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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4428-4438
Functional Cell Surface Expression of Band 3, the Human Red
Blood Cell Anion Exchange Protein (AE1), in K562
Erythroleukemia Cells: Band 3 Enhances the Cell Surface Reactivity
of Rh Antigens
By
Roland Beckmann,
Jonathan S. Smythe,
David J. Anstee, and
Michael
J.A. Tanner
From the Department of Biochemistry, University of Bristol, UK; and
the International Blood Group Reference Laboratory, Bristol, UK.
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ABSTRACT |
Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe
retroviral vector. Stable K562 clones expressing band 3 were isolated
by flow cytometry, and surface expression was quantified by
immunoblotting. The function of band 3 expressed at the cell surface
was demonstrated in chloride transport assays. K562 cells expressing
band 3 also displayed high levels of the Wrb blood group
antigen, confirming the role of band 3 in Wrb expression,
and an increase in the low levels of endogenous Rh antigen activity. We
also performed coexpression experiments with K562 clones that had
previously been transduced with cDNAs encoding RhD or RhcE
polypeptides. The transfection and expression of band 3 in these clones
substantially increased the levels of RhD and cE antigen activity
expressed on the cells and also increased the reactivity of the cells
with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The
increased reactivity of Rh antigens may result from cell surface or
intracellular interactions of band 3 with the protein complex which
contains the Rh polypeptides and RhGP, or from indirect effects of band
3 on the membrane environment. This work establishes a system for cell
surface expression of band 3 in a mammalian cell line, which will
enable further studies of the protein and its interactions with other
membrane components.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
BAND 3, THE HUMAN RED blood cell (RBC)
anion exchange protein (AE1), is the most abundant integral membrane
protein found in erythrocytes and a well-characterized
transporter.1-6 The expression of band 3 in heterologous
systems7 is of interest for several reasons. First, the
ability to express band 3 mutants is necessary for the development of
improved structural and functional models for the protein. Second, it
is important to understand how the biosynthesis of transport-active
band 3 is regulated, allowing the maturing RBC to overcome any
potential problems associated with the transient presence of a
pH-modifying transporter in intracellular membranes. Third, the study
of the interaction of band 3 with other proteins present in the
erythrocyte plasma membrane and in the skeleton is essential for our
understanding of erythrocyte maturation. Functional band 3 has been
expressed to the cell surface in Xenopus oocytes injected with
mouse band 3 poly-A+mRNA,8 mouse band 3 cRNA,9 and human band 3 cRNA.10
Transport-active human band 3 membrane domain has been expressed to the
cell surface in Saccharomyces cerevisiae,11 whereas
the full-length protein expressed in yeast is retained in internal
membranes.12 Functional cell surface expression of band 3 in mammalian cell lines has not been reported to date. Transfection of
HEK 293 cells by Ruetz et al13 resulted in the synthesis of
significant levels of functional band 3, but the protein was retained
in intracellular membranes. Transfected cells showed abnormal
morphology, possibly as a result of intracellular band 3 anion
transport activity. The work of Gomez and Morgans14 using
HEK 293 cells transfected with band 3 cDNA suggested that ankyrin binds
to band 3 in the endoplasmic reticulum of these cells and
may facilitate the exit of band 3 from this compartment, but not its
movement to the cell surface.
An important aspect of band 3 expression is its interaction with
glycophorin A (GPA). Evidence for this comes from the study of two rare
types of human RBCs; MkMk cells, which lack
glycophorins A and B, and homozygous En(a ) cells, which lack
only GPA.15,16 The band 3 found in these cells migrates
more slowly on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) than normal band 3, due to increased N-glycosylation.17-21 The increased glycosylation is
indicative of a slower movement of the protein through the Golgi
complex.22 Bruce et al21 also studied the anion
transport activity of band 3 in MkMk RBCs and
showed that the sulphate transport activity of the protein in these
cells is reduced to about 60% of that of normal RBC band 3. In
MkMk cells, the Vmax for sulphate
influx remained unaltered, but the apparent KM was
substantially higher than in normal cells. Furthermore, Groves and
Tanner23,24 showed that GPA increases band 3-mediated chloride transport in the Xenopus oocyte system by increasing the rate at which band 3 accumulates in the oocyte plasma membrane. For
these reasons, it has been suggested that GPA acts like a chaperone for
band 3 by forming an intracellular complex with the newly synthesized
protein. The formation of this complex is thought to promote structural
and conformational changes to band 3, making it fully transport active,
and increasing its rate of translocation to the cell
surface.7
Here we report the functional expression of human band 3 at the cell
surface in the human K562 erythroleukemia cell line. K562
cells25 do not express endogenous band 3, lack major
erythroid surface antigens such as ABH and
Wrb,26,27 and either do not express or express
only low levels of the Rh polypeptides28,29 and the
LW30 and Fy31 blood group active proteins.
However they do express GPA,32 RhGP,28 CD47,29 and, on induction,33 some other
erythroid proteins, including hemoglobin. The K562 cell line is
therefore potentially suitable as an expression system for band 3. We
transfected K562 cells with band 3 cDNA, using the pBabe retroviral
vectors,34 which have recently been used to express the RhD
and G or the c and E antigens in K562 clones.35 Our work
resulted in the isolation of stable K562 clones expressing high levels
of functional band 3 on the cell surface and thus presents a novel
expression system for band 3. We examined the effects of band 3 on the
expression of other antigens in K562 cells and showed that band 3 surface expression coincides with the appearance of high levels of the Wrb antigen. We also detected an increase in the low level
of endogenous Rh antigen activity. To further investigate the effect of
band 3 on the expression of Rh polypeptides, we studied K562 clones sequentially transfected with cDNAs encoding RhD or cE and then band 3 cDNA and found that the expression of band 3 substantially increases
the reactivity of the K562/Rh clones with antibodies to the RhD, c, and
E antigens, as well as antibody to the RhGP glycoprotein. This may
arise from indirect effects of band 3 on the membrane environment.
However, it also suggests the possibility that band 3 may interact with
the complex containing the Rh polypeptides and RhGP either at the cell
surface or during the folding and translocation of newly synthesized Rh
components to the cell surface.
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MATERIALS AND METHODS |
Monoclonal antibodies and RBCs.
Murine antibodies to band 3 (BRIC 6,36 IgG3;
BRIC 155,37 IgG2b; BRIC 169,36 IgG1, and BRIC
170, IgG1), GPA (R10, IgG1 and R18,38 IgG2b),
Wrb (BRICs 14,39 IgG2a and 201,40
IgG1), Rh polypeptides (BRIC 69,41 IgG1), CD47 (BRIC
126,41 IgG2b) and glycophorin C (GPC; BRIC 4,42
IgG1) were as described. Purified human anti-D, Brad 543
(IgG1), was used at 50 µg/ml 1, diluted in
phosphate-buffered saline (PBS)/1% bovine serum albumin/0.1% NaN3 (PBS-A). Human anti-c, MS37 (IgG3), was provided by Dr
K. Thompson (IGRL, Oslo, Norway). Murine anti-Fy3, CBC-512 (IgG) was
provided by Dr M. Uchikawa (Japanese Red Cross, Shibuya-ku, Japan).
Murine anti-RhGP, LA18.18 (IgG1), was provided by Professor A.E.G. Kr.
von dem Borne (C.L.B., Amsterdam, The Netherlands). Murine anti-LW,
BS46 (IgG1), was provided by Dr H.H. Sonneborn (Biotest AG, Dreieich,
Germany). RBCs of known phenotype were available in house.
Cloning of band 3 cDNA into the pBabe puro and pBabe neo retroviral
vectors.
Polymerase chain reaction (PCR) was used to amplify band 3 cDNA from
the BSXG1.B3 construct previously described.23 This band 3 cDNA encodes a common, asymptomatic band 3 polymorphism, Memphis I
(Lys56  Glu44,45). PCR reactions were
performed in a DNA thermal cycler (Perkin Elmer, Norwalk, CT) using
Pwo polymerase (Boehringer Mannheim, Lewes, UK). The primers
were based on the band 3 cDNA 5 and 3 coding nucleotides
and incorporated Mfe I and Sal I restriction sites,
respectively. PCR products were cloned into the pBabe puro retroviral
vector (pBp), kindly provided by Dr H. Land (ICRF, London, UK), using
the compatible EcoRI and Sal I restriction sites, to
yield the construct pBpB3. The band 3 cDNA was then subcloned into the
pBabe neo vector (pBn), also provided by Dr H. Land, using the
BstEII and Sal I restriction sites, thus generating the
construct pBnB3. The band 3 coding region was confirmed in all
constructs by DNA sequencing, using T7 DNA Sequenase 2.0 (Amersham,
Little Chalfont, UK) and a 377 Applied Biosystems automated DNA
sequencer (Warrington, UK), according to manufacturer's instructions.
Electroporation competent Escherichia coli XL-1 Blue cells
(Stratagene, La Jolla, CA) were prepared using a method similar to that
described by Hanahan,46 transformed with pBabe puro or neo
constructs, and used for subsequent DNA purification (Qiagen, Dorking,
UK). All constructs were linearized using the Sca I site in
pBabe before transfection.
Transfection of K562 cells.
Human erythroleukemic K562 cells were obtained from the European
Collection of Animal Cell Cultures (Porton Down, Salisbury, Wiltshire,
UK). Cells were cultured in Iscove's Modified Eagle's Medium
supplemented with 10% fetal bovine serum (IMEM/FBS), at 37°C, in a
5%CO2 humidified incubator. Approximately 2 × 105 cells were transfected by electroporation with
approximately 10 µg of vector DNA in a Biorad gene pulser (Hercules,
CA) at 200 V/1,000 µF. Electroporated cells were cultured in 10 mL of IMEM/FBS for 2 days and then transferred to 96-well plates (0.2 mL per
well), in medium supplemented with 3 µg/mL puromycin (Sigma, Poole,
UK), or 3 µg/mL puromycin and 2 mg/mL Geneticin G418 (Calbiochem, Beeston, UK) if subject to sequential transfection as described below.
After 2 to 3 weeks, antibiotic-resistant, stable transfected clones
from wells containing only a single discrete colony were transferred to
24-well plates and expanded before flow cytometric analysis. The clones
expressing the highest levels of band 3 were selected using the
anti-band 3, BRIC 6. We also transfected K562 clones already
expressing RhD or cE polypeptides (generated by retroviral transduction
of K562 cells with pBabe puro/Rh constructs as previously
described35) with the pBnB3 construct. These sequential transfections generated K562/RhD+B3 clones and K562/RhcE+B3 clones, expressing both band 3 and RhD or cE polypeptides, respectively. Untransfected K562 cells or K562 cells expressing Rh polypeptides were
transfected with empty pBabe puro or neo vectors to generate control
cell clones.
Flow cytometric analysis.
K562 clones transfected with band 3 cDNA were analyzed by flow
cytometry (FACStar Plus, Becton Dickinson, Mountain View, CA). Mean
fluorescence intensity (FL1) was used as a measure of antibody binding.
The specificity of the antibodies was confirmed using RBCs of the
appropriate phenotype (data not shown). A total of 2 × 105 K562 cells were washed once and suspended in 50 µL of
PBS-A. Cells were incubated with an equal volume of antibody for 1 hour at room temperature, then washed and resuspended in PBS-A. The cells
were then incubated with affinity-purified fluorescein isothiocyanate (FITC)-labeled (Fab)2 fragments of rabbit antihuman
IgG or rabbit antimouse IgG (50 µL, 1:20 dilution, DAKO, Glostrup,
Denmark), for 1 hour at room temperature. Cells were then washed once
in PBS-A, and the sample volume was adjusted to 300 µL before
analysis. A K562 clone transfected with empty pBp (K562/pBp) was used
as control for K562 clones transfected with band 3. K562/RhD or
K562/RhcE clones were transfected with empty pBabe neo vector, and the
resulting K562/RhD+pBn and K562/RhcE+pBn clones were used as controls
for the analysis of K562/RhD and K562/RhcE clones transfected with band
3.
Cell growth rates.
Clones were seeded at 5 × 104 cells per mL in 10 mL
of culture medium, and cell numbers were determined (using a
hemocytometer) over a 6-day period by counting four samples per clone
per time point. Specific growth rate constants µ = ln((Nt N0)/t) and doubling times td = ln2/µ
were calculated from plots of ln (mean cell count) versus time.
Immunoblotting.
K562 cells were prepared for immunoblotting by lysing whole cells in a
detergent buffer, followed by SDS-PAGE. Cells were first washed in PBS
pH 7.5 at 4°C, and centrifuged. For samples subjected to
chymotrypsin digestion, the cells were incubated with
N -p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated chymotrypsin (Sigma), at 2 mg/mL in PBS pH 7.5, at 4°C for 6 hours. Examination of the time course of digestion showed that there was no increase in
the degree of digestion after this time (data not shown).
Chymotrypsin-treated cells were washed three times in PBS pH 7.5, containing the protease inhibitors phenylmethyl sulfonyl fluoride
(PMSF) (2 mmol/L), 4-(2-aminoethyl)benzenesulphonyl fluoride
(AEBSF) (1 mmol/L), leupeptin (140 mmol/L), antipain (80 mmol/L) pepstatin A (44 mmol/L), bestatin (40 mmol/L), E-64 (14 mmol/L), and aprotinin (0.8 mmol/L) (PBS-PI), supplemented with 5 mg/mL
bovine serum albumin (BSA). Cells were then washed once in PBS-PI,
without BSA. For detergent extraction, 50 µL cell pellets of
chymotrypsin-treated cells or untreated cells were suspended in an
equal volume of PBS-PI, 100 µL of PBS-PI containing 1% Triton X-100
were added, and the suspensions were incubated on wet ice for 20 minutes. Samples were centrifuged at 13,000g for 10 minutes at
4°C to pellet out nuclei and cell debris. Ghosts of
chymotrypsin-treated and untreated RBCs (homozygous for the band 3 Memphis I mutation) were used as controls on immunoblots. Triton
extracts of K562 cells and RBC ghosts were both solubilized in SDS-PAGE
buffer, and some chymotrypsin-treated samples were treated with PNGase
F (Oxford Glycosystems, Oxford, UK) in NEBuffers (New England Biolabs,
Hitchin, UK), for 1 hour at 37°C, to remove N-glycan chains.
Samples were separated on 8% acrylamide gels,47 immunoblotted,37 and visualized using the ECL method
(Amersham) and Biomax MR film (Kodak, Rochester, NY). For quantitative
analysis, blots were scanned using a high resolution color scanner
(Sharp, Chiba-City, Japan) and IMAGEMASTER scanning software (Pharmacia LKB, Uppsala, Sweden).
Chloride-36 efflux assays.
Our transport studies were based on the method described by Dissing et
al.48 Approximately 5 × 108 K562 cells
were washed once and resuspended in degassed transport buffer (140 mmol/L KCl, 1 mmol/L MgCl2, 5 mmol/L glucose, 5 mmol/L KH2PO4, pH7.2), and cytocrits were determined
using microhematocrit tubes. For 36Cl loading, 200 µL of
cells were incubated in transport buffer (2% cytocrit) containing a
tracer quantity of 36Cl at 20°C for 15 minutes, with
occasional swirling, and then centrifuged at 1,600g for 5 minutes at 4°C. Loaded cells were resuspended at 2% cytocrit in
ice-cold chloride-free degassed wash buffer (140 mmol/L potassium
gluconate, 1 mmol/L MgSO4, 5 mmol/L glucose, 5 mmol/L
KH2PO4, pH 7.2), split into two and centrifuged to give two 100-µL cell pellets. One pellet was washed in ice-cold wash buffer containing 50 mmol/L
4,4'-dinitrostilbene-2,2-disulphonate (DNDS, a band 3 inhibitor) and the other pellet in ice-cold wash-buffer alone. Cell
pellets were rapidly resuspended in transport buffer at 2% cytocrit,
in the presence or absence of 50 mmol/L DNDS, at 5°C or 20°C.
At intervals, 500 µL aliquots of the constantly mixed cell suspension
were transferred into 1.5 mL microcentrifuge tubes, containing 500 µL
of ice-cold stop buffer48 (transport buffer supplemented
with 50 mmol/L DNDS and 2 mmol/L furosemide). The quenched samples were
immediately centrifuged at 6,000g for 30 seconds, and 500 µL
of the resulting supernatants were added to 10 mL of Emulsifier-Safe
(Packard Instrument B.V., Groningen, The Netherlands) for liquid
scintillation counting. The values for infinite time were established
by counting 0.25 mL of unquenched cell suspension, and background
counts were determined by counting three samples of transport buffer.
The Cl- efflux rate constant,
okCl, was calculated according to
okCl = ln[(cpmi cpm0)/(cpmi cpmt)] t1, where cpm0, cpmt, and cpmi are
counts per minute released into the medium surrounding the cells after
time zero, time t, and infinite time, respectively. Transport data were
obtained from plots of ln(1(cpmt/cpmi)) versus time, in which
cpmt and cpmi had been corrected for cpm0. On these plots,
okCl equals the gradient of linear
regression lines. The relative chloride efflux,
oMCl, was calculated according to the
equation oMCl = (mcv/A) × okCl × [Cl]i, where mcv is the mean cell
volume (determined as below) and A the mean cell surface area (which
was calculated assuming that K562 cells are spherical). The
intracellular chloride concentration [Cl]i is 84 ± 2 mmol/L for K562
cells incubated in the transport buffer we have used.48
Determination of mean cell volume.
Approximately 5 × 107 cells were washed once and
resuspended in 0.5 mL of transport buffer. Cell suspensions were
counted in triplicate, using a Haematology Analyser (Sysmex, Milton
Keynes, UK). The cytocrit of the same suspensions was then immediately determined and the mean cell volume calculated, allowing for 2% trapped medium.48
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RESULTS |
Analysis of K562 clones transfected with band 3 cDNA by flow cytometry.
Several K562 clones transfected with the pBpB3 construct were generated
and analyzed by flow cytometry, using a panel of monoclonal antibodies
recognizing band 3, Wrb, GPA, GPC, Rh polypeptides, CD47,
and the RhGP, LW, and Fy glycoproteins. A K562 clone transfected with
empty pBp vector (K562/pBp) was used as control; flow cytometric
analysis showed almost identical expression of cell surface antigens
for this clone and untransfected K562 cells (data not shown).
Table 1 shows a comparison of
antibody-binding to one K562/band 3 clone (K562/B3 clone 1) and
K562/pBp cells. The tabulated results are the absolute values obtained
on the same day for antibody-binding to the two clones, and the average increase in antibody-binding to K562/B3 clone 1 relative to K562/pBp, measured daily over 3 consecutive days. The histograms obtained on day
1 are shown in Fig 1. Most significantly,
an antibody to an extracellular domain of band 3 (BRIC 6) bound much
more strongly to K562/B3 clone 1 than to K562/pBp cells, with a 10-fold
increase in mean fluorescence intensity. In addition, K562/B3 clone 1 showed high levels of de novo expression of the Wrb
antigen; this was observed for all of the analyzed K562 clones which
expressed band 3 to the cell surface (data not shown). One anti-Wrb (BRIC 14) showed a 42-fold increase in binding to
K562/B3 clone 1 cells over K562/pBp cells, while another antibody (BRIC
201) showed an eightfold increase. Antibodies to an intracellular
epitope of band 3 (BRIC 169, used as negative control), to GPC (BRIC
4), to Fy3 (CBC-512), and to CD47 (BRIC 126) showed only minimal
differences in binding between K562/B3 clone 1 and control cells; these
differences are probably a reflection of a slightly higher affinity of
K562/B3 clone 1 for the secondary antibody. Two antibodies to GPA (R10 and R18) bound strongly to both K562/B3 clone 1 and K562/pBp cells. Both of these antibodies showed a small (twofold) increase in binding
to K562/B3 clone 1 relative to the control clone. However, this
increase was not found in all K562/band 3 clones tested and may be due
to the heterogeneity of K562 cells with regard to GPA expression.49,50 For K562/B3 clone 1, we also observed a
threefold increase in the background expression of Rh polypeptides
(BRIC 69), and a twofold increase in binding of antibodies to the RhGP and LW glycoproteins (LA18.18 and BS46, respectively). Similar observations were made for most of the K562/band 3 clones. To further
investigate the effect of band 3 expression on the expression of Rh
components, we used K562 clones sequentially transfected with cDNAs
encoding RhcE or D and then band 3 cDNA; the results of these
experiments are described later.
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| Fig 1.
FACS analysis of K562/pBp and K562/B3 clone 1. All data
are from Table 1, day 1. Antibodies used in each histogram were (1)
BRIC 6, anti-band 3; (2) BRIC 14, anti-Wrb; (3) BRIC 201, anti-Wrb; (4) R10, anti-GPA; (5) R18, anti-GPA; (6) BRIC 4, anti-GPC; (7) BRIC 69, anti-Rh30; (8) LA18.18, anti-RhGP; (9) BS46,
anti-LW; (10) BRIC 126, anti-CD47, and (11) CBC-512, anti-Fy3. GPA,
glycophorin A; GPC, glycophorin C; Rh30, Rh polypeptides; RhGP, Rh
glycoprotein.
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Morphology and cell growth rates of K562 clones.
Under a light microscope, K562 cells expressing band 3 appear to be
morphologically very similar to both K562 cells and K562/pBp cells,
which are themselves morphologically indistinguishable. However, the
flow cytometric results showed that the K562/band 3 clones are slightly
more heterogenous in cell size and granularity, as measured by forward
and side scatter (data not shown). The mean cell volume of K562/pBp
cells was found to be 2.61 × 103 µm3
and that of K562/B3 clone 1 cells was 2.36 × 103 µm3. Assuming that K562 cells are
spherical, the mean cell surface area is 1,967 µm2 for
K562/pBp and 1,840 µm2 for K562/B3 clone 1. We also
measured the growth rates of untransfected K562 cells, K562/pBp cells,
and three K562 clones expressing band 3 at the cell surface
(Table 2). The doubling time of band
3-expressing clones was found to be about twofold greater than that of
K562 and K562/pBp cells.
Immunoblotting and quantification of expressed band 3.
The band 3 protein expressed in K562 cells was characterized by
SDS-PAGE and immunoblotting of K562/B3 clone 1 Triton X-100 extracts.
Solubilized RBC ghosts and Triton extracts of K562/pBp cells were used
as controls. Immunoblotting was performed with antibodies to band 3, BRIC 155 (recognizing an intracellular epitope near the C-terminus) and
BRIC 170 (directed against an intracellular epitope towards the
N-terminus), which have been shown to detect erythrocyte band
3.37 The results obtained for untreated K562/B3 clone 1 cells, untreated K562/pBp cells, and ghosts of untreated RBCs are shown
in Fig 2. Both BRIC 155 and BRIC 170 detected band 3 polypeptide, with an apparent molecular weight of
approximately 100 kD in erythrocyte membranes, and an
apparent molecular weight of approximately 90 kD in K562/B3 clone 1 samples. No corresponding band was detected in K562/pBp samples. The
same observations were made for all other analyzed K562/band 3 clones
(data not shown). Quantitative scanning densitometry was used to
calculate that the total amount of band 3 expressed in 55 µL of
packed K562/B3 clone 1 cells is equivalent to that found in 1 µL of
packed RBCs. To determine the surface fraction of band 3 expressed in
K562 cells, we performed immunoblots of K562/B3 clone 1 cells which had
been chymotrypsin-treated before Triton extraction; ghosts of
chymotrypsin-treated erythrocytes were used as a control (see Fig 3). Chymotrypsin cleaves whole band 3 protein in the third extracellular loop at amino acid residues 553 and
558,51 giving a 60-kD N-terminal fragment and a 35-kD
C-terminal fragment. In RBCs homozygous for the band 3 Memphis I
mutation, present in K562/B3 clone 1 and in our RBC controls, the
N-terminal chymotrypsin fragment has a reduced mobility on SDS-PAGE and
runs at approximately 63 kD.52 The cell surface fraction of
the band 3 expressed in K562/B3 clone 1 (determined by scanning
densitometry of the bands shown in lane 2, Fig 3) was calculated to be
47% of the total expressed protein. The amount of cell-surface band 3 expressed in approximately 115 µL of packed K562/B3 clone 1 cells is
therefore equal to that found in 1 µL of packed RBCs. Mature
erythrocytes have a mean cell volume of 80 µm3 and a mean
cell surface area of 142 µm2, while the mean cell volume
for K562/B3 clone 1 is 2,360 µm3 and the mean cell
surface area 1,840 µm2. Thus, the cell surface area per
unit packed cell volume is 2.3 times larger in erythrocytes than in
K562/B3 clone 1 cells. The density of cell surface band 3 molecules can
therefore be estimated to be 50 times greater in RBCs than in K562/B3
clone 1.
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| Fig 2.
Immunoblotting of K562 cells for band 3, 8% Laemmli gel.
Lanes 1 and 6, K562/pBp; lanes 2 and 4, homozygous Memphis RBC ghosts;
lanes 3 and 5, K562/B3 clone 1. BRIC 155 is a murine monoclonal
antibody directed against an intracellular site at the C-terminus of
band 3, while BRIC 170 binds to the N-terminal cytosolic domain of band
3.
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| Fig 3.
Immunoblotting of chymotrypsin-treated K562 cells for
band 3, 8% Laemmli gel. Lanes 1 and 4, ghosts of chymotrypsin-treated
homozygous Memphis RBCs; lanes 2 and 3, chymotrypsin-treated K562/B3
clone 1 cells; lane 5, PNGase-treated ghosts of chymotrypsin-treated
homozygous Memphis RBCs.
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Figure 3 also shows that both the C-terminal 35-kD chymotrypsin
fragment (lane 3) and the N-terminal 63-kD chymotrypsin fragment (lane
2) of band 3 expressed in K562 cells migrated at a slightly lower
apparent molecular weight than the corresponding band 3 fragments from
RBCs. In addition, the N-terminal 63-kD fragment ran as a doublet
rather than as a single band. The possible reasons for these
observations are discussed later. However, it should be noted that the
deglycosylated C-terminal chymotrypsin fragment of RBC band 3 (the
PNGase-treated sample in lane 5) ran at a lower apparent molecular
weight than the C-terminal fragment of band 3 from K562 cells. At least
some of the bands seen in Fig 3, which do not correspond to whole band
3 or to the band 3 chymotrypsin fragments discussed above, probably
represent proteolytic degradation products of band 3.
Chloride-36 transport in K562 clones.
DNDS-sensitive chloride efflux was measured for K562/B3 clone 1 at
20°C and 5°C, using K562/pBp as control.
Figure 4 shows the chloride efflux at
20°C; the gradients of the linear regression lines are equivalent
to efflux rate constants. In the absence of DNDS, efflux was much
faster for K562/B3 clone 1 than for K562/pBp (14-fold). The increased
efflux in K562/B3 clone 1 was inhibited by 50 mmol/L DNDS. A fraction
of the slow efflux found in K562/pBp was also sensitive to the
inhibitor; this efflux is mediated by transporters other than band
3.53 We therefore subtracted the efflux rate constant for
K562/pBp without DNDS (okCl = 0.158 × 102, r2 = .98) from the efflux rate constant for K562/B3 clone 1 without DNDS
(okCl = 2.224 × 102, r2 = .97) to calculate the
efflux rate constant for band 3-mediated efflux in K562/B3 clone 1 (okCl = 2.066 × 102). Because of the very fast efflux found for
K562/B3 clone 1 at 20°C, we also measured transport at 5°C. At
this temperature, efflux in the absence of DNDS was much slower than at
20°C (14 times) for both K562/B3 clone 1 (okCl = 0.156 × 102, r2 = .99) and K562/pBp
(okCl = 0.012 × 102, r2 = .85). The band
3-mediated efflux in K562/B3 clone 1 was calculated as at 20°C
(okCl = 0.144 × 102). Table 3 compares
the data for band 3-mediated chloride transport in K562/B3 clone 1 cells and RBCs at 5°C and 20°C; data for RBCs are according to
Brahm.54 The relative chloride efflux
oMCl was calculated as described in
Materials and Methods. For K562/B3 clone 1, oMCl = 1.55 × 1016 mmol Cl per
µm2 cell surface per second at 5°C, and
oMCl = 2.23 × 1015 mmol Cl per
µm2 cell surface per second at 20°C. At both
temperatures, the relative chloride flux is approximately 41 times
greater in RBCs than in K562/B3 clone 1. This is consistent with our
immunoblotting data, which suggest that the number of band 3 molecules
per unit surface area is 50 times greater in RBCs than in K562/B3 clone
1 (see above).
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| Fig 4.
Chloride efflux in K562 clones at 20°C. The gradients
of linear regression lines are equivalent to efflux rate constants
(okCl). r2 values are
K562/B3 clone 1 DNDS, .97; K562/B3 clone 1 + DNDS, .96; K562/pBp DNDS, .98; K562/pBp + DNDS, .88. The rate constant for band
3-mediated efflux in K562/B3 clone 1 was calculated as the difference
between the rate constants for K562/B3 clone 1 DNDS and K562/pBp DNDS.
|
|
Flow cytometric analysis of K562/RhD and K562/RhcE clones transfected
with band 3 cDNA.
To examine the potential interactions of band 3 and Rh polypeptides, we
investigated the effects of band 3 expression on K562 cells previously
transfected with cDNAs encoding Rh polypeptides and already expressing
substantial levels of the polypeptides on the cell surface. Two K562
clones transduced with RhD or RhcE cDNAs in pBp (as previously
described35) were transfected with the pBnB3 construct,
thus generating K562/RhD+B3 clones and K562/RhcE+B3 clones. As a
control, we transfected the same two K562/Rh clones with empty pBn
vector, yielding 12 K562/RhD+pBn and 11 K562/RhcE+pBn control clones.
Antibodies to band 3, Rh polypeptides, RhGP and the RhD antigen were
used to compare the expression of these antigens in the 12 K562/RhD+pBn
clones with their expression in several K562/RhD+B3 clones. Care was
taken to perform all measurements on the same day and under the same
conditions. An analogous experiment was performed to compare antigen
expression in the 11 K562/RhcE+pBn clones with that in several
K562/RhcE+B3 clones, using anti-Rhc instead of anti-RhD. Flow
cytometric results for the experiments are listed in
Table 4, and typical histograms are shown
in Fig 5. We found that cotransfection of a
K562/RhD clone with empty pBn vector alone did not result in any
noticable changes in the antigen activity of Rh polypeptides, Rh
glycoprotein, and RhD antigen for any of the resulting K562/RhD+pBn
clones. However, K562/RhD+B3 clone 1 (which binds 12-fold more
anti-band 3 than an average K562/RhD+pBn clone) showed a substantial
increase in binding of antibodies to the Rh proteins (×3.6 for Rh
polypeptides, ×5.9 for RhGP, ×2 for RhD). Similar results
were obtained for K562/RhcE+B3 clone 1 (which shows 11-fold greater
binding of anti-band 3 than an average control clone); binding of
antibodies to Rh polypeptides was raised 5.8-fold, antibodies to RhGP
8.8-fold, and antibodies to Rhc threefold. The other clones in which
band 3 was coexpressed with Rh polypeptides also showed substantially enhanced binding of antibodies to the appropriate Rh polypeptides and
RhGP. Band 3 expression did not have a similar effect on LW antigen
activity in the cotransfected clones (data not shown).
View larger version (42K):
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| Fig 5.
FACS analysis of K562 clones transfected with cDNAs
corresponding to Rh and/or band 3. (1) to (3) are data for
K562/pBp and K562/B3 clone 1, Table 1, day 1. (4) to (6) are data for a
K562/RhD+pBn clone (with expression levels close to the calculated
average) and K562/RhD+B3 clone 1; all data are from Table 4. (7) to
(9) are data for a K562/RhcE+pBn clone (with expression levels close
to the calculated average) and K562/RhcE+B3 clone 1; all data are
from Table 4. Antibodies used were: BRIC 6 (anti-band 3) in histograms
1, 4, and 7; BRIC 69 (anti-Rh30) in histograms 2, 5, and 8; LA18.18
(anti-RhGP) in histograms 3, 6, and 9. Rh30, Rh polypeptides; RhGP, Rh
glycoprotein.
|
|
| |
DISCUSSION |
The purpose of this work was to develop a system for the heterologous
expression of erythrocyte band 3 which would allow us to study (1) the
biosynthesis of band 3, (2) its interactions with other erythrocyte
proteins, and (3) its structure and function, by expressing band 3 mutants. Stable cell surface expression of functional band 3 in a cell
line with protein expression similar to that found in maturing RBCs
would clearly be useful for such investigations. However, previous cell
surface expression of band 3 in mammalian cells has not been reported.
In this study, we used the pBabe retroviral vectors to transfect K562
erythroleukemia cells with cDNA corresponding to the human AE1
gene. Flow cytometric analysis was used to compare cell surface
expression of band 3 in the resulting K562 clones with expression in
control clones, transfected with empty pBabe. Monoclonal antibody BRIC
6 (which binds to an extracellular domain of band 3) detected high
levels of band 3 expression in K562 cells transfected with band 3 cDNA, but no expression was detected in control cells. The expressed band 3 protein was also detected by immunoblotting, with monoclonal antibodies
to intracellular epitopes. Our results suggest that approximately half
of the total band 3 protein expressed in K562/B3 clone 1 is present at
the cell surface. The amount of band 3 expressed per unit area of
plasma membrane was estimated to be 50 times less in this clone than in
RBCs. A simple chloride efflux assay was used to confirm the
functionality of expressed band 3. The transport was found to be
DNDS-sensitive, confirming it to be band 3-mediated. In K562/B3 clone
1, the band 3-mediated chloride flux was 41 times smaller than in
RBCs, in agreement with our immunoblotting data. In conclusion, our
results suggest that there are approximately 150 transport-active sites
present per µm2 of K562/B3 clone 1 plasma membrane,
assuming a density of 7,000 molecules of band 3 per µm2
of erythrocyte membrane.9
Immunoblots showed that the band 3 expressed in K562 cells was not
identical to the erythrocyte protein in that it migrated at an
apparently lower molecular weight on SDS-PAGE (approximately 90 kD
rather than 100 kD). Blotting for band 3, cleaved by chymotrypsin in
the third extracellular loop, showed that the C-terminal 35-kD fragment
of band 3 from K562 cells had an apparently lower molecular weight than
the corresponding fragment found in RBC ghost samples. The presence of
a smaller N-glycan chain may explain why the fragment from K562 cells
ran faster than that from RBCs. PNGase treatment of the RBC sample
showed that the deglycosylated C-terminal chymotrypsin fragment of RBC
band 3 migrated faster than the K562 cell band 3 fragment. This
suggests that the protein expressed in K562 cells is N-glycosylated to
some extent. We were unable to successfully carry out PNGase-treatment
of the band 3 expressed in K562 cells because of difficulties caused by
endogenous proteolysis of the band 3 in the samples. The N-terminal
63-kD chymotrypsin fragment of band 3 from K562 cells also migrated at
a slightly lower apparent molecular weight than the corresponding
erythrocyte protein fragment. The reason for this observation may be
that the band 3 expressed in K562 cells is not posttranslationally
processed in the same way as RBC band 3, for example differently
phosphorylated55 at Tyr8/Tyr21 or
differently acetylated.56 In addition, the N-terminal K562
cell band 3 fragment migrated as a doublet rather than a single band.
Although the examination of the time course of chymotrypsin-digestion
showed that cleavage of the protein into two fragments was complete, it
is possible that a proportion of the band 3 expressed in K562 cells
undergoes only a single cleavage at residue 558, rather than cleavage
at both residues 553 and 558. Incomplete digestion at residue 553 could
be the result of different N-glycosylation of some of the protein. We also cannot eliminate the possibility that some of the band 3 expressed
in K562 cells is proteolytically cleaved at the N-terminus.
The flow cytometric analysis of K562/band 3 clones demonstrated that
all clones expressing band 3 also displayed high levels of
Wrb antigen which is not expressed on untransfected K562
cells and K562/pBp cells. The Wrb antigen57 is
a major blood group antigen which requires both GPA (residues 61-70)
and band 3 (Glu658) for expression. The appearance of the
Wrb antigen in K562/band 3 cells demonstrates that the
expressed band 3 and the endogenous GPA present in these cells are able to interact as they do in RBCs. The two antibodies to Wrb
we used (BRICs 14 and 201) have previously been shown to recognize different epitopes within the antigen.40 The observation
that both of these antibodies bind to K562/band 3 cells confirms that the band 3 and GPA interact normally in K562 cells. The large difference in reactivity of the two anti-Wrb antibodies
with K562/B3 clone 1 may reflect differences in the accessibility of
the two epitopes to the antibodies in the transfected K562 cells. The
appearance of Wrb antigen in K562 cells transfected with
band 3 cDNA also provides direct evidence that the band 3 polypeptide
is required for formation of the Wrb epitopes.
Fluorescence-activated cell sorting (FACS) analysis with BRIC 69 (which
binds to Rh polypeptides) demonstrated that K562/band 3 clones
consistently showed an increase in the low level of endogenous Rh
antigen reactivity. For K562/B3 clone 1, this increase was threefold
relative to K562/pBp. The Rh polypeptides are the products of at least
two highly homologous genes RHD and
RHCE.58-60 The RHD gene gives rise to the D
antigen, while the RHCE gene gives rise to the allelic antigens
C/c and E/e, which are likely to be located on the same polypeptide
chain.35 The antibody BRIC 69 recognizes both the RhD and
RhcE polypeptides. The expression of Rh antigens on RBCs requires the
presence/association of the Rh polypeptides with the RhGP glycoprotein,
and there is evidence that the two groups of protein form a complex in
the membrane.61,62 The Rh polypeptides are also thought to
interact with other proteins, including CD47 and LW.58 K562
cells have substantial amounts of intrinsic RhGP expression, but only
very low levels of Rh polypeptide expression. To investigate the effect
of band 3 expression in K562 cells on expression of the Rh system
antigens, we used two K562 clones previously transduced with pBpRhcE or
pBpRhD constructs which express substantial amounts of RhD or cE
polypeptides respectively.35 We cotransfected these
K562/RhD and K562/RhcE clones with the pBnB3 construct. The resulting
K562/RhD+B3 clones showed a substantial increase in binding of
anti-band 3 in conjunction with marked increases in binding of
antibodies to Rh polypeptides, RhGP, and RhD antigen. The binding of
antibodies to the Rh components was increased in a similar manner in
the K562/RhcE+B3 clones. It is unlikely that the increased antigen
activity of Rh components in the K562/band 3 clones is a result of
their reduced growth rates, as the increased reactivity was
specifically observed for the Rh proteins and not for any of the
endogenously expressed antigens GPC, CD47, or Fy3. The increased
reactivity of the band 3-expressing clones with antibodies to Rh could
originate in several ways. Band 3 may alter the membrane environment or
interact with the proteins of the Rh complex in the plasma membrane, so
that conformational or other rearrangements of the Rh protein complex result in increased reactivity with anti-Rh antibodies and BRIC 69. Alternatively, intracellular interactions of band 3 with the Rh
proteins could increase the amount of Rh components present at the cell
surface, by increasing the efficiency of folding of the Rh proteins, or
by enhancing their translocation to the plasma membrane. All of these
possibilities would be consistent with evidence that Rh antigen
expression is reduced in RBCs which are heterozygous for the mutated
band 3 present in Southeast Asian Ovalocytosis (deletion
of residues 400-408).63,36 We have so far been unsuccessful
in resolving the above possibilities by using immunoblotting to
quantitatively detect the polypeptide chains of the Rh proteins in the
transfected K562 cells with the antibody reagents available to us. More
work will be necessary to establish the extent to which interactions
occur between band 3 and the Rh protein complex, and the expression
system presented here will be useful for these studies.
Under a light microscope, band 3-expressing K562 cells look
morphologically similar to nontransfected K562 cells and K562 cells
transfected with empty pBabe vectors, but FACS analysis demonstrated
that the band 3-expressing clones are slightly more heterogenous in
cell size and granularity than clones not expressing band 3. To further
characterize the effects of band 3 expression on K562 cells, we
measured growth rates of untransfected K562 cells, K562 cells
transfected with empty pBabe vector, and three K562/band 3 clones. The
doubling time of K562/band 3 clones was about twofold greater than that
of nonexpressing clones. The increased demand on the protein synthetic
apparatus of the cells may contribute to this effect, but it is likely
that band 3 expression also has toxic effects on K562 cells. The
transport activity of band 3 located transiently in the membranes of
intracellular organelles could result in changes to the pH and ion
contents of these compartments, adversely affecting their function. It
is not clear whether the transport activity of internally localized,
newly synthesized band 3 is regulated in K562 cells, but further
investigations may yield information on this question.
| |
ACKNOWLEDGMENT |
We thank G.K. Jones for help with chloride transport assays, L.J. Bruce
for ghosts of chymotrypsin-treated and untreated RBCs, H. Land for the
pBabe puro and pBabe neo retroviral vectors, and K. Thompson and M. Uchikawa for monoclonal antibodies.
| |
FOOTNOTES |
Submitted June 4, 1998;
accepted August 4, 1998.
Supported in part by the Wellcome Trust. R.B. was the recipient of a
University of Bristol Postgraduate Scholarship.
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.
Address reprint requests to Michael J.A. Tanner, PhD,
Department of Biochemistry, School of Medical Sciences, University of
Bristol, Bristol BS8 1TD, UK; e-mail: M.Tanner{at}bris.ac.uk.
| |
REFERENCES |
1.
Reithmeier RAF:
The erythrocyte anion transporter (band 3).
Curr Opin Struct Biol
3:515, 1993
2.
Wang DN:
Band 3 protein: Structure, flexibility and function.
FEBS Lett
346:26, 1995
3.
Delaunay J:
Genetic disorders of the red cell membrane.
FEBS Lett
369:34, 1995[Medline]
[Order article via Infotrieve]
4.
Lux SE, Palek J:
Disorders of the red cell membrane, in
Handin RI,
Lux SE,
Stossel TP
(eds):
Blood: Principles and Practice of Haematology. Philadelphia, PA, Lippincott, 1995, p 1701.
5.
Delaunay J, Alloisio N, Morle L, Baklouti F, DellaVenezia N, Maillet P, Wilmotte R:
Molecular genetics of hereditary spherocytosis.
Ann Genet
39:209, 1996[Medline]
[Order article via Infotrieve]
6.
Tanner MJA:
The structure and function of band 3 (AE1): Recent developments.
Mol Membr Biol
14:155, 1997[Medline]
[Order article via Infotrieve]
7.
Tanner MJA, Bruce LJ, Groves JD:
The expression of the erythrocyte anion transporter (band 3, AE1), in
Hamasaki N,
Mihara K
(eds):
Membrane Proteins: Structure, Function and Expression Control. Basel, Switzerland, Karger, 1997, p 353.
8.
Morgan M, Hanke P, Grygorczyk R, Tintschl A, Fasold H, Passow H:
Mediation of anion transport in oocytes of Xenopus laevis by biosynthetically inserted band 3 protein from mouse spleen erythroid cells.
EMBO J
4:1927, 1985[Medline]
[Order article via Infotrieve]
9.
Bartel D, Lepke S, Layh-Schmitt G, Legrum B, Passow H:
Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein-encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulphonate binding site.
EMBO J
8:3601, 1989[Medline]
[Order article via Infotrieve]
10.
Garcia AM, Lodish HF:
Lysine 539 of human band 3 is not essential for anion transport or inhibition by stilbene disulphonates.
J Biol Chem
264:19607, 1989[Abstract/Free Full Text]
11.
Groves JD, Falson P, Le Maire M, Tanner MJA:
Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.
Proc Natl Acad Sci USA
93:12245, 1996[Abstract/Free Full Text]
12.
Sekler I, Kopito RR, Casey JR:
High level expression, partial purification and functional reconstitution of human AE1 anion exchanger in Saccharomyces cerevisiae.
J Biol Chem
270:21028, 1995[Abstract/Free Full Text]
13.
Ruetz S, Lindsey AE, Ward CL, Kopito RR:
Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment.
J Cell Biol
121:37, 1993[Abstract/Free Full Text]
14.
Gomez S, Morgans C:
Interaction between band 3 and ankyrin begins in early compartments of the secretory pathway and is essential for band 3 processing.
J Biol Chem
268:19593, 1993[Abstract/Free Full Text]
15.
Cartron J-P, Rahuel C:
Human erythrocyte glycophorins: Protein and gene structure analysis.
Transfus Med Rev
6:63, 1992[Medline]
[Order article via Infotrieve]
16.
Tanner MJA:
Molecular and cellular biology of the erythrocyte anion exchanger (AE1).
Semin Hematol
30:34, 1993[Medline]
[Order article via Infotrieve]
17.
Gahmberg C, Myllyla G, Leilola J, Pirkola A, Nordling S:
Absence of the major sialoglycoprotein in the membrane of human En(a-) erythrocytes and increased glycosylation of band 3.
J Biol Chem
251:6108, 1976[Abstract/Free Full Text]
18.
Tanner MJA, Anstee DJ:
The membrane change in En(a-) erythrocytes.
Biochem J
153:271, 1976[Medline]
[Order article via Infotrieve]
19.
Tanner MJA, Jenkins RE, Anstee DJ, Clamp JR:
Abnormal carbohydrate composition of the major membrane-penetrating protein of En(a-) human erythrocytes.
Biochem J
155:701, 1976[Medline]
[Order article via Infotrieve]
20.
Tokunaga E, Sasakawa S, Tamaka K, Kawamata H, Giles CM, Ikin EW, Poole J, Anstee DJ, Mawby WJ, Tanner MJA:
Two apparently healthy Japanese individuals of type MkMk have erythrocytes which lack both blood group MN and Ss-active sialoglycoproteins.
J Immunogenetics
6:383, 1979[Medline]
[Order article via Infotrieve]
21.
Bruce LJ, Groves JD, Okubo Y, Thilaganthan B, Tanner MJA:
Altered band 3 structure and function in glycophorin A- and B-deficient (MkMk) red blood cells.
Blood
84:916, 1994[Abstract/Free Full Text]
22.
Wang WC, Li N, Aoki D, Fukudo MN, Fukuda M:
The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex.
J Biol Chem
266:23185, 1991[Abstract/Free Full Text]
23.
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]
24.
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]
25.
Gahmberg CG, Andersson LC:
K562  a human leukemia cell line with erythroid features.
Semin Hematol
18:72, 1981[Medline]
[Order article via Infotrieve]
26.
Benz EJ, Murnane MJ, Tonkonow BL, Berman BW, Mazur EM, Cavallesco C, Jenko T, Snyder EL, Forget BG, Hoffman R:
Embryonicfetal erythroid characteristics of a human leukemic cell line.
Proc Natl Acad Sci USA
77:3509, 1980[Abstract/Free Full Text]
27.
Horton MA, Cedar SH, Edwards PA:
Expression of red cell specific determinants during differentiation in the K562 erythroleukaemia cell line.
Scand J Haematol
27:231, 1981[Medline]
[Order article via Infotrieve]
28.
Wiener E, Shiels A, Wickramasinghe SN, Anstee DJ, Avent ND:
Ara-C treatment of K562 cells increases the expression of Rh polypeptides.
Transfus Med
4:44, 1994 (abstr)
29.
Suyama K, Lunn R, Haller S, Goldstein J:
Rh(D) antigen expression and isolation of a new Rh(D) cDNA isoform in human erythroleukemia K562 cells.
Blood
84:1975, 1994[Abstract/Free Full Text]
30.
Anstee DJ, Holmes CH, Judson PA, Tanner MJA:
Use of monoclonal antibodies to determine the distribution of red cell surface proteins on human cells and tissues, in
Agre PC,
Cartron J
(eds):
Protein Blood Group Antigens of the Human Red Cell: Structure, Function and Clinical Significance. Baltimore, MD, John Hopkins University, 1992, p 170.
31.
Chaudhuri A, Polyakova J, Zbrzezna V, Pogo AO:
The coding sequence of Duffy blood group gene in humans and simians: Restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy-negative individuals.
Blood
85:615, 1995[Abstract/Free Full Text]
32.
Andersson LC, Gahmberg CG, Teerenhovi L, Vuopio P:
Glycophorin A as a cell surface marker of early erythroid differentiation in acute leukemia.
Int J Cancer
24:717, 1979[Medline]
[Order article via Infotrieve]
33.
Alitalo R:
Induced differentiation of K562 leukemia cells: A model for studies of gene expression in early megakaryoblasts.
Leuk Res
14:501, 1990[Medline]
[Order article via Infotrieve]
34.
Morgenstern JP, Land H:
Advanced mammalian gene transfer: High titre retroviral vectors with multiple drug selection markers and a complementary free packaging cell line.
Nucleic Acids Res
18:3587, 1990[Abstract/Free Full Text]
35.
Smythe JS, Avent ND, Judson PA, Parsons SF, Martin PG, Anstee DJ:
Expression of RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of Rh blood group antigens.
Blood
87:2968, 1996[Abstract/Free Full Text]
36.
Smythe JS, Spring FA, Gardner B, Parsons SF, Judson PA, Anstee DJ:
Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes.
Blood
85:2929, 1995[Abstract/Free Full Text]
37.
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 human tissues.
Biochem J
258:211, 1989[Medline]
[Order article via Infotrieve]
38.
Anstee DJ, Edwards PAW:
Monoclonal antibodies to human erythrocytes.
Eur J Immunol
12:228, 1982[Medline]
[Order article via Infotrieve]
39.
Ridgwell K, Tanner MJA, Anstee DJ:
The Wrb antigen, a receptor for Plasmodium falciparum malaria, is located on a helical region of the major membrane sialoglycoprotein of human red blood cells.
Biochem J
209:273, 1983[Medline]
[Order article via Infotrieve]
40.
Poole J, Banks JA, Bruce LJ, Ring SM, Tanner MJA, Levene C:
A novel glycophorin A polymorphism affecting Wrb expression.
Transfusion
35:S160, 1995 (abstr)
41.
Avent ND, Judson PA, Parsons SF, Mallinson G, Anstee DJ, Tanner MJA, Evans PR, Hodges E, Maciver AG, Holmes C:
Monoclonal antibodies that recognise different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.
Biochem J
251:499, 1988[Medline]
[Order article via Infotrieve]
42.
Anstee DJ, Parsons SF, Ridgwell K, Tanner MJA, Merry AH, Thomson EE, Judson PA, Johnson P, Bates S, Fraser ID:
Two individuals with elliptocytic red cells apparently lack three minor erythrocyte membrane sialoglycoproteins.
Biochem J
218:615, 1984[Medline]
[Order article via Infotrieve]
43.
Hadley AG, Zupanska B, Kumpel BM, Leader KA:
The functional activity of FC gamma RII and Fc gamma RIII on subsets of human lymphocytes.
Immunology
76:446, 1992[Medline]
[Order article via Infotrieve]
44.
Yannoukakos D, Vasseur C, Driancourt C, Blouquit Y, Delaunay J, Wajcman J, Bursaux E:
Human erythrocyte band 3 polymorphism (band 3 Memphis): Characterization of the structural modification (Lys56Glu) by protein chemistry methods.
Blood
78:1117, 1991[Abstract/Free Full Text]
45.
Jarolim P, Rubin HL, Zhai S, Sahr KH, Liu SC, Mueller TJ, Palek J:
Band 3 Memphis: A widespread polymorphism with abnormal electrophoretic mobility of band 3 protein caused by substitution AAGGAG (LysGlu) in codon 56.
Blood
80:1592, 1992[Abstract/Free Full Text]
46.
Hanahan D:
Studies on transformation of Escherichia coli with plasmids.
J Mol Biol
166:557, 1983[Medline]
[Order article via Infotrieve]
47.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
48.
Dissing S, Hoffman R, Murnane MJ, Hoffman JF:
Chloride transport properties of human leukemic cell lines K562 and HL60.
Am J Physiol
247:C53, 1984[Abstract/Free Full Text]
49.
Alder S, Ciampi A, McCulloch EA:
A kinetic and clonal analysis of heterogeneiety in K562 cells.
J Cell Physiol
118:186, 1984[Medline]
[Order article via Infotrieve]
50.
Hering S, Bottger V, Jantscheff P, Micheel B:
Differences of K562 leukemia cell clones in the pattern of monoclonal antibody binding and NK cell susceptibility.
Biomed Biochim Acta
45:673, 1986[Medline]
[Order article via Infotrieve]
51.
Jennings ML, Adams MF:
Modification by papain of the structure and function of band 3, the erythrocyte anion transport protein.
Biochemistry
20:7118, 1981[Medline]
[Order article via Infotrieve]
52.
Mueller TJ, Morrison M:
Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte.
J Biol Chem
252:6573, 1977[Abstract/Free Full Text]
53.
Law F-Y, Steinfeld R, Knauf PA:
K562 cell anion exchange differs markedly from that of mature red blood cells.
Am J Physiol
244:C68, 1983[Abstract/Free Full Text]
54.
Brahm WA:
Temperature-dependent changes of chloride transport kinetics in human red cells.
J Gen Physiol
70:283, 1977[Abstract/Free Full Text]
55.
Low PS, Geahlen RL, Mehler E, Harrison ML:
Extracellular control of erythrocyte metabolism mediated by a cytoplasmic tyrosine kinase.
Biomed Biochim Acta
49:S135, 1990[Medline]
[Order article via Infotrieve]
56.
Kaul RK, Murthy SN, Reddy AG, Steck TL, Kohler H:
Amino acid sequence of the N-terminal 201 residues of human erythrocyte membrane band 3.
J Biol Chem
258:7981, 1983[Abstract/Free Full Text]
57.
Bruce LJ, Ring SM, Anstee DJ, Reid ME, Wilkinson S, Tanner MJA:
Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in band 3: A site of interaction of band 3 and glycophorin A under certain circumstances.
Blood
85:541, 1995[Abstract/Free Full Text]
58.
Anstee DJ, Tanner MJA:
Biochemical aspects of the blood group Rh (Rhesus) antigens.
Baillieres Clin Haematol
6:401, 1993[Medline]
[Order article via Infotrieve]
59.
Cartron J-P, Agre P:
Rh blood group antigens: Protein and gene structure.
Semin Hematol
30:193, 1993[Medline]
[Order article via Infotrieve]
60.
Cartron J-P:
Defining the Rh blood group antigens.
Blood Rev
8:199, 1994[Medline]
[Order article via Infotrieve]
61.
Eyers SAC, Ridgewell K, Mawby WJ, Tanner MJA:
Topology and organization of human Rh (Rhesus) blood group-related polypeptides.
J Biol Chem
269:6417, 1994[Abstract/Free Full Text]
62.
Cherif-Zahar B, Raynal V, Gane P, Mattei MG, Bailly P, Gibbs B, Colin Y, Cartron J-P:
Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency.
Nat Genet
12:168, 1996[Medline]
[Order article via Infotrieve]
63.
Booth PB, Serjeantson S, Woodfield DG, Amato D:
Selective depression of blood group antigens associated with hereditary ovalocytosis among melanesians.
Vox Sang
32:99, 1977[Medline]
[Order article via Infotrieve]
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Top
Abstract
Introduction