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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2146-2151
Complete Deficiency of Glycophorin A in Red Blood Cells From Mice
With Targeted Inactivation of the Band 3 (AE1) Gene
By
Hani Hassoun,
Toshihiko Hanada,
Mohini Lutchman,
Kenneth E. Sahr,
Jiri Palek,
Manjit Hanspal, and
Athar H. Chishti
From the Laboratory of Tumor Cell Biology, Department of Biomedical
Research, St. Elizabeth's Medical Center, Tufts University School of
Medicine, Boston, MA.
 |
ABSTRACT |
Glycophorin A is the major transmembrane sialoglycoprotein of red
blood cells. It has been shown to contribute to the expression of the
MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai
virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane.
Several lines of evidence suggest a close interaction between
glycophorin A and band 3 during their biosynthesis. Recently, we have
generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell
membrane proteins. In this report, we show that the band 3  / red
blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin
C. In contrast, the band 3 / red blood cells are completely
devoid of glycophorin A (GPA), as assessed by Western blot and
immunocytochemistry techniques, whereas the polymerase chain reaction
(PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase
experiments show that GPA is not incorporated in the membrane and is
rapidly degraded in the cytoplasm. Based on these findings and other
published evidence, we propose that band 3 plays a chaperone-like role,
which is necessary for the recruitment of GPA to the red blood cell
plasma membrane.
| |
INTRODUCTION |
AMONG THE TRANSMEMBRANE
sialoglycoproteins of the red blood cell, glycophorin A (GPA) was the
first protein to be sequenced and has been the focus of extensive
investigations in recent years (for reviews see Chasis and
Mohandas1 and Fukuda2). This 36-kD
protein represents the major sialoglycoprotein of the red blood cell
membrane displaying about one million copies per cell. Its orientation
places its N-terminal domain outside the cell, with a single
transmembrane domain, thus connected to the cell interior via its
C-terminal cytoplasmic domain.3,4 Despite its extensive
characterization, the functional significance of GPA remains poorly
understood. Although GPA contributes to the expression of blood group
antigens, may modulate red blood cell membrane mechanical
properties,1,2 and serves as an attachment site for the
malaria parasite Plasmodium falciparum5,6 and Sendai
virus,7 its complete loss in human red blood cells is not
associated with any detectable alterations in shape, function, or
lifespan.8,9 Similarly, red blood cells with genetic
variants of GPA have been described and appear to exhibit normal
physiologic properties.10,11
To investigate the functional roles of GPA, previous studies have
focused on the putative interaction of GPA with the band 3 anion
transporter of the red blood cell membrane. Band 3, which is present in
a stoichiometrically comparable amount ( 1.2 × 106 copies per red blood cell), consists of two structural
domains as defined by limited digestion with selective proteases (for review, see Tanner12 and Low13). The N-terminal
43-kD segment constitutes the cytoplasmic domain, which interacts with
several proteins including glycolytic enzymes,14-16
hemichromes,17-19 protein 4.2,12,13 and
ankyrin.20-23 The interaction of band 3 with ankyrin provides a mechanism for coupling the membrane skeleton to the lipid
bilayer. In contrast, the C-terminal 52 kD domain of band 3 forms
multiple membrane-spanning segments through the lipid bilayer and
facilitates the efflux of HCO3 from the red
blood cell in exchange for Cl (for review, see
Jennings24).
Although a direct interaction between GPA and band 3 has not yet been
documented, several previous studies suggest that the two proteins do
interact in vivo. Evidence supporting this claim was initially provided
by the observation that the glycosylation of band 3 is altered in GPA
deficient En(a) red blood cells, as well as in other GPA
mutant red blood cells.25-27 These results suggested that
GPA directly or indirectly modulates the posttranslational modification
of band 3 in vivo. In addition, anti-GPA antibodies reduce the
rotational diffusion of band 3 suggesting that the two proteins may
associate in the red blood cell membrane.28 This view is
further strengthened by the observation that the binding of anti-GPA
antibodies to its extracellular domain rigidifies the red blood cell
membrane29 and leads to the immobilization of both GPA and
band 3 as measured by in situ FRAP (fluorescence recovery after
photobleaching) technique.30 Furthermore, reconstitution of
purified GPA and band 3 in nonionic detergents indicates that GPA may
directly associate with band 3, albeit weakly.31 Recently, GPA was shown to facilitate the expression of band 3 in Xenopus oocytes and enhance its anion transport function.32 In
addition, immunologic studies also support the view of a close GPA-band 3 interaction: a monoclonal antibody raised against band 3 was shown to
coprecipitate GPA from red blood cell membranes.33-35 Finally, the expression of the antithetical antigens Wra
and Wrb, which represent alternative polymorphisms of band
3, requires an interaction between band 3 and GPA involving residues of
the transmembrane domain of GPA.36 The presence of
additional blood group epitopes, which are dependent on the interaction
of GPA with band 3, was also suggested,37,38 and the
existence of one such epitope was recently documented.39
Taken together, these observations strongly suggest that GPA and band 3 interact at the red blood cell membrane.
Using gene targeting techniques, we and others have recently generated
mice lacking the band 3 (AE1) protein in red blood cells.40,41 The development of band 3 / mice
provides a unique opportunity to study the molecular basis of band
3-GPA interaction in vivo. Here we show that the membrane of mature
erythrocytes obtained from band 3 / mice is completely
devoid of GPA, as assessed by Western blot and immunofluorescence
techniques. Furthermore, consistent with the presence of GPA mRNA, the
GPA protein is synthesized in band 3 / erythroblasts, but
fails to assemble on the plasma membrane. Based on these observations,
and other published studies,32-39 we propose that a complex
between band 3 and GPA is formed before the delivery of these proteins
to the plasma membrane and that the band 3 protein plays an essential
role in the recruitment of GPA to the red blood cell membrane.
| |
MATERIALS AND METHODS |
Reagents.
All reagents for electrophoresis and Western blot analysis were
purchased from Bio-Rad Laboratories (Richmond, CA). Enzyme-coupled antibodies were purchased from Zymed, Inc, San Francisco,
CA. The enhanced chemiluminescence (ECL) kit, purchased from Amersham, Arlington Heights, IL, was used for Western blot analysis.
Western blotting.
Peripheral blood was collected in EDTA by tail bleeding of normal and
band 3 / adult littermates. The cells were washed three
times in phosphate-buffered saline (PBS) (5.0 mmol/L
Na2HPO4, pH 8.0, 0.5 mmol/L EGTA, 150 mmol/L
NaCl) at 4°C. Washed red blood cells were lysed in 10 volumes of
ice cold lysis buffer (5.0 mmol/L Na2HPO4, pH
8.0, 0.5 mmol/L EGTA, 2.0 mmol/L phenylmethyl sulfonyl fluoride
[PMSF]). Ghosts were prepared by repeated washing of the pelleted
membranes in lysis buffer at 4°C. The ghosts were then immediately
solubilized by boiling in Laemmli sodium dodecyl sulfate (SDS) sample
buffer and processed for electrophoresis (10% acrylamide gels) and
Western blot analysis using the ECL kit, as described by the
manufacturer. Antibodies against protein 4.2 were obtained from
Catherine Korsgren of our department, and glycophorin C from Dr Philip
Low of Purdue University (West Lafayette, IN). Details of
the antibodies against band 3, dematin, and p55 have been published
before.40,42,43 Rabbit antibodies against adducin were
raised against purified human erythrocyte adducin (our unpublished
data). Polyclonal serum recognizes both  and  subunits of adducin
in erythrocyte ghosts.
Immunocytochemistry of red blood cells.
Blood was collected in Heparin after cardiac puncture from normal and
band 3 / adult mice. The samples were centrifuged at
8,000 rpm onto Alcian Blue-coated coverslips using cytospin 3 (Shandon,
Philadelphia, PA). The cells were then fixed for 15 minutes using a 4%
paraformaldehyde solution, pH 7.4 (Baxter, Deerfield, IL).
This was followed by three washes in PBS. The red blood cells were then
permeabilized by incubation for 5 minutes in a PBS solution containing
0.1% Triton X-100 (Sigma, St Louis, MO). After washing
with PBS, the cells were blocked overnight by incubation in a PBS
solution containing 1.0% bovine serum albumin and 1.0% normal goat
serum at 4°C. The coverslips were washed again at room temperature,
and incubated with 200 µL of PBS solution containing the glycophorin
A polyclonal antibody. These rabbit antibodies were raised against the
cytoplasmic domain of human GPA (a gift from Dr M. Fukuda, La Jolla
Cancer Research Foundation, La Jolla, CA). The coverslips
were incubated overnight at 4°C with serum diluted to 1:50, washed
three times with PBS, and the signal was detected with the goat
antirabbit/rhodamine-conjugated secondary antibody (1:100 dilution in
PBS). As controls, both normal and band 3 / red blood
cells were treated with the secondary antibody alone. The coverslips
were mounted onto slides using Fluoro Guard AntiFade reagent (Bio-Rad
Laboratories, Melville, NY). Photographs were taken on the Nikon
confocal microscope under 60X oil immersion lens.
Isolation of RNA and reverse transcriptase-polymerase chain reaction
(RT-PCR).
RNA was prepared from the spleens of normal and band 3 null
littermates. Spleens were removed and minced in a small volume of cold
PBS on ice. Total RNA was then isolated using the Ultraspec isolation
system (Biotecx Laboratories, Houston, TX). cDNA was synthesized from
1.0 µg of total RNA using the Moloney murine leukemia virus
(M-MLV) reverse transcriptase (Promega Corp, Madison, WI), with random hexamer oligonucleotide primers under standard reaction conditions.44 An aliquot of each cDNA was then
amplified by PCR using primers specific for mouse GPA45
(sense primer 5 -CCCAGTATGACCGAGAGCACA-3 and antisense
primer 5-TCTTCATTAGGAGTCTGCTCA-3), which yielded a
product of 497 bp. An aliquot of each cDNA was also used to amplify
band 3 (sense primer 5-CTCAGCCAGTCACAGAG-3 and antisense
primer 5-GCTCCACATAGACCTGACC-3) and globin (sense
primer 5-TGGTGCACCTGACTGATG-3 and antisense primer
5-GTGGTACTTGTGAGCCAA-3) specific sequences. The expected PCR products using the band 3 and the globin primers are 362 bp and
420 bp in length, respectively. An aliquot of each PCR reaction was
analyzed by electrophoresis on a 1.5 % (wt/vol) agarose gel.
Biosynthesis of GPA in erythroblasts.
Band 3 / spleens were placed in ice cold Iscove's
modified Dulbecco's medium (IMDM, GIBCO Laboratories, Grand Island,
NY). A single cell suspension of spleen was obtained by disrupting the
tissue with tweezers and passing through a polyethylene mesh (Spectramesh, Spectrum Medical Industries, Los Angeles, CA) of pore
size 202 µm. The cell suspension was subjected to a discontinuous Percoll gradient (Percoll; Pharmacia Fine Chemicals, Piscataway, NJ)
consisting of 45%, 65%, 70%, 77%, and 90% Percoll in
IMDM.46 Fraction 4 consisted almost entirely of late
(polychromatophilic and orthochromatic) erythroblasts as judged by
staining with Wright-Giemsa and benzidine-hematoxylin. Morphologically
identical control erythroblasts were similarly obtained from spleens of
Balb/c mice 25 days after infection with Friend anemia virus
(FVA).47 Control and B3/ erythroblasts were
metabolically labeled with [35S]methionine (30 µCi/mL,
1,000 Ci/mmol, ICN Biomedicals, Irvine, CA) for 60 minutes and chased
with unlabeled methionine (0.4 mmol/L) for different time periods.
[35S]methionine-labeled cells were lysed in hypotonic
buffer (10 mmol/L Tris-HCl, pH 7.5, 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 1 mmol/L PMSF, 1 mmol/L leupeptin, and 10 µg/mL
aprotinin) and then disrupted with 10 strokes of a tight-fitting Dounce
homogenizer. An appropriate volume of 2.0 mol/L sucrose was added
immediately to restore isotonicity. The homogenate was centrifuged at
800g for 5 minutes to remove nuclei. The resulting supernatant
was centrifuged at 18,000g for 20 minutes to separate the
insoluble plasma membrane fraction from the soluble fraction containing fragments of endoplasmic reticulum (ER). The latter could be purified by centrifugation of the soluble fraction at 100,000g for 2 hours followed by fractionation on a concanavalin A column, but in the present study, this purification step was omitted because of the limited quantity of erythroblasts. GPA was immunoprecipitated from both
the soluble and membrane fractions using rabbit polyclonal antibodies
against the cytoplasmic domain of GPA.
| |
RESULTS AND DISCUSSION |
A large body of evidence supports the notion that band 3 may interact
with GPA in the red blood cell membrane.12,32-39 However, a
limitation to the in vivo documentation of such an interaction is the
lack of vertebrate models displaying homozygous loss of band 3 in human
red blood cells. The recent development of band 3 /
animal models40,41,48 allowed us to investigate the consequences of band 3 deficiency on the assembly of membrane proteins
in murine red blood cells. We first investigated the presence of GPA on
the red blood cell membranes of band 3 / mice by Western
blot analysis. Polyclonal antibodies raised against the cytoplasmic
domain of human GPA were used to detect the level of GPA in the murine
red blood cell membranes. It should be noted that the cytoplasmic
domains of human and mouse GPA share 46% sequence identity (Lasergene
MEGALIGN program, Irvine, CA), and as a consequence, the
predicted antigenic indexes of the cytoplasmic domains of human and
murine GPA are remarkably similar suggesting conservation of the
immunoreactive epitopes in the two species (data not shown). Therefore,
antibodies raised against human GPA would be expected to cross-react
with murine GPA. Using these antibodies, we examined red blood cell
membranes of band 3 / mice and their wild-type
littermates for the presence of GPA. The samples analyzed on
polyacrylamide gels were purposely overloaded to detect trace amounts
of GPA. As shown in Fig 1, Western blotting failed to detect any measurable amount of GPA in the red blood cell
membrane of band 3 / mice. In contrast, the dimeric form of GPA was detected in the red blood cell membranes of wild-type littermates (Fig 1). The absence of GPA in band 3 / red
blood cells was confirmed in three additional mice of different ages ranging from 1 to 4 months (data not shown). It is relevant to mention
here that the anti-GPA antibodies used in this study would not detect
the presence of glycophorin B (GPB) and glycophorin E (GPE), as these
proteins lack the cytoplasmic domain of GPA.1,2 Therefore,
the status of GPB and GPE in band 3 / red blood cells remains to be investigated.
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| Fig 1.
Western blot analysis of the red blood cell membrane
proteins. Red blood cells were collected from both band 3 +/+ and
band 3 /mice and washed in PBS. Ghosts were prepared by lysis as described in Materials and Methods. Ghosts membranes were solubilized and analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). After transfer to nitrocellulose, Western
blot analysis was performed using the ECL chemiluminescence kit as
suggested by the manufacturer. Both band 3 +/+ and band 3 /
red blood cell membranes contain protein 4.1, glycophorin C, p55,
dematin, and adducin, while the complete absence of protein 4.2 and GPA is noted. The GPA panel shows the position of the dimer. No monomeric GPA was detected (data reviewed, but not shown). It should be noted
that each membrane fraction used for Western blotting was isolated from
the same number of normal and band 3 / red blood cells. However,
a more precise quantitative method such as enzyme-linked immunosorbent
assay (ELISA) will be required to compare the absolute amounts of
membrane proteins in normal and band 3 null red blood cells.
|
|
To confirm the absence of GPA in band 3 / red blood
cells, these cells were examined by indirect immunofluorescence after a
brief exposure to Triton X-100 to allow permeabilization and penetration of the antibodies into the cells. As shown in
Fig 2, no GPA signal was detected in band 3 / red blood cells, whereas the GPA epitopes were present
in the band 3 +/+ red blood cells. Having established the absence of
GPA expression on the membrane of band 3 / red blood
cells, we next examined whether the GPA mRNA is present in the
cytoplasm of early erythroblasts isolated from the spleens of band 3 / mice. Total RNA was reverse transcribed and GPA cDNA
was amplified by PCR using GPA-specific primers (see Materials and
Methods). As shown in Fig 3, a comparable
amount of GPA mRNA is present in normal and band 3 /
erythroblasts, although a more precise quantification will be required
to determine the absolute amounts of GPA mRNA in normal and band 3 null
red blood cells. The positive and negative controls used for the PCR amplification consisted of globin and band 3 mRNA, respectively (Fig 3).
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| Fig 2.
Immunocytochemistry of red blood cells. Red blood cells
from band 3 +/+ and band 3 / mice were fixed onto Alcian
Blue-coated coverslips with 4% paraformaldehyde. After
permeabilization of the cells in a PBS solution containing 0.1%
Triton-X 100 and blocking in a PBS solution with 1.0% bovine serum
albumin and 1.0% goat serum, the cells were incubated with a rabbit
antibody raised against the cytoplasmic domain of human GPA. The signal
was detected with a goat antirabbit/rhodamine-conjugated secondary
antibody. In contrast to band 3 +/+ red blood cells, band 3 /red blood cells do not display any detectable level of GPA on
their surface.
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| Fig 3.
RT-PCR of mRNA isolated from spleen erythropoietic cells.
Total RNA was isolated from the spleens of normal and band 3 null mice
as described in Materials and Methods. cDNA was synthesized using
random hexamer primers and M-MLV reverse transcriptase. Specific GPA
primers were used to amplify a small fragment of the GPA cDNA. Small
segments of band 3 and globin cDNA were also amplified as negative
and positive controls, respectively. The results confirm the presence
of GPA mRNA indicating that the transcription of the gene takes place
in band 3 / erythroblasts.
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|
The complete absence of GPA protein in band 3 / red blood
cells, despite an apparently normal transcription of its gene, suggested that the loss of GPA in the red blood cell membranes may be
due to an abnormal synthesis and turnover of GPA polypeptides in vivo.
To test this hypothesis, we measured the rate of synthesis, the
stability, and the membrane incorporation of GPA in murine erythroblasts. Control and band 3 / erythroblasts were
metabolically labeled with [35 S]methionine for 60 minutes and pulse-chased with unlabeled methionine for specified time
intervals. The soluble and membrane fractions were separated and
immunoprecipitated with anti-GPA antibodies. In normal erythroblasts,
GPA immunoprecipitation showed that the newly synthesized protein in
the soluble fraction is, as expected, approximately 5.0 kD shorter than
the protein assembled in the membrane fraction
(Fig 4). This size difference presumably
reflects the degree of glycosylation: the newly synthesized GPA is
cotranslationally inserted into the ER membrane where it gets
N-glycosylated. Complete glycosylation takes place by the addition of
O-linked sugars during its transit to the plasma
membrane.2,49 In band 3 / erythroblasts, newly synthesized GPA is also detected in the soluble fraction. The
amount of GPA synthesized in band 3 / cells during the
60-minute labeling period (ie, at time 0 minute of chase) is decreased
compared with the amount in band 3 +/+ cells, but reaches normal levels after 60 minutes of chase. These results suggest that the synthesis of
GPA is delayed in band 3 / cells. The increase in the
amount of radiolabeled GPA after 60 minutes of chase likely represents the completion and release of nascent polypeptides synthesized during
the pulse labeling period. Once the synthesis is complete, the newly
synthesized GPA assembles stably in the membrane of band 3 +/+ cells.
In contrast, the newly synthesized GPA in band 3 / cells
is not incorporated into the membrane and is presumably degraded in the
cytoplasm (Fig 4). At this stage, the basis for the delayed synthesis
of GPA in band 3 / erythroblasts is not known. A
reasonable speculation is that the presence of band 3 has a modulatory
effect on the synthesis and turnover of GPA in vivo.
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| Fig 4.
Synthesis, stability, and membrane incorporation of GPA
in mouse erythroblasts. Band 3 +/+ and band 3 / erythroblasts
were labeled with [35S]methionine for 60 minutes and then
chased with unlabeled methionine for 0, 60, and 120 minutes.
Thereafter, the cells were lysed and separated into soluble (ER) and
insoluble (membrane) fractions. The identity of the membrane fraction
derived from band 3 / erythroblasts was confirmed by
immunoprecipitation of protein 4.1 (data reviewed, but not shown).
Equal volumes of each sample were immunoprecipitated using anti-GPA
antibodies and the immunoprecipitates were analyzed by SDS-PAGE. The
immunoprecipitation conditions were similar to those described
previously.46 The gels were processed for fluorography. The
immunoprecipitated bands in the autoradiogram correspond to the
position of GPA dimer. The absence of GPA in the membrane fraction of
band 3 / erythroblasts shows that despite the synthesis of GPA in
band 3/ erythroblasts, GPA is not recruited to the plasma
membrane and is presumably degraded rapidly in the cytoplasm. The
results of immunoprecipitated GPA were confirmed by three independent
experiments. GPa, partially glycosylated glycophorin A; GPA, completely
glycosylated glycophorin A.
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Band 3 serves as a major attachment site between the
spectrin-actin-based skeleton and the plasma membrane. This physical membrane-skeleton coupling is largely achieved by the formation of a
complex between band 3, ankyrin, spectrin, and protein 4.2 (for
review, see Tanner12 and Low13). In addition,
it is believed that band 3 also plays a role in the attachment of the
spectrin-actin junctional complexes to the membrane via its binding to
protein 4.1.50,51 Because spectrin-actin
junctional complexes also contain proteins such as adducin, dematin,
and p55, we examined the presence of these proteins in band 3 / red blood cell membranes. As shown in Fig 1, Western
blotting demonstrated the presence of protein 4.1, glycophorin C, p55, dematin, and adducin in band 3 / red blood cell
membranes. These results indicate that the presence of band 3 is not
required for the assembly of the junctional complex proteins on the red
blood cell membrane and supports the notion that the ternary complex consisting of glycophorin C, protein 4.1, and p55 provides an independent site for the attachment of spectrin-actin junctional complexes in vivo.43,52 In contrast, the concomitant loss
of protein 4.2 in band 3 / red blood cells suggests that
another dominant site for the attachment of the spectrin-ankyrin
complex to the plasma membrane may be provided by the ternary complex consisting of band 3, GPA, and protein 4.2.
Because of the unavailability of mature red blood cells completely
deficient in the band 3 protein, previous studies have focused on
studying the effects of GPA loss on the expression, biosynthesis, and
processing of band 3. The complete loss of GPA in naturally occurring
En(a-) and MkMk red blood cells is
not associated with any significant phenotypic abnormality.8-10 Nevertheless, the loss of GPA was shown to
modulate the extent of the N-glycan chain on band 3 in GPA-deficient
red blood cells.8,25,53 These observations led to the
suggestion that the absence of GPA may delay the export of band 3 from
the golgi apparatus, thus extending the length of band 3 glycosylation during erythropoiesis. Similarly, the sulfate transport properties of
band 3 were shown to be impaired in red blood cells lacking GPA,
suggesting a reduced affinity of band 3 for sulfate anions in the
absence of GPA.53 Finally, in the Xenopus oocyte
expression system, it was shown that GPA facilitates the expression of
band 3 on the membrane and enhances the anion transport function of band 3 in a heterologous expression system.54 Together,
these previous studies clearly suggest an important role for GPA in the
regulation of band 3 functions in vivo and provide convincing evidence
for the physical interaction of these proteins in the red blood cell
membrane. However, it is relevant to mention here that human
erythroleukemic K562 cells do not synthesize band 3 (AE1) protein, yet
they express a surface membrane glycoprotein that is identical or
closely similar to GPA.55,56 This observation suggests that
other alternate mechanisms may exist in K562 cells that compensate for
the absence of band 3. Indeed, K562 cells are reported to express a
band 3-like glycoprotein termed GP105 that may associate with GPA and
functionally substitute for the AE1 protein.55,56
In conclusion, the recent development of band 3 / mice by
us and others40,41 allowed us to study the effect of band 3 loss on GPA and other red blood cell membrane proteins. Our findings showing complete loss of GPA in band 3 / mice provide,
for the first time, an unequivocal proof that the biosysthesis and
stability of GPA is dependent on the presence of band 3 in vivo. Our
results also suggest that band 3, GPA, and protein 4.2 may form a
ternary complex in the red blood cell membrane. Based on these findings and other published evidence,32-39 we propose that band 3 not only interacts with GPA in the membrane of mature red blood cells, but also plays an essential role in the processing and transport of
intracellular GPA during erythropoiesis. This putative
"chaperone-like" function of band 3 is consistent with similar
paradigms established for other membrane proteins.57,58 The
loss of GPA and protein 4.2 in band 3 / red blood cells
raises interesting questions about the role of band 3 and protein 4.2 on the kinetics of transport, assembly, expression, and processing of
GPA in the red blood cell membrane.
| |
FOOTNOTES |
Submitted June 30, 1997;
accepted October 31, 1997.
H.H. and T.H. contributed equally to this report.
Supported by Grants No. HL 51445 and CA66263 (to A.H.C.) and KO8
HL02720 (to H.H.) from the National Institutes of Health, Bethesda,
MD. A.H.C. is an established investigator of the American Heart Association.
Address reprint requests to Athar H. Chishti, PhD, ACH4
Building, St. Elizabeth's Medical Center, 736 Cambridge St, Boston, MA
02135.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Iva Smockova for her technical help in the
biosynthetic experiments, Jennifer Wu for her expertise in alignment analysis and editorial assistance, Donna-Marie Mironchuk for the art
work, and Dr Shih-Chun Liu for helpful discussions.
| |
REFERENCES |
1.
Chasis JA,
Mohandas N:
Red blood cell glycophorins.
Blood
80:1869,
1992[Free Full Text]
2.
Fukuda M:
Molecular genetics of the glycophorin A gene cluster.
Semin Hematol
30:138,
1993[Medline]
[Order article via Infotrieve]
3.
Furthmayr H:
Structural comparison of glycophorins and immunochemical analysis of genetic variants.
Nature
271:519,
1978[Medline]
[Order article via Infotrieve]
4.
Siebert PD,
Fukuda M:
Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: Nucleotide sequence and mRNA structure.
Proc Natl Acad Sci USA
83:1665,
1986[Abstract/Free Full Text]
5.
Perkins M:
Inhibitory effects of erythrocyte membrane proteins on the in vitro invasion of the human malarial parasite (Plasmodium falciparum) into its host cell.
J Cell Biol
90:563,
1981[Abstract/Free Full Text]
6.
Gratzer WB,
Dluzewski AR:
The red blood cell and malaria parasite invasion.
Semin Hematol
30:232,
1993[Medline]
[Order article via Infotrieve]
7.
Wybenga LE,
Epand RF,
Nir S,
Chu JW,
Sharom FJ,
Flanagan TD,
Epand RM:
Glycophorin as a receptor for Sandai virus.
Biochemistry
35:9513,
1996[Medline]
[Order article via Infotrieve]
8.
Tanner MJA,
Anstee DJ:
The membrane change in En(a-) human erythrocytes.
Biochem J
153:271,
1976[Medline]
[Order article via Infotrieve]
9.
Dahr W,
Uhlenbruck G:
Studies on the membrane glycoprotein defect of En(a-) erythrocytes. I. Biochemical aspects.
J Immunogenet
3:329,
1976[Medline]
[Order article via Infotrieve]
10.
Tokunaga E,
Sasakawa S:
Two apparently healthy Japanese individuals of type Mk Mk have erythrocytes which lack both blood group MN and Ss-active sialoglycoproteins.
J Immunogenet
6:383,
1979[Medline]
[Order article via Infotrieve]
11.
Reid ME,
Anstee DJ,
Jensen RH,
Mohandas N:
Normal membrane function of abnormal beta-related erythrocyte sialoglycoproteins.
Br J Haematol
67:467,
1987[Medline]
[Order article via Infotrieve]
12.
Tanner MJA:
Molecular and cellular biology of the erythrocyte anion exchanger (AE1).
Semin Hematol
30:34,
1993[Medline]
[Order article via Infotrieve]
13.
Low PS:
Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions.
Biochim Biophys Acta
864:145,
1986[Medline]
[Order article via Infotrieve]
14.
Kliman HJ,
Steck TL:
Association of glyceraldehyde-3-phosphate dehydrogenase with the human red cell membrane.
J Biol Chem
255:6314,
1980[Abstract/Free Full Text]
15.
Tsai I,
Prasanna Murthy SN,
Steck TL:
Effect of red cell membrane binding on the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase.
J Biol Chem
257:1438,
1982[Abstract/Free Full Text]
16.
Yu J,
Steck TL:
Associations of band 3, the predominant polypeptide of the human erythrocyte membrane.
J Biol Chem
250:9176,
1975
17.
Waugh SM,
Walder JA,
Low PS:
Partial characterization of the copolymerization reaction of erythrocyte membrane band 3 with hemichromes.
Biochemistry
26:1777,
1987[Medline]
[Order article via Infotrieve]
18.
Kannan R,
Labotka R,
Low PS:
Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes.
J Biol Chem
263:13766,
1988[Abstract/Free Full Text]
19.
McPherson RA,
Sawyer WH,
Tilley L:
Rotational diffusion of the erythrocyte integral membrane protein band 3: Effect of hemichrome binding.
Biochemistry
31:512,
1992[Medline]
[Order article via Infotrieve]
20.
Bennett V,
Stenbuck PJ:
The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes.
Nature
280:468,
1979[Medline]
[Order article via Infotrieve]
21.
Davis L,
Lux SE,
Bennett V:
Mapping the ankyrin-binding site on the human erythrocyte anion exchanger.
J Biol Chem
264:9665,
1989[Abstract/Free Full Text]
22.
Hargreaves WR,
Giedd KN,
Verkleij A,
Branton D:
Reassociation of ankyrin with band 3 in erythrocyte membranes and in lipid vesicles.
J Biol Chem
255:11965,
1980[Abstract/Free Full Text]
23.
Thevenin BJM,
Low PS:
Kinetics and regulation of the ankyrin-band 3 interactions of the human red blood cell membrane.
J Biol Chem
265:16166,
1990[Abstract/Free Full Text]
24.
Jennings ML:
Structure and function of the red blood cell anion transport protein.
Annu Rev Biophys Chem
18:397,
1989[Medline]
[Order article via Infotrieve]
25.
Gahmberg CG,
Myllyla G,
Leikola J,
Pirkola A,
Nordling S:
Absence of the major sialoglycoprotein in the membrane of human En (a-) erythrocyte and increased glycosylation of band 3.
J Biol Chem
251:6108,
1976[Abstract/Free Full Text]
26.
Sheetz MP,
Sawyer D:
Triton shells of intact erythrocytes.
J Supramol Struct
8:399,
1978[Medline]
[Order article via Infotrieve]
27. Dahr W: Immunochemistry of sialoglycoproteins in human red blood
cell membranes, in Vengelen-Tyler V, Judd WJ (eds): Recent Advances In
Blood Group Biochemistry. Arlington, VA, American Association of Blood
Banks, 1986, p 23
28.
Nigg EA,
Bron EA,
Girardel M,
Cheryl RJ:
Band 3-glycophorin A association in erythrocyte membranes demonstrated by combining protein diffusion measurements with antibody-induced crosslinking.
Biochemistry
19:1887,
1980[Medline]
[Order article via Infotrieve]
29.
Chasis JA,
Reid ME,
Jensen RH,
Mohandas N:
Signal transduction by glycophorin A: Role of extracellular and cytoplasmic domains in a modulatable process.
J Cell Biol
107:1351,
1988[Abstract/Free Full Text]
30.
Knowles DW,
Chasis JA,
Evans EA,
Mohandas N:
Cooperative action between band 3 and glycophorin A in human erythrocytes: Immobilization of band 3 induced by antibodies to glycophorin A.
Biophys J
66:1726,
1994[Medline]
[Order article via Infotrieve]
31.
Dahr W,
Wilkinson S,
Issitt PD,
Beyreuther K,
Hummel M,
Morel P:
High frequency antigens of the human erythrocyte membrane sialoglycoproteins. III. Studies on the EnaFR, Wrb and Wra antigens.
Biol Chem Hoppe Seyler
367:1033,
1986[Medline]
[Order article via Infotrieve]
32.
Groves JD,
Tanner MJA:
Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.
J Biol Chem
267:22163,
1992[Abstract/Free Full Text]
33.
Telen MJ,
Chasis JA:
Relationship of the human erythrocyte Wrb antigen to an interaction between glycophorin A and band 3.
Blood
76:842,
1990[Abstract/Free Full Text]
34.
Ring SM,
Tippett P,
Swallow DA:
Comparative immunochemical analysis of the Wra and Wrb red cell antigens.
Vox Sang
67:226,
1994[Medline]
[Order article via Infotrieve]
35.
Wainwright SD,
Tanner MJA,
Martin GEM,
Yendle JE,
Holmes C:
Monoclonal antibodies to the membrane domain of the human erythrocyte anion transport protein. Localization of the C-terminus of the protein to the cytoplasmic side of the red cell membrane and distribution of the protein in some tissues.
Biochem J
258:211,
1989[Medline]
[Order article via Infotrieve]
36.
Huang CH,
Reid ME,
Xie SS,
Blumenfeld OO:
Human red blood cell wright antigens: A genetic and evolutionary perspective on glycophorin A-band 3 interaction.
Blood
87:3942,
1996[Abstract/Free Full Text]
37.
Leddy JP,
Wilkinson SL,
Kissel GE,
Passador ST,
Falany JL,
Rosenfeld SI:
Erythrocyte membrane proteins reactive with IgG (warm-reacting) anti-red blood cell autoantibodies: II. Antibodies coprecipitating band 3 and glycophorin A.
Blood
84:650,
1994[Abstract/Free Full Text]
38.
Bruce LJ,
Ring SM,
Anstee DJ,
Reid ME,
Wilkinson S,
Tanner,
MJA:
Changes in the blood group antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: A site of interaction between band 3 and glycophorin A under certain conditions.
Blood
85:541,
1995[Abstract/Free Full Text]
39. (abstr)
Jarolim P,
Moulds JM,
Moulds JJ,
Rubin HL,
Dahr W:
MARS and AVIS blood group antigens: Polymorphism of glycophorin A affects band 3-glycophorin A interaction.
Blood
88:182a,
1996
40.
Southgate CD,
Chishti AH,
Mitchell B,
Yi SJ,
Palek J:
Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe hemolytic anemia despite a normal membrane skeleton.
Nat Genet
14:227,
1997
41.
Peters LL,
Shivdasani RA,
Liu S,
Hanspal M,
John KM,
Gonzalez JM,
Brugnara C,
Gwynn B,
Mohandas N,
Alper SL,
Orkin SH,
Lux SE:
Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton.
Cell
86:917,
1996[Medline]
[Order article via Infotrieve]
42.
Chishti AH,
Faquin W,
Wu CC,
Branton D:
Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity.
J Biol Chem
264:8985,
1989[Abstract/Free Full Text]
43.
Alloisio N,
Venezia ND,
Rana A,
Andrabi K,
Texier P,
Gilsanz F,
Cartron J-P,
Delaunay J,
Chishti AH:
Evidence that red blood cell protein p55 may participate in the skeleton-membrane linkage that involves protein 4.1 and glycophorin C.
Blood
82:1323,
1993[Abstract/Free Full Text]
44. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A
laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989
45.
Gu H,
Planas J,
Gomez R,
Wilson DJ:
Full length mouse glycophorin A gene constructed using recombinant polymerase chain reaction.
Biochem Biophys Res Commun
177:202,
1997
46.
Hanspal M,
Palek J:
Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors.
J Cell Biol
105:1417,
1987[Abstract/Free Full Text]
47.
Hanspal M,
Hanspal J,
Kalraiya R,
Liu S,
Sahr KE,
Howard D,
Palek J:
Asynchronous synthesis of membrane skeletal proteins during terminal maturation of murine erythroblasts.
Blood
80:530,
1992[Abstract/Free Full Text]
48.
Inaba M,
Yawata A,
Koshino I,
Sato K,
Takeushi M,
Takakuwa Y,
Manno S,
Yawata Y,
Kanzaki A,
Sakai J,
Ban A,
Ono K,
Maede Y:
Defective anion transport and marked spherocytosis with membrane instability caused by hereditary total deficiency of red cell band 3 in cattle due to a nonsense mutation.
J Clin Invest
97:1804,
1996[Medline]
[Order article via Infotrieve]
49.
Jokinen M,
Ulmanen I,
Anderson LC,
Kaarianen L,
Gahmberg CG:
Cell-free synthesis and glycosylation of the major human red cell sialoglycoprotein, glycophorin A.
Eur J Biochem
114:393,
1981[Medline]
[Order article via Infotrieve]
50.
Pasternack GR,
Anderson RA,
Leto TL,
Marchesi VT:
Interactions between protein 4.1 and band 3: An alternative binding site for an element of the membrane skeleton.
J Biol Chem
260:3676,
1985[Abstract/Free Full Text]
51.
Lombardo CR,
Willardson BM,
Low PS:
Localization of the protein 4.1 binding site on the cytoplasmic domain of erythrocyte membrane band 3.
J Biol Chem
267:9540,
1992[Abstract/Free Full Text]
52.
Marfatia SM,
Cabral JHC,
Kim AC,
Byron O,
Chishti AH:
The PDZ domain of human erythrocyte p55 mediates its binding to the cytoplasmic carboxyl terminus of glycophorin C. Analysis of the binding interface by in vitro mutagenesis.
J Biol Chem
272:24191,
1997[Abstract/Free Full Text]
53.
Bruce LJ,
Groves JD,
Okubo Y,
Thilaganathan B,
Tanner MJA:
Altered band 3 structure and function in glycophorin A- and B-deficient (Mk Mk) red blood cells.
Blood
84:916,
1994[Abstract/Free Full Text]
54.
Groves JD,
Tanner MJA:
The effects of glycophorin A on the expression of the human red cell anion transporter (band 3) in Xenopus oocytes.
J Membr Biol
140:81,
1994[Medline]
[Order article via Infotrieve]
55.
Marshall LM,
Thureson-Klein A,
Hunt RC:
Exclusion of erythrocyte-specific membrane proteins from clathrin-coated pits during differentiation of human erythroleukemic cells.
J Cell Biol
98:2055,
1984[Abstract/Free Full Text]
56.
Gahmberg CG,
Jokinen M,
Andersson LC:
Expression of the major red cell sialoglycoprotein, glycophorin A, in the human leukemic cell line K562.
J Biol Chem
254:7442,
1979[Abstract/Free Full Text]
57.
Watanabe M,
Blobel G:
High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein.
Proc Natl Acad Sci USA
92:10133,
1995[Abstract/Free Full Text]
58. Randall LL, Topping TB, Hardy SJS: in Morimoto RI,
Tissieres A, Georgopoulos C (eds): The Biology of Heat Shock Proteins and Molecular Chaperons. Plainview, NY, Cold Spring
Harbor, 1994, p 385
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Abstract
Introduction
Abstract