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Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-09-2824.
RED CELLS
From the Department of Biochemistry, University of
Bristol; the Department of Haematology, University of Cambridge; the
Bristol Institute for Transfusion Sciences, United
Kingdom; Unidade de Hematologia Molecular, Servico de
Hematologia, Centro Hospitalar de Coimbra, Portugal;
Jackson Laboratory, Bar Harbor, ME; Life Sciences Division, University
of California, Lawrence Berkeley National Laboratory; INSERM U473 and
Service d'Hématologie, d'Immunologie et de
Cytogénétique, Hôpital de Bicêtre,
Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre,
France; and the New York Blood Center, New York.
We have studied the membrane proteins of band 3 anion
exchanger (AE1)-deficient mouse and human red blood cells. It
has been shown previously that proteins of the band 3 complex are
reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These
observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3( The band 3 anion exchanger (AE1) is the major
integral protein of the RBC membrane. This multifunctional protein has
3 domains, a membrane-spanning domain that carries out
chloride/bicarbonate exchange, a short C-terminal cytoplasmic domain,
and a large N-terminal cytoplasmic domain (reviewed by
Tanner1,2). The C-terminal cytoplasmic domain of band 3 binds carbonic anhydrase II (CAII), forming a metabolon that channels
HCO Rh proteins form another major integral protein complex in the RBC
membrane. The existence of the Rh protein complex (Rh-associated glycoprotein [RhAG], Rh polypeptides, glycophorin B (GPB), CD47, LW)
was suggested by the absence or deficiency of these proteins in human
RBCs with the Rhnull phenotype (reviewed by
Cartron9). RhAG is sequence-related to the Rh polypeptides
but is N-glycosylated.10,11 RhAG and the Rh polypeptides
are predicted to contain 12 membrane spans and are thought to form
heterotetramers comprising 2 RhAG and 2 Rh polypeptides.12
Rh-like proteins have a broad species distribution, but their function
remains controversial. RhAG and Rh have a distant sequence relationship
with the Amt/MEP family of ammonium/methylammonium transporters in
micro-organisms and plants.13 Recent evidence suggests the
Amt/MEP proteins are channels that allow the facilitated diffusion of
NH3 and methylamine as neutral species across
membranes.14,15 RhAG (and RhCG, the mammalian nonerythroid
homolog of RhAG) have been reported to carry out efflux transport of
ammonium ions from complementation studies using these proteins with
yeast mutants defective in all 3 endogenous MEP
transporters.16 However, other workers14 have
pointed out inconsistencies in the interpretation of these results and
suggest they can be explained by the transport of the neutral ammonia
species. It has also been reported that RhAG enhances
ammonium/methylammonium ion influx into Xenopus
oocytes.17 These investigators suggest that RhAG mediates
an NH CD47 (integrin-associated protein [IAP])19-21 is a
5-spanning membrane protein with a large, highly glycosylated,
extracellular immunoglobulin domain. It has broad tissue distribution
(reviewed by Brown and Frazier22) and associates with
integrins in many cell types, where it has a role in cell signaling and
activation. However, the function of CD47 in mature RBCs is unclear
because RBCs lack integrins. The LW glycoprotein (ICAM-4) is a cell
adhesion molecule that binds the integrinVLA-4 on hemopoietic cells and Serologic studies on Southeast Asian ovalocytosis RBCs (which are
heterozygous for a mutant band 3 containing a 9-amino acid deletion1,2) showed that these cells had depressed blood group Rh antigen expression24 and gave the first
indication of an interaction between the Rh complex and band 3. Later
studies using transfected K562 cell lines also support the presence of associations between band 3 and Rh complexes.25,26 Human
protein 4.2( We have, therefore, examined the proteins of the Rh complex in mouse
band 3( The band 3( RBC membrane protein analysis
Protein concentration of RBC membranes was estimated using the Bradford
assay, and equal amounts (5 µg) of protein were loaded per track of
each gel. Quantification of the band 3( For flow cytometry, RBCs were washed 3 times with phosphate-buffered
saline (PBS), pH 7.5, supplemented with 1% bovine serum albumin,
and 5 × 106 cells were incubated with appropriate mAb.
Unlabeled mAbs were used as neat culture supernatants, except that
purified antibody was used for Brad 3 (20 µg/mL) and BS46 (50 µg/mL). Some antibodies were directly labeled with fluorescein
isothiocyanate (FITC) and were used at 1:50 dilution (BRIC6 and
BRIC256) or 1:30 dilution (BRIC69, LA1818, and Brad 3). Binding of
unlabeled mAbs was detected using FITC-conjugated goat antimouse or
goat antihuman (Fab')2 fragments (DAKO, Glostrup, Denmark).
Samples were analyzed using a FACScalibur instrument and CellQuest
software (Becton Dickinson, Mountain View, CA), and the mean
fluorescence intensity FL1 was used as a measure of antibody binding.
Coimmunoprecipitation of band 3 and Rh proteins from human RBC
membranes
Analysis of band 3(  /) membranes had no
protein 4.2 (Table 1; Figure 2), as previously
reported.28,32
Band 3( We also analyzed 2 other proteins, not known to be associated with the
band 3 complex. The total amount of aquaporin 1 (AQP1) was slightly
reduced (approximately 75% of controls), and the N-glycosylated
fraction of AQP1 in the band 3( Human Coimbra RBC membranes contain traces of band 3 Coimbra The patient homozygous for the band 3 Coimbra mutation was reported to completely lack RBC band 3.29 We analyzed RBC membrane proteins from this patient using a blood sample drawn 6 months after transfusion. Immunoblotting of membranes with monoclonal antibodies directed at the N-terminal or C-terminal regions of band 3 (BRIC170 and BRIC155) suggested that these cells contain traces of band 3 (approximately 2% of controls) (Table 2; Figure 3A). Because this result was at variance with the original data on the RBCs taken from cord blood,29 flow cytometry using 4 different anti-band 3 mAbs was used to determine whether this band 3 originated from residual transfused RBCs. Any residual healthy RBCs would have normal levels of band 3 and would be clearly distinguished on flow cytometry by the much larger amount of anti-band 3 bound. Results (Table 3; Figure 4) showed only one population of RBCs was present in the band 3 Coimbra sample. Event counts for the Coimbra samples indicated fewer than 1 residual transfused healthy cell in 20 000 band 3 Coimbra RBCs, using 3 different anti-band 3 antibodies (BRIC6, BRIC90, and BRIC200). These 3 different anti-band 3 antibodies reacted with band 3 Coimbra cells at levels similar to those of the negative control antibody (Table 3). However one anti-band 3 antibody (BRIC71) showed low reactivity that was greater than the negative control antibody (Table 3). Taken together with the immunoblotting results, this suggests that a small amount of the mutant band 3 is present in the band 3 Coimbra RBC membranes. The mutant band 3 retains the BRIC71 epitope but is misfolded so that it no longer displays the epitopes for the other 3 anti-band 3 mAbs used in the flow cytometry. All 4 mAb epitopes are located on the third extracellular loop of band 3, and all except the BRIC71 epitope are sensitive to chymotrypsin cleavage of RBCs,40 indicating that the BRIC71 epitope is presented by a different part of the loop. These results confirm that the band 3 remaining in the blood sample originates from the patient's own RBCs and not from residual transfused healthy RBCs.
Analysis of band 3 Coimbra RBC membranes Other membrane proteins in the band 3 Coimbra RBCs were also analyzed. Proteins associated with the GPC complex (GPC, protein 4.1, p55) were present in equivalent amounts in band 3 Coimbra and healthy RBCs (Table 2; Figure 3B). Flow cytometry using an anti-GPC mAb (BRIC4) also showed the same amount of GPC was present in control and band 3 Coimbra RBCs (Table 3). Therefore, as with the mouse membranes, we chose to use the proteins of the GPC complex to normalize the quantitative SDS-gel data (Table 2). Protein 4.2 was undetectable, and GPA was reduced (approximately 29% of controls) in band 3 Coimbra RBCs (Table 2; Figure 3A) in agreement with the previous report.29All proteins of the Rh complex were markedly reduced in the band 3 Coimbra RBC membranes, RhAG (approximately 7% of controls), Rh polypeptides (approximately 21% of controls), CD47 (approximately 2.5% of controls), LW (approximately 7% of controls), and GPB (approximately 56% of controls) (Table 2; Figure 3C). The amount of spectrin and actin in band 3 Coimbra membranes, assessed from either protein-stained or immunoblotted SDS-PAGE gels, was also reduced (approximately 63% and 71% of controls respectively) (Table 2; Figure 3A-B). AQP1 was reduced (approximately 59% of controls) (Table 2; Figure 3B), and the N-glycosylated fraction of AQP1 migrated with a faster mobility on SDS-PAGE gels than in healthy cells, suggesting that it was hypoglycosylated (Figure 3B). The amount of glucose transporter (GLUT 1) was increased in band 3 Coimbra RBCs (approximately 182% of controls), as were the GPI-linked protein, decay-accelerating factor ([DAF] CD55) (approximately 163% of controls), Lutheran protein (Lu) (approximately 169% of controls), and lymphocyte function-associated antigen 3 ([LFA-3] CD58) (approximately 320% of controls) (Table 2; Figure 3D). The lymphocyte homing receptor, CD44 (approximately 115% of controls), was present at close to normal amounts (Table 2; Figure 2D). Flow cytometric analysis of band 3 Coimbra RBCs Flow cytometry was performed on band 3 Coimbra RBCs using a range of mAbs and in general gave results that support the SDS-PAGE and immunoblotting data (Table 3; Figure 4). However, there were some exceptions. The main differences between the flow cytometry and SDS-PAGE data were with anti-GPA mAbs. Surprisingly, flow cytometric analysis of the variant RBCs (Table 3) showed more reactivity with 2 anti-GPA monoclonals (BRIC256 and R10) than would be expected from the reduced amount of GPA in these cells indicated by the immunoblotting data (Table 2). GPA associates with band 3 in healthy RBCs, and these results probably reflect an increased accessibility of GPA epitopes or altered glycosylation of GPA in band 3 Coimbra RBCs. The deficiency of nearly all the major integral membrane proteins in the band 3 Coimbra RBCs drastically changes the surface organization of the cells and, therefore, the accessibility of epitopes to antibodies. The R10 epitope is closer to the GPA N-terminus than the BRIC256 epitope,41 and the greater reactivity of R10 compared with BRIC256 may reflect a more accessible location of the R10 epitope in the variant RBCs. Given that flow cytometry is performed on intact RBCs and antibody binding depends on the presentation and steric accessibility of the antigenic sites, flow cytometry results from the healthy and band 3-deficient cells cannot be directly compared because of the very different surface organizations of the 2 cell types. This effect, combined with the intrinsic nonlinearity of the fluorescence signal, interferes with quantitative analysis by flow cytometry. Immunoblotting data more accurately reflect the quantitative amounts of the proteins present in the band 3 Coimbra RBC membranes. Unexpectedly, one Wrb mAb (BRIC14) was as reactive with band 3 Coimbra RBCs as with control RBCs, though the other (BRIC201) did not react with the variant RBCs (Table 3). The Wrb antigen depends on an interaction between band 3 and GPA.42 We presume that the small amount of band 3 in band 3 Coimbra RBCs is sufficient to form some BRIC14 epitope, and the amount of this epitope is overestimated by flow cytometry because of its increased accessibility in the variant cells (as discussed above for the R10 and BRIC256 epitopes). Flow cytometry also showed that the band 3 Coimbra RBCs were more reactive with anti-CD47 than would be expected from the immunoblotting data, as previously observed with CD47-deficient RBCs.27Coimmunoprecipitation of band 3 and Rh components from RBC membranes We sought direct evidence for the association of band 3 with the Rh complex in mature RBC membranes from coimmunoprecipitation studies using healthy human membranes. Band 3 was immunoprecipitated with the mouse monoclonal anti-band 3 BRIC169 from membranes that were completely solubilized using sodium deoxycholate. Immunoprecipitated material was separated by SDS-PAGE, and the protein components associated with band 3 were examined by immunoblotting using rabbit polyclonal antibodies or a sheep polyclonal anti-band 3. All the components examined were efficiently solubilized by sodium deoxycholate (Figure 5). RhAG and Rh were present in the band 3 immunoprecipitate, relative to band 3, in amounts similar to those present in the original membranes (Figure 5), but little CD47 appeared to be present in the band 3 immunoprecipitate. However, rabbit polyclonal anti-CD47 bound an additional sharp band (probably the immunoprecipitated mouse H-chains) in the region of the CD47 band that made it difficult to determine whether CD47 was completely absent. There was also poor recovery of protein 4.2 in the band 3 immunoprecipitate from the deoxycholate-solubilized membranes (Figure 5). Band 4.2 is known to be associated with band 3,4 and the low recovery likely results from the dissociation of protein 4.2 from band 3 by deoxycholate treatment. This probably also accounts for the poor recovery of CD47 in the band 3 immunoprecipitate because band 4.2 appears to be a major site of association of CD47 in the membrane.27 We were unable to determine whether LW was present in the immunoprecipitate because we lacked a suitable nonmurine anti-LW. As expected, no GPC or actin was detected in the immunoprecipitate (Figure 5), confirming that specific coimmunoprecipitation of Rh and RhAG with band 3 was obtained. In addition, no AQP1 was detected in the band 3 immunoprecipitate (Figure 5). Results show that band 3 is associated with the core proteins of the Rh complex (RhAG and Rh) in the RBC membrane but is not associated with AQP1. Parallel experiments were carried out using Triton X-100 instead of sodium deoxycholate (data not shown), but efficient solubilization of the membrane components required the additional presence of high salt concentrations (0.5 M KCl). We recovered less RhAG in band 3 immunoprecipitates from Triton-solubilized membranes than deoxycholate-solubilized membranes. Band 3 association with the Rh proteins may be destabilized by the high salt concentration used, or Triton X-100 may not be a suitable detergent for maintaining this association.
The most significant finding of this study is that the band 3 and
the Rh complexes are associated in the RBC membrane in a single
complex, which we term the band 3 macrocomplex. This is demonstrated by
the major deficiencies in the proteins of the Rh complex when band 3 is
absent from human or mouse RBCs and by the coimmunoprecipitation of
band 3 and Rh complex proteins (RhAG and Rh) from human RBC membranes,
and it confirms earlier indirect evidence for this
association.24-27 The number of tetrameric Rh complexes
(105 per cell43) is similar to the number of
band 3 tetramers that linked the cytoskeleton to the membrane. This,
together with observations that the Rh antigens are linked to the RBC
skeleton, suggests that the band 3 macrocomplex is formed around the
ankyrin-associated tetrameric fraction of band 3 that binds the
spectrin-actin skeleton.27 A schematic illustration of the
organization of this macrocomplex is illustrated in Figure
6A.
A major difference between mouse and human band 3-deficient RBCs is
that the mouse cells retain almost normal levels of CD47, whereas CD47
is almost completely absent from the human cells. A similar difference
is shown by mouse and human protein 4.2( Other less marked differences between the mouse and human band
3-deficient RBCs most likely reflect the dissimilar natures of the
defects that lead to band 3 deficiency in the 2 cell types. Disruption
of the band 3 gene in band 3( Another difference between the mouse and human band 3-deficient cells is that although both show a similar marked reduction in RhAG protein, the mouse cells contain almost no Rh polypeptide, whereas the human cells contain reduced but still significant amounts (21% normal) of Rh polypeptide. The fact that mice have only the RhCcEe gene, whereas humans have both the RhCcEe and the RhD genes, combined with the fact that band 3 is thought to interact more tightly with RhCcEe than with RhD polypeptides,26 may partly explain this difference, but the difference may also result from the presence of residual band 3 Coimbra in the cells. Surprisingly, there is more Rh polypeptide than RhAG in the band 3 Coimbra RBCs, suggesting that the Rh polypeptide can exist independently of RhAG in these membranes. Aquaporin 1 (AQP1) was altered in mouse and human band 3-deficient RBCs. In healthy RBCs, AQP1 migrates as 2 separate bands because only one of the subunits in the aquaporin tetramer is N-glycosylated.46 The total amount of AQP1 was reduced in the band 3-deficient cells, and the N-glycosylated aquaporin band in human and mouse band 3-deficient RBCs was noticeably sharper than in healthy cells, indicating altered AQP1 N-glycan processing. Both effects suggest that in healthy RBCs the intracellular biosynthetic pathways of band 3 and AQP1 are connected and that AQP1 movement to the cell surface is linked with band 3 in some way. One possible mechanism for this interaction is suggested by the observation that the C-termini of both proteins can bind the PDZ domain of the homo-oligomeric protein PICK1.47 PICK1 is involved in the targeting and clustering of synaptic proteins (reviewed by Deken et al48). AQP1 and band 3 may become associated during biosynthesis by the binding of both proteins to PICK1 (or a PDZ protein with similar specificity because it is unknown whether PICK1 is present in erythroid precursors). However, this association probably only occurs during the biosynthesis of the 2 proteins in erythroid precursors because our coimmunoprecipitation results suggest band 3 and AQP1 do not interact in the mature RBC membrane. This is consistent with observations that the lateral mobility of AQP1 in the RBC membrane is not affected by antibody-induced immobilization of band 3 or GPA.49 We used the proteins of the GPC complex (GPC, protein 4.1, and p55) to
normalize the levels of membrane proteins between the variant cells and
control RBCs. One group of proteins was present at significantly higher
levels in the band 3 Coimbra RBCs than the proteins of the GPC complex.
These were GLUT1, the cell adhesion protein Lu, and the GPI-anchored
proteins LFA-3 (CD58) and DAF (CD55). Cell adhesion protein CD44 was
also slightly increased. The presence of large amounts of bound
immunoglobulin on the mouse membranes (see "Materials and methods")
precluded the use of murine mAbs to identify all the corresponding
membrane proteins in the mouse band 3( The presence of all these components within one structural
macrocomplex makes it likely that the individual components have linked
functional or regulatory roles, and we can speculate on the possible
nature of this function. CAII and band 3 are part of a metabolon at the
cytoplasmic surface of the RBC membrane that accepts CO2 on
entry into the cell and forms bicarbonate, which is then channeled
through band 3 to leave the cell in exchange for
chloride.3 H2O may be supplied from outside
the cell through AQP1.55 The efflux of bicarbonate through
band 3-mediated chloride-bicarbonate exchange acidifies the cell and
causes the release of O2 from hemoglobin by the Bohr
effect. This provides the link between coordinated
CO2 uptake and O2 release by the RBC, which is
essential to meet the respiratory demands of tissues and which is
reversed in the pulmonary capillary system. Because the protons
produced by the export of HCO It has been suggested that the Rh proteins function as
CO2 channels in RBCs.14,18 Data showing that
CO2 transport into RBCs is inhibited by more than 90% by
treatment with the band 3-specific inhibitor
4,4'-di-isothiocyanato-stilbene-2,2'disulfonate (DIDS) also supports
the view that CO2 transport is mediated by a protein
closely associated with band 3.56 We speculate that the Rh
proteins might be relatively nonspecific channels for neutral small
molecules and might act as gas channels for O2 and
CO2 given that they are optimally located to channel
CO2 to and from CAII, and O2 to and from
hemoglobin in the local area around band 3. We hypothesize that the
band 3 macrocomplex acts as an O2/CO2 gas
exchange metabolon (Figure 5B), extending the model of a metabolon between band 3 and CAII that has been suggested by
others.3,55 Membrane localization and channeling offered
by this metabolon would provide the short paths for O2,
CO2, H+, and HCO Why should the RBC require the accelerated gas exchange provided by this putative metabolon? The movement of RBCs through the tissue and lung capillaries is rapid (transit time, 0.3-1 second in humans, depending on cardiac output). Cells in the tissue surrounding the microcapillary endothelium signal their requirement for O2 from RBCs by their release of CO2. To satisfy this, O2 must be released from RBCs in the vicinity of the cells that signal their need for it. Thus, RBC CO2 uptake and O2 release must be complete within the brief time required for the RBC to move past a single cell of the capillary endothelium. Slower RBC gas exchange would result in the inappropriate and potentially detrimental delivery of O2 to cells further along the capillary that might not have any requirement for O2. This would be especially important during vigorous physical exercise, when capillary transit time is decreased but CO2 production and O2 demand in muscle tissues are increased. Although there is constant movement of RBCs within the microcapillaries, intimate contact between the surfaces of RBCs and endothelial cells (to facilitate gas exchange between the 2) can be maintained over longer periods by tank-treading of the RBC membrane, which is known to occur during the movement of RBCs in narrow vessels.57 This interfacial contact may also be facilitated by transient adhesive interactions between RBC and the endothelium and local regulation of the RBC skeleton, both of which could be mediated by RBC adhesion proteins, such as CD4727 and LW. Our understanding of the properties of the RBC is based mainly on in vitro studies, under conditions in which the cell is relatively inert. Further studies that focus on the properties of RBCs during their movement through the microcirculation are likely to illuminate unexpected features of the RBC membrane and to rationalize the reasons for the complex organization of the membrane proteins and the skeleton in this "simple" cell.
We thank the family studied here for their kind cooperation. We thank P. Agre for anti-AQP1, S. A. Baldwin for anti-GLUT1, V. M. Holers for anti-Crry, D. Shotton for anti-spectrin, J. Poole and I. Skidmore for serology testing, P. G. Martin for Rh genotyping, and J. S. Smythe for helpful advice on FACS analysis.
Submitted September 23, 2002; accepted January 3, 2003.
Prepublished online as Blood First Edition Paper, January 16, 2003; DOI 10.1182/blood-2002-09-2824.
Supported in part by grants from the Wellcome Trust, the Institut National de la Santé et de la Recherche Médicale (Unité 473) alone or jointly with the Association Française contre les Myopathies (project no. 4MR09F), and the National Institutes of Health (grants HL64885, DK56267, DK26263, DK32094, and HL31579) and by the Director, Office of Health and Environment Research Division, US Department of Energy (contract DE-AC03-76SF00098).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael J. A. Tanner, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, United Kingdom; e-mail: m.tanner{at}bristol.ac.uk.
1. Tanner MJA. The structure and function of band 3 (AE1): recent developments [review]. Mol Membr Biol. 1997;14:155-165[Medline] [Order article via Infotrieve]. 2. Tanner MJA. Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders. Curr Opin Hematol. 2002;9:133-139[CrossRef][Medline] [Order article via Infotrieve]. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
V integrins on nonhematopoietic cells.
-actin (Abcam, Cambridge, United Kingdom). Rabbit
polyclonal anti-Rh, anti-p55, anti-4.1, anti-4.2 and
anti-CD47,
