 °C.

 


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  		TABLE II
Summary of the binding-diffusion quenching parameters for GPA variant red cell membranes

Parameters and S.D. values were obtained from fitting the data to Equations 1 and 2 for Kd for I- and Kd for , respectively. All fluorescence quenching measurements were performed under the same conditions, as described in the legend to Fig. 5.

 
Competitive inhibition experiments of the I– quenching reaction with were also performed. The data were analyzed as described by Pan and Cherry (21) by determining the Kd' for I– binding in the presence of different concentrations of . A plot of Kd' against [] then yields the dissociation constant for . The values thus obtained showed no differences in normal, En(a–), MkMk, and MiV red cell membranes (Table II). This further confirms that the increased quenching efficiencies observed in En(a–), MkMk, and MiV red cell membranes are caused only by an increase in Kq.

Because the fluorescence lifetime of band 3-bound EMA is ~10–9 s (30), the I– ion must reach the fluorophore within this time interval for quenching to occur. In the binding-diffusion model, a mechanism whereby a bound I– ion dissociates and diffuses to the probe would require a very high off-rate constant, in the order of 109 s–1. Therefore, we have previously argued that bound I– collides with the probe as a result of flexing motions of the band 3 protein (21). Thus, Kq can be regarded as an indication of the local flexibility in the region of the band 3-bound EMA site and I–-binding site.

Fluorescence Polarization Measurements Show Enhanced Flexibility of the Anion Transport Domain in GPA-deficient Membranes—The results of fluorescence polarization experiments for EMA-labeled normal and GPA-deficient red cell membranes are shown in Fig. 6. Whereas the fluorescence polarization value for normal cell membranes was 0.37 ± 0.02, the polarization values for En(a–), MkMk, and MiV cell membranes were 0.29 ± 0.01, 0.27 ± 0.03, and 0.30 ± 0.02, respectively. The decreased fluorescence polarization values indicate more local flexibility of band 3 in red cell membranes lacking GPA compared with normal membranes. These results are consistent with the increased Kq values obtained for the GPA-deficient red cell membranes (Fig. 5 and Table II).



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  		FIG. 6.
Fluorescence polarization of band 3-bound EMA in normal and variant red cell membranes. The measurements were carried out at 14 °C in buffer B. The excitation and emission wavelengths were 510 and 547 nm, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There is evidence to suggest that GPA plays a role in both the biosynthesis and folding of band 3 in the red blood cell (20). Many studies have shown that GPA facilitates the movement of band 3 to the cell surface, suggesting that it may have a chaperone-like role in enhancing trafficking through internal compartments during band 3 biosynthesis (3, 4, 31). This effect has been shown not to require the GPA dimer because the GPA monomers also enhance band 3 anion transport in Xenopus oocytes (10). Other studies have indicated that these two proteins can interact in the mature red cell (1, 2, 5–9). The relationship between band 3 and GPA appears to be a reciprocal one because GPA in band 3–/– mice does not reach the red cell membrane despite the presence of normal amounts of GPA mRNA (32). Similarly, plasma membrane expression of GPA is increased by the coexpression of band 3 in the K562 cell line (10).

Two sites on GPA involved in interactions with band 3 have recently been defined by expression studies in Xenopus oocytes (12). In addition, a known site of interaction between band 3 and GPA in mature red cells results in the formation of the Wrb blood group antigen. Expression of the Wrb antigen involves Glu658 of band 3 and residues 58–70 of GPA (7). Glu658 is at the extracellular side of putative transmembrane span 8 of band 3, suggesting that GPA interacts close to span 8 of band 3. Other evidence has shown that GPA enhances the surface movement of C-terminal (but not N-terminal) fragments of band 3 (33). The minimum fragment of band 3 on which GPA acts to enhance band 3 surface presentation is the portion encompassing putative transmembrane spans 9–12 (33).

In a previous study of band 3 in MkMk red cells (which lack both GPA and GPB), we suggested that the increased size of the N-glycan chain on band 3 and the reduced sulfate transport activity in these cells result from the absence of GPA rather than GPB (20). The present study demonstrates similar changes in anion transport in En(a–) red cells, but not in S–s–U– red cells. This confirms that it is the absence of GPA (and not GPB) that affects the function of band 3. En(a–) cells also show an increase in the size of the N-glycan chain as reported previously (16, 17). These same anion transport and N-glycan size changes are found in band 3 in MiV cells, indicating that the site of interaction between band 3 and GPA is in the GPA region comprising residue 59 to the C terminus, consistent with other observations (12).

In this study, we have shown that both sulfate and iodide transport are reduced in MkMk, En(a–), and MiV red cells and that chloride transport is reduced in En(a–) red cells, although all these red cells contain the same number of band 3 molecules as normal red cells. The absence of GPA clearly results in a structural change that alters the specific activity of the anion transport site of band 3. Although the GPA-deficient red cells have larger band 3 N-glycan chains, this is unlikely to be the cause of the change in the specific anion transport activity of band 3 because it is known that deglycosylation of band 3 has no effect on the rate of anion transport (34) or the affinity of the protein for stilbene disulfonates (35). Our data shows that band 3 can exist in two states: a high activity conformation when GPA is present and a lower activity conformation when GPA is absent. The change in band 3 conformation affects anion binding and anion translocation. Interestingly, the nature of the reduced anion transport in the absence of GPA differed between the monovalent and divalent anions. The reduced sulfate transport resulted from a reduction in the apparent affinity of band 3 for this divalent anion (increased apparent Km), and the reduced iodide transport resulted from a decrease in the maximum rate of transport for this monovalent anion (reduced Vmax). GPA-deficient cells also showed reduced efflux transport of chloride, a physiological substrate for band 3.

Fluorescence quenching of the probe EMA provides an alternative method of investigating the transport domain of band 3. The finding that the Kd for I– and for another monovalent anion () is unchanged in the GPA-deficient red cells is consistent with the unchanged Km for iodide transport. The quenching experiments reveal, however, that there is a change in the transport domain of band 3 in the GPA-deficient red cells that results in an increase in Kq. This parameter has been interpreted as reflecting local flexibility of band 3. To test this proposal, we measured fluorescence polarization of the EMA probe and found it to be decreased in the GPA-deficient cell membranes (Fig. 6). A decrease in fluorescence polarization is evidence for an increase in the wobbling motion of the probe in its binding site. Thus, both the fluorescence quenching and fluorescence polarization experiments indicate that there is more local motion in the transport domain of band 3 in the GPA-deficient cells compared with normal cells. It is reasonable to suppose that the same structural change is responsible for both the increase in local flexibility and the decrease in Vmax for monovalent anion transport, although the precise relationship between these two effects is unclear. Although EMA is an inhibitor of anion transport, the present experiments demonstrate that the fluorescence properties of the bound probe can reflect subtle structural changes that influence anion transport activity. At present, it unclear whether the altered structure and increased flexibility of band 3 result from changes in the folding of band 3 that are mediated directly or indirectly by GPA during band 3 biosynthesis or result from GPA changing the structure of mature band 3 in the red cell membrane.

In earlier studies of band 3 in normal red cells, we found that sulfate was not able to inhibit I– quenching of EMA-labeled band 3.2 This has prevented the application of the quenching assay for the measurement of divalent anion binding affinity. This is probably due to the different ways in which band 3 interacts with monovalent and divalent substrate anions and suggests that the structures of the monovalent and divalent anion-binding sites also differ in band 3 in normal cells. Divalent anion transport occurs by a proton-anion cotransport mechanism (36) and also has other characteristics that differ significantly from monovalent anion transport (reviewed by Passow (37)).

It has been shown previously that coexpression of GPA with band 3 enhances band 3-mediated chloride transport induced in Xenopus oocytes and that increased movement of band 3 to the oocyte cell surface is at least partly responsible for this effect (3). A recent study showed that two separate effects of GPA are responsible for increasing band 3-mediated transport in Xenopus oocytes: an increase in the amount of band 3 moved to the oocyte cell surface and an increase in the intrinsic anion transport activity of the protein (12). These two effects are mediated by different regions of the GPA molecule (12). Analysis of the effects of GPA or GPB mutants on band 3 expression (including naturally occurring GPA/GPB hybrid molecules like MiV) showed that the C-terminal cytoplasmic tail of GPA enhances trafficking of band 3 to the cell surface, whereas extracellular GPA residues 68–70 increase the specific chloride transport activity of band 3. Deletion of Phe68–Glu70 of GPA reduces the enhancement by GPA of band 3 chloride transport by 80% when the two proteins are coexpressed in Xenopus oocytes without affecting the amount of band 3 expressed at the cell surface (12). The present work shows the same two effects on band 3 expression in human red cells. Normal red cells (which contain GPA) have enhanced band 3 anion transport activity and normal N-glycosylation. In GPA-deficient cells, the size of the N-glycan chain on band 3 is altered, indicating altered treatment of band 3 by the Golgi apparatus of these cells (20). In addition, band 3 anion transport activity is reduced in GPA-deficient red cells; however, the total number of band 3 molecules/mature red cell is normal. Unlike the transient expression of band 3 in Xenopus oocytes, the amount of band 3 in the mature red cell reflects the steady state and is probably limited by some structural feature of the red cell membrane controlling the number of band 3-binding sites.

This study shows that the structure and function of band 3 are altered in red cells that lack GPA or in GPA mutants lacking residue 59 to the C terminus. Fluorescence quenching and polarization experiments indicated that the EMA-binding pocket, which is probably the anion access channel, has greater flexibility in GPA-deficient red cells. These GPA-deficient red cells show reduced band 3 anion transport activity, and the altered structure of band 3 affects monovalent and divalent anion transport in different ways. Taken together, the data indicate that band 3 can take up two conformations: one structure with high anion transport activity and another structure with lower anion transport activity. The formation of the high activity structure requires the presence of GPA, and GPA acts on band 3 either during band 3 biosynthesis or in the mature red cell membrane.


   FOOTNOTES
 
* This work was supported in part by grants from the Wellcome Trust (to M. J. A. T. and R. J. C.) and by National Institutes of Health Grant HL-66173 (to R. B. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Bristol Institute of Transfusion Sciences, Southmead Rd., Bristol BS10 5NB, UK. Tel.: 44-117-991-2126; Fax: 44-117-959-1660; E-mail: lesley.bruce{at}nbs.nhs.uk.

1 The abbreviations used are: GPA, glycophorin A; GPB, glycophorin B; EMA, eosin-5-maleimide; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Jp, Japan; No, Norway. Back

2 R. J. Cherry, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Yasuto Okubo (Osaka Red Cross Blood Center) and Joyce Poole (International Blood Group Reference Laboratory) for assistance in obtaining MkMk, En(a–), MiV, and S–s–U– red cells and Professor D. J. Anstee for supplying the monoclonal antibodies and control blood samples.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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