Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 336-341
RED CELLS
Surface expression of Rh-associated glycoprotein (RhAG) in
nonerythroid COS-1 cells
Kimita Suyama,
Hua Li, and
Alex Zhu
From the Lindsley F. Kimball Research Institute of The New York
Blood Center, New York.
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Abstract |
In the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50)
is thought to be involved in Rh30 (D, CE) expression by forming a
protein complex on the red cell surface. To obtain further insight into
the Rh complex, we chose nonerythroid COS-1 cells instead of
proerythroblast-like K562 cells, which produce endogenous Rh proteins
as cell host, for the expression of both RhAG and RhD. The RhAG cDNA
was subcloned into a retroviral vector, and a stable COS-1 cell line
was then established via retroviral transduction. Surface expression of
RhAG on the COS-1 cells was monitored by flow cytometry using mouse
monoclonal anti-RhAG(2D10). Under these conditions, we detected
significant expression of RhAG on the cell surface, compared to stable
COS-1 cells transduced with the vector alone. To confirm the results,
we isolated RhAG by immunoprecipitation from the lysate of the COS-1
cells, which were metabolically labeled with
[35S]-methionine. A strong band of the 32 kd on SDS-PAGE
was obtained, corresponding to the results obtained from other cultured
cells (K562 cell and others), which always produce partially
glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was
expressed without Rh30 and other Rh-related glycoproteins (LW,
glycophorin B) in nonerythroid cells. Using the same strategy, however,
we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the
surface expression of RhD antigen. (Blood. 2000;95:336-341)
© 2000 by The American Society of Hematology.
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Introduction |
The elucidation of the RhD antigenicity is of clinical
importance because of its involvement in transfusion medicine and
hemolytic disease of the newborn.1-3 Molecular analysis of
partial Ds in DIV, DV, DVI, Dc- and
DFR cells as well as the commonly occurring antigens (C/c, E/e, and D)
and the identification of these antigen-carrying membrane proteins
have been reported.4-11 Furthermore, a 30-epitope model was
recently established for D antigen using a number of partial D
phenotypes and human monoclonal anti-D antisera.12 However, the nature of RhD antigenicity is still unknown.
Expression of recombinant RhD cDNA in eukaryotic cells would greatly
contribute to our understanding of RhD antigenicity and biological
function of this protein if it exists. Although RhD cDNA and RhAG cDNA
can be transiently expressed in COS-1 cell lysates, surface expression
of these proteins was not successful.13-15 Iwamoto et al
tried expression of RhD or RhcE antigen on both nonerythroid cell, 293, and erythroid cell, human erythroleukemia (HEL) together with RhAG.
However, they could not detect RhD or RhcE antigen on them and
suggested that a second coexpressing factor will be needed to express
Rh antigens.16 Because Rh antigenicity is believed to be
erythroid cell-specific and -conformational, the correct environment
may be important for the proper folding and expression of the Rh
epitopes. Smythe et al reported the surface expression of RhD, Rhc, and
RhE antigens in K562 cells transfected with RhD or RhcE cDNA,
respectively.17 Also, Liu et al demonstrated that D
epitopes were expressed by retroviral vectors containing mutant RhD
cDNA.18 Recently, Zhu et al showed clearly endogenous expression of D antigen on K562 cells with human monoclonal anti-D and
reported the expression of D antigen epitopes in K562 cells using RhD
fusion proteins.19 Because K562 cells resemble
proerythroblasts and express D antigen on their cell surfaces
endogenously, it is likely that they produce the correct environment
for the expression.20
The presence of RHAG-like sequences in nematode and the sponge
is reported.3,21 The human RhAG protein is known to share a
certain degree of homology (20% to 27% identical) with the Mep/Amp family of NH4+ transporter, permeases that have
been identified from bacteria to yeast and plant (but not
animals).22 In fact, Caenorhabditis elegans is
found to possess at least 4 ammonium transporter genes and 2 RHAG-like sequences. Interestingly, Cherif-Zahar et al proposed that mutant alleles of RHAG are likely candidates for
suppressors of the RH locus, accounting for most cases of Rh
deficiency (Rhnull, regulator type).23 It has
been suggested that the deficiency of Rh30 proteins on red cells from
individuals in Rhnull amorph type results in homozygosity
for a silent allele at the RH locus and is associated with the lack or
reduced expression of Rh-related glycoproteins. Huang et al and
Cherif-Zahar et al demonstrated the molecular defects in the RHCE gene
from Rhnull amorph type.24,25 Therefore, at
least these 2 proteins (Rh30 and RhAG) seem to be closely interacting
with each other. Moore and Green also immunoprecipitated RhAG
glycoprotein with human anti-D and anti-c antibodies.26 Moreover, Hartel-Schenk et al coprecipitated Rh components as a 170-kd
complex by density ultracentrifugation of
[3H]-palmitate-labeled Rh30 proteins.27
Rh expression in nonerythroid cells like the COS-1 cell is useful for
reconstitution of Rh complex because K562 cell possesses Rh30 and
Rh-related glycoproteins (Rh50, LW, CD47, and glyco- phorin B)
endogeneously. To shed light onto the physiologic role of RhAG and
functional role of RhAG in Rh30 expression, we produced a stable COS-1
cell line with RhAG cDNA using a retroviral vector. We report in this
paper surface expression of RhAG glycoprotein in stable COS-1 cells
transfected with the retroviral vector containing RhAG cDNA.
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Materials and methods |
Materials
COS-1 cells were obtained from American Type Culture Collection;
culture media and G418 were purchased from Life Technologies Inc; pLNCX
vector, PT67 cells, and CalPhos Maximizer Transfection Kit were
obtained from Clontech Laboratories Inc; [3H]-mannose was
purchased from ICN and L-[35S]-methionines from Amersham
for metabolical labeling grade and from Dupont NEM for translation
grade. Transcription and translation-coupled reticulocyte lysate system
and canine pancreatic microsomal membranes were obtained from Promega,
restriction enzymes (Xba I, Hind III, Hpa I) from New England BioLabs,
goat F(ab')2 anti-human immunoglobulin G (IgG) conjugated
with phycoerythrin and goat F(ab')2
anti-mouse IgG conjugated with phycoerythrin from Biosource
International, and polybrene from Sigma. Purified mouse monoclonal
anti-RhAG(2D10) was kindly given by Dr Albert von dem Borne (Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service,
Amsterdam, Netherlands) and human monoclonal anti-D (LOR15C9) by Dr A. Blancher (Hopital Purpan, Toulouse, France).
Methods
Construction of retroviral expression vectors.
The pcDNA3RhAG vector previously constructed was cut with Xba I
restriction enzyme and filled in with Klenow DNA polymerase. Then, RhAG
cDNA was released from the vector using Hind III restriction enzyme and
purified. The retroviral expression vector pLNCX was digested with Hind
III followed by Hpa I and purified. The RhAG cDNA released was ligated
with the Hind III/Hpa I-cleaved pLNCX, and the ligates were transformed
into DH5
cells (Figure 1). After confirming the orientation and the sequence by dideoxy- chain termination method, the plasma vector was referred to as pLNCXRhAG. The
pcDNA3RhD vector was also cut with Bam H I/Xba I, and the released RhD
cDNA was filled in with T4 DNA polymerase and purified. The pLNCX
vector was cut with Hpa I, dephosphorylated with calf intenstine
phosphatase, and then purified. This vector was ligated with the
purified RhD cDNA, and the newly produced vector was referred to as
pLNCXRhD. Packaging cell line (PT67 cell) was transfected with the
constructed plasmid vector (10 µg/100 mm plate, pLNCX, or pLNCXRhAG,
or pLNCXRhD) using CalPhos Maximizer Transfection Kit and cultured for
48 hours; the produced viruses then were collected from the culture
medium of the transfected PT67 cells. The virus-containing medium was
used for transduction of actively dividing COS-1 cells. The medium was
filtered through 0.45-µm cellulose acetate filter and diluted to 10%
and then added to COS-1 cells (5 × 105 cells/100 mm
plate). The cells were incubated for 24 hours after addition of 4 µg/mL polybrene. They were then re-placed into fresh regular medium
and cultured for 24 hours. By antibiotic selection with G418 (0.5 mg/mL), the stable COS-1 cells with RhAG or RhD cDNA were produced.
Construction of pR-RhD was described previously.19
Expression of RhAG in vitro.
Expression of RhAG in vitro was performed with a transcription and
translation-coupled reticulocyte lysate system (TNT-T7) following the
method described previously and using pLNCXRhAG as expression
vector.14 The reaction mixtures were incubated at 30°C
for 90 minutes, diluted 1:10 in lysis buffer (1% Triton X-100 + 1 mM
TPCK + 1 mM phenylmethylsulfonyl fluoride + 100 u/mL aprotinin in 20 mM
tris, pH 7.4) and incubated with 2D10 overnight at
4°C. The immune complexes were eluted from protein A-Sepharose beads by boiling for 5 minutes in 1% sodium dodecyl sulfate (SDS) with
8 mol/L urea and 5% 
-mercaptoethanol in 0.16 mol/L tris, pH 6.8, and run on SDS-PAGE (SDS-polyacrylamide gel electrophoresis) (12%).
Expression of RhAG and RhD polypeptides in stably transduced COS-1
cells.
Expression of RhAG and RhD proteins in stable COS-1 cells
(5 × 106 cells/60 mm plate) transduced with the
viruses containing pLNCX, or pLNCXRhAG, or pLNCXRhD was performed as
follows. Logarithmically growing stable COS-1 cells were labeled with
200 µCi/mL [35S]-methionine in 1mL methionine-free
medium for 2 hours at 37°C. As described above, the cells were
washed 3 times with ice-cold phosphate buffered saline (PBS), lysed
with lysis buffer, and centrifuged. The supernatants were
immunoprecipitated with rabbit polyclonal anti-Rh or 2D10, and the
immune complexes were eluted from protein A-Sepharose (anti-Rh) or
protein G-Sepharose(2D10) and run on SDS-PAGE (12%) under the reducing conditions.
[3H]-mannose incorpration into RhAG glycoprotein in
stable COS-1 cells transduced with the viruses containing pLNCXRhAG.
Logarithmically growing COS-1 cells (5 × 106 cells)
were incubated with [3H]-mannose (135 µCi/mL) in
glucose-free RPMI 1640 for 5 hours at 37°C. After washing 3 times
with PBS, the cells were lysed as described above. Supernatant (500 µL) from the lysate was incubated with 5 µL of 1:10 diluted 2D10
overnight at 4°C. The immune complexes extracted by protein
G-Sepharose were run on SDS-PAGE (12%) under the reducing conditions,
followed by fluorography.
Fluorescence-activated cell sorting (FACS).
Stable COS-1 cells (5 × 105 cells) were incubated
with 0.1 mL of 1:100 diluted 2D10 (1 mg/mL) for 30 minutes at 37°C
and washed 3 times in PBS with 0.5% bovine serum albumin. The cells
were reincubated with phycoerythrin-conjugated goat
F(ab')2 anti-mouse IgG for 30 minutes at room temperature,
washed 3 times, and resuspended in PBS. The cells were analyzed by FACS
IV (Becton Dickinson, Sunnyville, CA). Data were presented
as fluorescence intensity versus cell frequency.
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Results |
Expression of RhAG glycoprotein in vitro
Previously, we were able to detect RhAG polypeptide by
immunoprecipitation in COS-1 cells transiently transfected with
pSVLRhAG vector using purified mouse monoclonal anti-RhAG(2D10), but
surface expression of the RhAG antigen on the cells was not successful when examined by flow cytometry.14 Now, as shown in Figure
1, we constructed a retroviral vector (pLNCXRhAG) carrying the RhAG cDNA. After confirming the authenticity of the vector by checking the
orientation and the DNA sequence, the vector was used for expression of
the RhAG glycoprotein in a transcription and translation-coupled reticulocyte lysate system. [35S]-methionine-labeled RhAG
glycoprotein was immunoprecipitated with 2D10. RhAG polypeptides
immunoprecipitated from reticulocyte lysate without microsomal
membranes resided in the 30-kd region of a 12% acrylamide SDS-PAGE, as
shown in Figure 2. Addition of microsomal
membranes to this reticulocyte system caused partial glycosylation of
the protein, as we expected, producing 32-kd protein.
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| Fig 2.
Expression of RhAG glycoprotein in a transcription and
translation-coupled reticulocyte lysate system.
Autoradiogram of [35S]-methionine-labeled proteins
separated by 12% SDS-PAGE (for details, see Materials and Methods).
Lane 1, pLNCXRhAG vector; lane 2, pLNCXRhAG vector with microsomal
membranes. Arrow indicates nonglycosylated RhAG; arrow head,
glycosylated RhAG.
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Expression of RhAG and RhD proteins in stable COS-1 cells
COS-1 cells were transduced with the viruses containing pLNCX, or
pLNCXRhAG, or pLNCXRhD; the stable COS-1 cell lines were then
established by antibiotic selection with G418. Expression of RhAG
glycoprotein in stable COS-1 cells transduced with the viruses
containing pLNCXRhAG was demonstrated by immunoprecipitating the [35S]-methionine labeled proteins in the supernatants
of the cell lysates with 2D10, and the immunoprecipitates were analyzed
by SDS-PAGE. The stable COS-1 cells exhibited a 32-kd band, which signifies expression of RhAG glycoprotein (Figure
3A). This partially glycosylated protein
was similar to those produced in a microsomal in vitro translation
system (Figure 2) and in K562 cells endogenously, as reported
previously.14 To confirm whether the 32-kd band is a
glycoprotein, RhAG was immunoprecipitated from the lysate of the stable
COS-1 cells metabolically labeled with [3H]-mannose using
2D10, and the immune complex was analyzed by SDS-PAGE (Figure
4). Again,
[3H]-mannose-labeled RhAG migrated to the 32-kd region
of the gel as single band, confirming that the band at 32 kd is RhAG
glycoprotein. Therefore, bands at a higher molecular weight region
(>40 kd) seen in Figure 3A are not RhAG-related proteins. These
results suggest that the stable COS-1 cells in tissue culture cannot
glycosylate the RhAG to the same extent (40 to 100 kd in size) as is
found in human red cells. Expression of RhD protein was also
accomplished, as evident from the presence of the immunoprecipitated
32-kd proteins in the lysates of COS-1 cells transduced with pLNCXRhD
using rabbit polyclonal anti-Rh, which specifically recognizes Rh30
proteins but not in the lysates of COS-1 cells transduced with the
pLNCX vector alone (Figure 3B).
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| Fig 3.
Autoradiogram of immunoprecipitates obtained from stable
COS-1 cells transduced with the viruses containing RhAG or RhD RNA
using 2D10 or rabbit polyclonal anti-Rh.
Logarithmically growing COS-1 cells were incubated with
[35S]-methionine for 2 hours, and the cell lysates were
subjected to immunoprecipitation with 2D10 or rabbit polyclonal
anti-Rh; the immunoprecipitates were then separated by 12% SDS-PAGE
under reducing conditions (for details, see Materials and Methods). (A)
Lane 1, stable COS-1 cells transduced with the viruses containing
vector alone; lane 2, stable COS-1 cells transduced with the viruses
containing pLNCXRhAG. Arrow shows RhAG glycoprotein. (B) Lane 1, stable
COS-1 cells transduced with the viruses containing vector alone;
lane 2, stable COS-1 cells transduced with the viruses containing
pLNCXRhD. Arrowhead shows RhD polypeptides.
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| Fig 4.
Autoradiogram of immunoprecipitates obtained from stable
COS-1 cells transduced with the viruses containing pLNCXRhAG using
2D10.
Logarithmically growing COS-1 cells were incubated with
[3H]-mannose for 5 hours, and the cell lysates were
subjected to immunoprecipitation with 2D10; the immunoprecipitates were
then separated by SDS-PAGE under reducing conditions (for details, see
Materials and Methods). Lane 1, stable COS-1 cells transduced with the
viruses containing pLNCX vector alone; lane 2, stable COS-1 cells
transduced with the viruses containing pLNCXRhAG. Arrow shows RhAG
glycoprotein.
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Surface expression of RhAG glycoprotein on the stable COS-1 cells
transduced with the retroviruses containing pLNCXRhAG
Surface expression of RhAG glycoprotein on stable COS-1 cells
transduced with the viruses containing pLNCXRhAG was determined by flow
cytometry using 2D10. As shown in Figure 5,
the cells sensitized with this antibody followed by incubation with
phycoerythrin-conjugated mouse anti-IgG, exhibited a significant
fluorescence intensity when compared with the control stable COS-1
cells transduced with the viruses containing pLNCX vector alone.
Because G418-resistant COS-1 cells were not cloned, the broad range of
fluorescence was observed. This is probably due to different transport
efficiency of RhAG to plasma membrane by each cell. After one-time cell
sorting, we achieved sharper and higher distribution of the
fluorescence (data not shown). This indicates, for the first time,
expression of RhAG glycoprotein on the membranes of the cultured
nonerythroid cells without any Rh30 proteins and Rh-related
glycoproteins. Surface expression of RhD protein on the stable COS-1
cells transduced with the viruses containing pLNCXRhD was also
determined by flow cytometry using human monoclonal anti-D (LOR15C9),
which shows broad D specificity and also recognizes denatured D
polypeptide. However, surface expression of RhD protein on the cells
was not recognized (data not shown). Other human monoclonal anti-D
(LOR1, SAL-20, LOR-12E2) also did not bind to the cells (data not
shown). Furthermore, a new stable COS-1 cell line obtained by
transfecting the stable COS-1 cells carrying RhAG cDNA with RhD
cDNA-containing plasmid vector (pR-RhD) was established. However, this
cell line could not express any D epitopes, suggesting that the
presence of both RhD protein and RhAG glycoprotein is not enough to
produce D antigen on COS-1 cells and that other factors might be
involved in expression of RhD antigen. However, as described in
"Materials and Methods," all the stable cell lines were
established after 2-week growth in antibiotics rather than grown from
individual colonies. Therefore, the level of protein expression is
likely to vary among cells. In the case of the COS-1 cells coexpressing RhAG and RhD, it may be worthwhile
using cell sorting in flow cytometry analysisto examine the possibility that only a very few
transducted cells are expressing RhD antigens.
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| Fig 5.
FACS profile of stable COS-1 cells transduced with the
viruses containing pLNCXRhAG using 2D10.
COS-1 cells were incubated with 2D10 (10 µg/mL) containing 0.5%
bovine serum albumin in PBS. After washing, the cells were stained with
phycoerythrin-conjugated goat F(ab')2 anti-mouse IgG and
then analyzed by flow cytometry (for details, see Materials and
Methods). The solid line represents control cells (COS-1 cells
transduced with the viruses containing vector alone), and the dotted
line, COS-1 cells transduced with the viruses containing pLNCXRhAG.
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| |
Discussion |
Rh30 and RhAG proteins are thought to be the same protein family
because of a significant amount of homology.28-32 Rh30
expression was successful using the retroviral gene transfer technology
to generate stable K562 clones expressing the Rh blood group antigens (D, G, c, E).17 Recently, expression of the RhD antigen in
K562 cells has been reported by a number of groups.17,19,33
It is worth noting that wild-type K562 cells produce a low level of RhD
antigens on the cell surface, and appropriate conditions are critical
for the detection by flow cytometry analysis.19 The
presence of endogenous Rh proteins in K562 cells supports the notion
that K562 cells contain the proper cellular mechanism and necessary
components for the surface expression of RhD.20 On the
other hand, the use of K562 cells in the study of de novo synthesis and
potential function of the Rh complex may thus be limited. Thus, we have
been implored to express Rh30 and RhAG in COS-1 cells.
This paper mainly describes expression of RhAG on COS-1 cells.
We constructed RhD- and RhAG-expressing vectors using a retroviral
vector (pLNCX) as shown in Figure 1 and produced stable COS-1 cells
with RhD cDNA or RhAG cDNA. RhAG was expressed on the cells when
determined by flow cytometry (Figure 5). However, RhD was not detected
on the cells, although RhD protein was immunoprecipitated by rabbit
polyclonal anti-Rh or human monoclonal anti-D (LOR15C9) from the
lysates of the stable COS-1 cells transfected with RhD cDNA (Figure 3).
Therefore, RhAG was simply expressed, but RhD was not expressed on the
cell surface regardless of the identical experimental conditions, such
as the same expression vector (pLNCX) and the same host cells (COS-1
cells). This means that expression of RhAG does not require Rh30
and other Rh-related glycoproteins. However, in humans,
CD47 has a very wide tissue distribution and is expressed as different
splicing isoforms. Therefore, the presence of a protein homologous to
CD47 in COS-1 cells (green monkey kidney cells) may not be neglected.
These results support that the expression of RhAG glycoprotein in red
cells does not require the coexpression of Rh30 polypeptides, because
RhnullU+ erythrocytes have RhAG glycoprotein but not
Rh30.34 Moreover, recent studies indicated that K562 cells
carried about 60 × 103 to
90 × 103 copies of RhAG/cell.25
Although the number of RhD protein expressed on K562 cells is not known
but is probably extremely low, it is likely that most of the RhAG
proteins on intact K562 cells are not complexed with the protein. The
studies with transduced COS-1 cells confirm these observations.
However, maximum expression of RhAG may require the presence of Rh30
and other Rh-related glycoproteins. We have shown previously that
endogenous RhAG of K562 cells is underglycosylated.14 This
is most likely explained by the very low level of Rh30 protein
expressed at the cell surface of these cells, which correlates well
with the studies of Ridgwell et al34showing
underglycosylation of RhAG in RhnullU+ red cells
lacking Rh30. The expression level of RhAG glycoprotein on some
Rhnull cells, however, is always very low.23,25
Therefore, the extent of RhAG glycosylation should depend on the
transit time in the Golgi and would be modulated by the presence or
absence of the Rh30 protein. However, transduction of the
RhAG-expressing COS-1 cells with RhD vector did not alter glycosylation
of RhAG (data not shown). Recently, it was also reported that
Rhnull amorph-type individuals bearing normal RHAG
gene and mutant RHCe gene have RhAG glycoprotein but not RhCe
protein on red cells.24,25 On the other hand, expression of
RhD protein seems to be dependent on the presence of RhAG glycoprotein,
because Rhnull regulator-type individuals bearing normal
Rh30 gene and mutant RHAG gene do not carry both Rh30
and RhAG proteins on their red cells. However, surface expression of
RhD was not recognized even in stable COS-1 cells carrying both RhD and
RhAG cDNAs. COS-1 cells transduced with RhD vector grew normally,
suggesting that Rh30 proteins are not toxic to the cells (data not
shown). For the RhD expression, another Rh-related glycoprotein
(glycophorin B, LW, CD47) may be required, or unknown factors including
cytoskeletal protein(s) of red cells, which is thought to interact with
Rh30 proteins, may be involved. However, it is clear that neither LW
nor glycophorin B are necessary, because variant phenotypes
(LW-negative, and S-s-) express normally the Rh antigens,
notwithstanding that LW-negative and S-s- red cells are deficient in LW
glycoprotein and glycophorin B, respectively. Band 3 might
be one of the factors, because Beckmann et al reported that band
3 transduced in K562 cells enhances the cell surface reactivity
of Rh antigens.35
Mallinson et al suggested that 2D10 epitope is chymotrypsin-sensitive
and N-glycan-dependent, because the epitope is destroyed by
endoglycosidase F.36 However, the partially glycosylated RhAG protein was immunoprecipitated by 2D10, indicating that 2D10 epitope may not be carbohydrate-dependent and may have been destroyed by protease contaminants in their glycosidase preparations. RhAG glycoproteins obtained from in vitro expression (Figure 2), intact K562
cells,14 stable COS-1 cells transduced with RhAG cDNA
(Figures 3 and 4), and 2-phase liquid culturewhich was recently
established by us to produce erythroid cells from the progenitor cells
of fresh peripheral blood37are always immunoprecipitated
as a 32-kd protein. Only red cells possess highly
heterogeneous RhAG glycoproteins (40 to 100 kd in size)
and the maximum copies of Rh30. Higher glycosylation of
RhAG may be necessary for the maximum transport of both RhAG and Rh30
to plasma membranes. Stable COS-1 cells expressing RhAG glycoprotein
were established and will be useful for studying
physiologic functions such as ion transport (eg,
NH4+, Ca++).
| |
Acknowledgments |
We are grateful to Dr Albert von dem Borne (Amsterdam, Netherlands) for
the generous gift of mouse monoclonal anti-RhAG(2D10) and Dr Antoine
Blancher for human monoclonal anti-D (LOR15C9). We thank Ruth
Croson-Lowery and Edward Beharry at the New York Blood Center for their
excellent technical assistances in flow cytometric analysis. We would
like to thank Andrea Molinaro of the Laboratory of MicroChemistry for
her outstanding work in DNA sequencing. We also thank Tellervo Huima
for her excellent photography.
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Footnotes |
Submitted March 29, 1999; accepted September 1, 1999.
Reprints: Kimita Suyama, The New York Blood Center, 310 East
67th St, New York, NY 10021; e-mail: ksuyama{at}nybc.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction
