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INTRODUCTION |
As one of the most important blood group systems involved in blood
transfusion and hemolytic disease, the Rh antigens are present on the
erythrocyte surface as a complex including Rh30 proteins (RhD and RhCE)
and Rh50 glycoprotein (see recent reviews in Refs. 1 and 2).
Hartel-Schenk and Agre (3) first demonstrated that the Rh polypeptides
migrated through sucrose gradients as a complex with an apparent
Mr of 170,000. Based on proteolytic digestion
and immunoblotting, Eyers et al. (4) proposed a model that
consists of two of each Rh30 and Rh50 molecules, with their N-terminal
portions forming the core of the complex. The genes encoding RhD and
RhCE are located at chromosome 1p34-p36 (5), whereas RH50
gene is at chromosome 6p11-21 (6). These three genes share not only a
similar exon-intron composition but also homology in their deduced
amino acid sequences (92% identity between RhD and RhCE, and 36%
identity between Rh30 and Rh50) (2). Therefore, it appears that all
three of these molecules belong to a family of structurally related
membrane proteins.
The multiprotein complex provides the basis for enormously complicated
D antigenic epitopes, which have been serologically classified into
nine epitopes, epD1 to epD9 (7). Most of the information regarding the
molecular basis for D epitopes has been derived from genetic analysis
of RhD variants where part of the RhD gene is missing or replaced by
the highly homologous RhCE sequence (2). Thus, DVI
phenotype, characterized as lack of epD1, 2, 5, 6, 7, and 8, contains
rearranged RHD gene where its exons 4-6 are replaced with
RHCE equivalents (8). In DIVa phenotype that
lacks epD 1, 2, 3, and 9, there is a rearrangement of exon 3 and part
of exon 7 of the RHD gene (9). These observations suggest
that serologically defined D epitopes are organized into overlapping
motifs. A similar notion was deduced from the analysis of
phage-displayed anti-RhD antibodies (10).
With the expression of Rh30 on the surface of K562 cells (11), it is
now possible to elucidate the molecular basis for the D epitopes using
the approach of molecular biology. However, it has been reported (12)
that K562 cells express endogenous RhD protein, as detected by reverse
transcription-PCR1 and
surface binding of anti-RhD. On the other hand, the attempts to express
RhD on the surface of nonerythroid cells, which do not produce
endogenous RhD protein, have met little success (13, 14). It is
possible that multiple proteins are required during RhD synthesis for
the formation of the RhD complex on the cell surface recognized by
specific antibodies. Therefore, to express a high level of RhD protein,
which is biochemically distinguishable from its endogenous counterpart
in K562 cells and useful in the study of the molecular basis for D
epitopes, we expressed RhD as a fusion protein attaching part of Duffy
protein (Fy) to the N terminus of RhD. Here, we present the data about
the surface expression of RhD fusion proteins in K562 cells and the
comparison of its antigenic profile with that of its endogenous
counterpart in K562 cells and mature erythrocytes. Furthermore, this
expression system was used in determining the amino acid residues
involved in the binding of the monoclonal anti-RhD, LOR15C9 (15).
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MATERIALS AND METHODS |
Construction of Expression Plasmids for RhD Fusion
Proteins--
The coding sequence for RhD was subcloned from
pcDNA3-RhD into pREP4 vector at KpnI and XbaI
restriction sites, generating the plasmid pR-RhD. Similarly, the
plasmid pR-Fy was created by subcloning the Duffy (Fy) coding region
from pcDNA1-Fy into pREP4 at HindIII and
NotI. The plasmid pR-DD1 coding for the full-length Fy and
RhD proteins tandemly linked (Fig. 1) was
constructed as follows. Using pR-RhD as a template and P-1 (see Table
I) and REP-R (reverse primer located downstream from the insert in the vector) as primers, the first PCR reaction was carried out according to
previously described conditions (16). Using the first PCR product as a
mega-primer, plus template pR-Fy and primer REP-F (a forward primer
located upstream from the insert) the second PCR reaction was
performed, generating a DNA fragment designated as DD1-PCR product
(Fig. 2). As detailed in the Fig. 2
legend, the plasmid pR-DD1 was constructed via a three-fragment
ligation at three restriction sites, AseI, BbsI,
and BglII. The sequence between BbsI and
BglII in pR-DD1 was verified by an automated DNA sequencer.
By applying a similar strategy, the plasmids pR-DD2 and pR-DD3, which
code the fusion proteins DD2 and DD3, respectively (Fig. 1) were
constructed. In the construction of these two plasmids, the primers P-2
and P-3 (see Table I) were used in the two-step PCR amplification, and
the restriction site HindIII, rather than BbsI,
was used in the three-fragment ligation.

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Fig. 1.
Schematic representation of RhD fusion
proteins. The gray and black lines represent
Fy and RhD protein portions, respectively. The numbers in
parentheses indicate amino acid residues of Fy and RhD. The
membrane topologies of Fy and RhD fusion proteins were proposed based
on hydropathy analysis. A, DD1. B, DD2.
C, DD3.
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Fig. 2.
Construction of the plasmid pR-DD1.
pR-Fy was digested with AseI and BbsI to isolate
a fragment containing the 5' region of the Fy gene. A second DNA
fragment containing the 3' region of the RhD gene was derived from a
double digestion (BglII and AseI) of the plasmid
pR-RhD. Furthermore, a third fragment containing the junction of Fy and
RhD was obtained by digesting DD1-PCR product (see text) with
BbsI and BglII. Finally, the ligation of these
three resulted in the plasmid pR-DD1.
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Substitution of Specific Regions in the RhD Exon 7 with
Corresponding Sequence from RhCE--
Two plasmids, pR-DD2A and
pR-DD2B expressing DD2 proteins mutated in regions A and B of RhD exon
7, respectively, were constructed. The plasmid pR-DD2 was used as a
template in two PCR reactions with two sets of primers (see Table I):
P-A and REP-R; P-Ac and REP-F. The two fragments thus generated were
then linked by PCR because of their 17-nucleotide overlapped region
(underlined sequence in P-A and P-Ac in Table I). The final PCR product
was digested with BglII and NotI and subcloned
into pR-DD2, generating the plasmid pR-DD2A. pR-DD2A has the same
sequence as pR-DD2 except for the region A in RhD exon 7 (Fig.
3), as confirmed by double strand DNA
sequencing. A second plasmid, pR-DD2B, coding for DD2 mutated in region
B of RhD exon 7 (Fig. 3), was constructed by using primers P-B and P-Bc
(see Table I) according to the same procedure described above.

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Fig. 3.
Sequence alignment of exon 7 of RhD and RhCE
genes. Exon 7 DNA sequences of RhD and RhCE are aligned, with the
amino acid sequences listed above and below the corresponding
cDNAs. All residues in RhCE that are identical to those of RhD are
indicated by asterisks. The numbers at the
beginning and the end of sequences indicate nucleotide or amino acid
residues of these two proteins. The boxed regions, A and
B, are two major clusters that differ in exon 7 between RhD
and RhCE and were chosen for mutations in the plasmids pR-DD2A and
pR-DD2B.
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Cell Culture and Transfection--
K562 erythroleukemic cells
were transfected with 1 µg of plasmid and 10 µl of LipofectAMINE
Reagent (Life Technologies, Inc.) according to the manufacturer's
instructions. After incubating the cells for 48 h at 37 °C and
5% CO2 in RPMI medium supplemented with 10% fetal bovine
serum (Life Technologies, Inc.), stably transfected cells were selected
by culturing diluted cells (1:10) in the presence of 200 µg/ml of
hygromycin (Boehringer Mannheim) for 2 weeks. The plasmids pREP4
("mock"), pR-RhD, pR-Fy, pR-DD1, pR-DD2, and pR-DD3 were used in
the K562 cell transfections.
Flow Cytometry Analysis of Transfected Cells--
K562 cells
(5 × 105) suspended in 50 mM phosphate
buffer with 0.59% saline (PBS) containing 0.5% bovine serum albumin
and 0.01% sodium azide were incubated with either human monoclonal
antibody (LOR15C9, 3 µg/ml) or a mouse monoclonal antibody
recognizing Fy6 (anti-Fy6, 9 µg/ml) at 37 °C for 30 min. After
washing the cells three times in the same buffer, the cells were
incubated with the appropriate secondary antibody, either sheep
anti-mouse IgG conjugated to fluorescein isothiocyanate (Sigma) or goat
anti-human IgG F(ab')2 conjugate to phycoerythrin
(Biosource) for 30 min at room temperature. The cells were then washed
and analyzed by flow cytometry (Becton Dickinson). The control cells
used in the study are K562 cells transfected with the vector, pREP4.
Confocal Microscope Analysis of Transfected K562
Cells--
Cells were fixed in a 1% paraformaldehyde solution for 20 min on ice. After washing with PBS buffer, the cells were incubated in
PBS containing 0.1% saponin for 30 min at room temperature. The cells
were then washed with the same PBS prior to antibody sensitization as
described under "Flow Cytometry Analysis of Transfected Cells." To
prepare slides, 1 × 105 cells were cytospun onto
poly-L-lysine coated slides, mounted with Vectashed, and
sealed. Slides were stored at
20 °C until viewed by confocal
microscopy (17). Photographs were taken with 2100 ASA Ektachrome film.
Immunoprecipitation of 35S-Labeled Cells--
Stable
transfected cells (1 × 107) were labeled for 2 h
with 200 µCi/ml of [35S]methionine (NEN Life Science
Products), washed three times with PBS, and lysed using 20 mM Tris-HCl buffer, pH 7.4, containing 1% Triton X-100,
0.25% SDS, l mM/liter tosylphenylalanyl chloromethyl ketone, 1 mM/liter phenylmethylsulfonyl fluoride, and 100 units/ml Trasylol. After preclearing the supernatant using normal
rabbit serum and protein A-Sepharose, the lysate was incubated
overnight at 4 °C with either rabbit anti-Rh polyclonal antiserum or
normal rabbit serum. The immune complexes were isolated by subsequent incubation with protein A-Sepharose, eluted, and separated by 12%
SDS-PAGE as described previously (14).
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RESULTS |
Flow Cytometric Analysis of K562 Cells Expressing RhD Fusion
Proteins--
To study the expression of RhD protein in K562
erythroleukemic cells and distinguish recombinant from endogenous RhD,
we constructed expression plasmids for RhD fusion proteins (DD1, DD2,
and DD3). As shown schematically in Fig. 1, the full-length RhD of 417 amino acid residues is linked at its N terminus either to the
full-length (338 amino acids) or to the N-terminal portion (97 amino
acids) of Fy, generating the fusion proteins, DD1 and DD2,
respectively. Further truncated from DD2, DD3 is composed of the amino
acid residues 1-64 from Fy and 32-417 from RhD. The DNAs coding for these fusion proteins were subcloned into the vector pREP4 generating pR-DD1, pR-DD2, and pR-DD3 plasmids, as detailed under "Materials and
Methods." The pREP4 was chosen as the expression vector for its
strong Rous sarcoma virus promoter and its multi-copy extra-chromosomal replication ability. Table I lists the
oligonucleotides used in the construction of these plasmids. After
stable transfected cell lines were established, the presence of
specific plasmid in the transfected cells was confirmed by PCR (data
not shown). Approximately 50 cells were used as a template in the PCR
with a specific Rh primer and a primer corresponding to the Rous
sarcoma virus promoter region to ensure that the amplified signal was from the plasmid rather than an endogenous sequence.
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Table I
Oligonucleotides used in the construction of plasmids
The numbers directly below the DNA codon represent the position of
corresponding amino acid residues. The arrows above the sequences
indicate the junction between Fy (5' region) and RhD (3' region). Note
that the P-3 includes an extra restriction site, SpeI,
between the Fy and RhD regions. P-A and P-B were designed based on the
coding sequence of RhCE, whereas P-Ac and P-Bc were derived from the
noncoding sequence. P-A and P-Ac are complementary to each other in the
region underlined. The same is true with P-B and P-Bc. The nucleotides
that are different from RhD are in bold type.
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To detect the expressed RhD on the K562 cell surface and distinguish it
from the endogenous protein in flow cytometry, we varied a number of
reaction parameters, including the amount of primary antibody, binding
temperature and time, washing conditions, and secondary antibodies.
Although incubation of anti-RhD antibody, LOR15C9, at higher
concentrations resulted in higher mean fluorescence intensity (MFI),
the MFI ratio for the expressed RhD antigen versus its
endogenous counterpart diminished. Thus at 3 µg/ml of purified LOR15C9, the MFI ratio was 3.18, whereas the ratio dropped to 1.28 when
the antibody concentration increased to 27 µg/ml. In addition, three
different fluorescence (fluorescein isothiocyanate, Texas Red, and
phycoerythrin) conjugated to secondary antibodies were tested. Under
the same conditions, the best result was obtained by using
phycoerythrin-conjugated secondary antibody, where the MFI is at least
six times higher than using fluorescein isothiocyanate conjugate
antibody. Following the conditions detailed under "Materials and
Methods," we obtained a profound shift in the fluorescence-activated cell sorter profile of expressed RhD protein over its endogenous counterpart on the K562 cell surface.
As shown in the first graph of Fig.
4A, the K562 cells transfected
with the vector pREP4 demonstrated stronger binding with LOR15C9
(peak e) than with control IgG (peak c) at the
same concentration. This corresponds to an increase in MFI value from
7.44 to 26.29 (Table II), indicating the
presence of endogenous RhD on the K562 cell surface. Therefore, serving
as a base line, peak e was added to each graph in Fig.
4A. Although no endogenous Fy antigen was detectable under
the applied conditions (the first graph in Fig. 4B), the
same reasoning and procedure were applied in constructing Fig.
4B. Cells transfected with either pR-RhD or pR-Fy
demonstrated significant shifts of their fluorescence-activated cell
sorter profile when treated with LOR15C9 or anti-Fy6, respectively,
suggesting the expression of both antigens on the transfected cell
surface. Among the three fusion protein-expressing cell lines, the
surface expression of DD2 and DD3 was positively identified by using
both LOR15C9 and anti-Fy6. For the pR-DD2 transfected cells, the
binding of LOR15C9 resulted in an increase of MFI from 26.29 to 70.02, whereas the binding of anti-Fy6 caused an increase from 9.39 to 32.25 (Table II). On the other hand, the surface expression of DD1 was not
detected using LOR15C9, although a slight increase in MFI (from 9.39 to
14.17) was observed by the binding of anti-Fy6.

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Fig. 4.
Flow cytometry analysis of stably transfected
K562 cells. Cells expressing mock, RhD, Fy, DD1, DD2, and DD3 were
incubated with either LOR15C9 (A) or anti-Fy6 (B)
and analyzed by flow cytometry. The results are shown as the
fluorescence intensity (x axis) plotted against the counts
of events (y axis). The name of each cell line is displayed
at upper right corner of each graph. The peak marked
c in the first graph of both panels is a control by using
normal IgG as a primary antibody. In the same graph, the peak marked
e was generated by using LOR15C9 (A) or anti-Fy6
(B) and thus displayed in all graphs as a reference.
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Table II
Mean fluorescence intensity and relative fluorescence derived from the
flow cytometry analysis shown in Fig. 5
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Confocal Microscope Analysis of Transfected Cells--
As shown in
Fig. 5, the presence of endogenous RhD
antigen in K562 cells was demonstrated by comparing panels 1 and 2 (control IgG and LOR15C9 as primary antibody,
respectively). Consistent with flow cytometric analysis, Fy antigen was
not detected (panel 3) in K562 cells using anti-Fy6.
Panels 4-7 in Fig. 5 depict the fluorescence of DD2- and
DD3-expressing cells treated with either LOR15C9 or anti-Fy6. Both cell
lines demonstrated strong binding with either antibody, and the
labeling seems more predominant on the edge of the cells. The data
provide further evidence that RhD fusion proteins, DD2 and DD3, were
expressed on the surface of transfected K562 cells.

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Fig. 5.
Confocal microscope analysis of transfected
K562 cell lines. All cell samples were fixed and then treated with
saponin and subjected to immunofluorescence analysis as described under
"Materials and Methods." Mock and DD2- and DD3-expressing cells
were labeled with LOR15C9 (panels 2, 4, and
6, respectively) or with anti-Fy6 (panels 3,
5, and 7, respectively). Mock cells were labeled
with normal IgG as a background control (panel 1).
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Immunoprecipitation of the Transfected K562 Cells--
Two
experiments were carried out to provide biochemical evidence for the
expression of RhD fusion proteins in transfected cells. Cells were
labeled with [35S]methionine and immunoprecipitated with
a rabbit polyclonal anti-Rh antibody. The immunoprecipitates were then
subjected to SDS-PAGE followed by autoradiography as shown in Fig.
6. The immunoprecipitate of cells
transfected with pR-RhD (lane 1) contained a band (marked a), which migrated as RhD protein (32 kDa). A band of 70 kDa
(marked b) was detected from pR-DD1 transfected cells
(lane 2), whereas a band (marked c) in the range
of 50-55 kDa was visualized from cells expressing DD2 and DD3
(lanes 3 and 4, respectively). The apparent
molecular masses of these bands are in agreement with estimated
molecular masses of RhD fusion proteins, DD1, DD2, and DD3. All of the
immunoprecipitates contained high molecular mass proteins as visualized
on the gel. Although the nature of these proteins have yet to be
identified, they may represent aggregates of RhD or the fusion proteins
or other components involved in the formation of an RhD antigen complex
in the cell membrane.

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Fig. 6.
Detection of RhD fusion proteins in
transfected K562 cell lysates. Cell lysates were prepared from
[35S]methionine-labeled cells and immunoprecipitated with
rabbit anti-Rh antiserum as described under "Materials and
Methods." Lanes 1-4 depict SDS-PAGE analysis of
immunoprecipitates isolated from cells expressing RhD, DD1, DD2, and
DD3 cells, respectively. Molecular mass standard (M) are
displayed in kDa on the left of the figure.
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A second approach involved a preparation of membrane fractions from
transfected cells following the procedure of Chaudhuri et
al. (18). This fraction was resuspended in the lysis buffer for
immunoprecipitation with rabbit polyclonal anti-Rh, followed by Western
blotting using anti-Fy6. As shown in Fig.
7, the samples from DD1-expressing cells
(lane 3) and DD2-expressing cells (lane 4)
contained distinctive bands marked b and c,
respectively, which are consistent with the estimated molecular masses
of DD1 and DD2 proteins. The high molecular mass smear visible in
lanes 3 and 4 may represent the aggregates
involving Rh fusion proteins. On the other hand, no visible bands were
detected from cells transfected with pREP4 (lane 2)
following the same procedure. This is expected because there is no
protein in the cells that can be recognized by both antibodies. Red
cell ghosts (10 µl) were directly loaded in lane 1 as a
control for the ECL Western blot using anti-Fy6.

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Fig. 7.
Detection of expressed fusion proteins using
two antibodies. The plasma membrane fractions were prepared from
5 × 107 culture cells, and the immunoprecipitation
was carried out by using rabbit anti-RhD antibody as described under
"Materials and Methods." The eluates from the Sepharose beads were
subjected to SDS-PAGE for ECL Western blotting with anti-Fy6.
Lane 1 is red cell ghosts control for immunoblotting with
anti-Fy6. Lanes 2-4 are immunoprecipitates from mock and
DD1- and DD2-expressing cells, respectively. The molecular mass
standards in kDa are listed on the left of the figure.
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Antigenic Profile of RhD on the Surface of K562
Cells--
Although the surface expression of RhD was confirmed by the
binding of LOR15C9, the extent to which the RhD on K562 cells resembles
its counterpart on red blood cells had yet to be established. Toward
that end, we were able to address this issue by flow cytometry analysis
using a series of monoclonal antibodies against different epitopes of
RhD (19) (Table III). The reactivity of
each antibody was first verified by using red blood cells (RhD+) under
our assay conditions. By comparing MFI values of K562 with background
(without primary antibody), we then showed that seven antibodies
reacted positively, whereas the remaining two (LOR11-12E2 and
LOR17-6C7) failed to bind to K562 cells. Furthermore, when
DD2-expressing cells were examined, the same antibody binding pattern
was observed. Although the two "negative" monoclonal reagents
remained negative, the rest showed a more than 3.5-fold increase
compared with K562 cells. Thus, the discrepancy in the RhD antigenic
profile between red cells and K562 cells is clearly demonstrated.
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Table III
Antigenic profile of RhD on the K562 cell surface
Nine different monoclonal reagents recognizing RhD proteins (G is an
RhD-associated antigen) were used in the flow cytometric analysis of
mock transfected K562 cells (K562), DD2-expressing cells (DD2), and
RhD-positive red blood cells (RBC). The reactions without antisera were
measured as background. H stands for highly reactive (MFI > 1,000).
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Substitution of Specific Regions in RhD Exon 7 with Corresponding
Sequence from RhCE--
Because LOR15C9 specifically recognizes RhD
exon 7 but not the highly homologous RhCE, it is reasonable to assume
that antibody-binding epitope(s) in RhD exon 7 reside in the regions
that differ between these two Rh proteins as shown in Fig. 3. The
alignment of exon 7 sequences of RhD and RhCE illustrates two major
clusters of sequence variation, designated as regions A and B, together
with two other single amino acid alterations at positions 314 and 342. Using synthetic oligomers and overlapping PCR, we generated a mutated
DD2 molecule named DD2A, where RhD sequence in region A was replaced
with corresponding RhCE sequence. Similarly, we constituted DD2B, where
region B was replaced with RhCE.
After confirming the surface expression of DD2A and DD2B on stably
transfected K562 cells by using anti-Fy6, we examined the possible
effect of mutations in RhD exon 7 on the binding of LOR15C9 in flow
cytometry analysis. As suggested in Fig.
8, the antibody binding was weaker to the
DD2A-expressing cells (4) than to the DD2-expressing cells (3). More
strikingly, mutation in region B (5) resulted in severe reduction in
antibody binding.

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Fig. 8.
Flow cytometry analysis using LOR15C9.
The data are plotted as fluorescent intensity against counts of events.
The K562 cells transferred with the vector pREP4 were incubated without
(1) or with (2) LOR15C9. Cells expressing DD2
(3), DD2A (4), or DD2B (5) were all
incubated with LOR15C9 prior to the binding a second antibody,
anti-human IgG (H and L chains) conjugated to phycoerythrin.
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DISCUSSION |
Our objective was to design a RhD fusion protein that can be
detected with antibodies against both RhD and the fused tag in flow
cytometry analysis and be easily distinguishable in size from
endogenous RhD on SDS-PAGE. Because the RhD molecule contains 12-transmembrane domains with both termini facing cytoplasma, the
fusion of a simple tag such as hexahistidine at either end would not
provide a marker detectable on the cell surface. Therefore, we explored
the possibility of using a type I transmembrane protein in the
construction of RhD fusion proteins. Fy protein, a blood group antigen
and a promiscuous chemokine receptor, has been expressed on the surface
of K562 cells that does not have detectable background (18, 20). The
extracellularly located N-terminal domain (64 residues) of the protein
is recognized by anti-Fy6. Furthermore, the antibody binding site has
been mapped to the region between residues 9-44 of Fy protein (21).
Taking advantage of these features, we constructed three plasmids
expressing RhD fusion proteins, DD1, DD2, and DD3 (Fig. 1). To maintain
the proposed membrane topology of RhD proteins, the full-length or
first 97 amino acids (the N-terminal extracellular and first
transmembrane domain) of Fy was conjugated to the N terminus of RhD
protein in the cases of DD1 and DD2. Furthermore, to examine whether
the transmembrane domain (66-86 amino acids) of Fy is required for membrane localization of DD2 and proper presentation of Fy6 and RhD
antigens, DD3 fusion protein was constructed. The N-terminal 32 amino
acids of RhD containing the first transmembrane domain was also removed
in the DD3 construct to maintain the proper conformation of RhD protein
in the cell membrane. In addition, two amino acid residues, Thr-Ser,
were engineered in the junction of Fy and RhD to provide the necessary
flexibility of the polypeptide to eliminate any possible steric
hindrance of neighboring domains recognized by antibodies. The same
strategy was used by Nunoki et al. (22) in constructing
hybrid potassium channels.
The data derived from flow cytometric and confocal microscope analyses
indicate that fusion proteins DD2 and DD3 are expressed on the K562
cell surface in conformations that are recognized by LOR15C9 and
anti-Fy6. This underscored the notion that these two fusion proteins
have the membrane topology as projected in Fig. 1. The expression of
these two fusion proteins was further confirmed by the
immunoprecipitation experiments. As shown in Fig. 6, both proteins
migrated to approximately the same position on SDS-PAGE, although the
calculated molecular mass of DD2 is approximately 7 kDa more than that
of DD3. This may be due to the fact that Rh protein, which constituted
mostly of DD2 and DD3, is known to migrate in SDS gel anomalously (23).
On the other hand, although the expression of DD1 (80 kDa) was
confirmed as shown in Figs. 6 and 7, its presence on the cell surface
was not definitely identified by flow cytometric analysis. Because DD1
is composed of 19 transmembrane domains (Fig. 1) based on the model for
Fy and RhD proteins, the fusion protein may be simply too big to be
transported to the cell surface.
As an important molecule in the Rh complex, Rh50 is endogenously
produced in K562 cell line and migrates as a protein of 32 kDa on
SDS-PAGE presumably due to incomplete
glycosylation.2 However, the
extent to which the Rh50 glycoprotein is required for the expression of
RhD and D epitope formation on the surface of K562 cells has yet to be
elucidated. In our immunoprecipitation experiment (Fig. 6, lanes
2-4), although no band around 32 kDa is visible, the possibility
that Rh50 was coprecipitated with RhD fusion proteins could not be
excluded. Compared with overexpressed fusion proteins, endogenous Rh50,
as well as endogenous RhD, could be simply too low in quantity to be
detected under experiment conditions. Nevertheless, RhD fusion proteins
with different molecular masses may prove useful in the study of
interactions between Rh50 and RhD.
Smythe et al. (11) reported the expression of RhD and RhcE
gene products by retroviral transduction of K562 cells. Their data
provide the first direct evidence that the putative RhD gene gives rise
to D and G antigens and that the putative RhcE gene gives rise to c and
E antigens. They concluded that retroviral delivery of the gene was of
critical importance in achieving expression of Rh antigens on K562
cells. Based on their conditions, anti-RhD bound more strongly to the
RhD-expressing K562 cells than to the control cells, with an increase
in MFI from 3.3 to 15.2, whereas the level of endogenous Rh antigen in
the untransduced K562 cells was minuscule (from 2.8 to 3.8). On the
other hand, based on our data, we were able to clearly demonstrate not
only endogenous RhD in K562 cells (increase in MFI from 7.44 to 26.29)
but also exogenous RhD expressed on the cell surface (increase in MFI
from 26.29 to 143.61). Our data suggest that successful expression and
detection of RhD protein on the K562 cell surface may have resulted
from optimized fluorescence-activated cell sorter conditions with a
highly specific and purified antibody, as well as an efficient transfection system.
Because the DD2 molecule contains the full-length RhD and the junction
between RhD and the Fy tag is intracellularly located, DD2 is more
likely than DD3 to retain the native D antigenic profile. In addition,
although RhD was also expressed under the same conditions, we believed
that the DD2 molecule would be preferable in the study of the molecular
basis for RhD antigenic epitopes. This is because the Fy portion at the
N terminus of DD2 can serve as a tag for confirming the cell surface
expression, regardless of any changes made in the RhD portion of the
fusion protein. Our mutagenesis study indicated that the binding of
LOR15C9 was adversely affected by both mutations, DD2A and DD2B, with
the latter being more severe. It is interesting to note that according
to the proposed membrane topology of RhD protein, region B is located
at the sixth extracellular loop of RhD, whereas region A is near the
intracellular end of a transmembrane domain. Thus, our data suggest
that region B may be directly involved in the binding with LOR15C9,
whereas mutations in region A may elicit a long distance effect on
antibody binding on the cell surface. In other words, the substitution
of region A in RhD with corresponding RhCE sequence may transform an
exofacial D epitope into "CE-like" conformation. Monoclonal
antibody LOR15C9 has been well characterized by Apoil et al.
(15). Their data indicate that the antibody recognizes not only RhD
molecule on the cell surface in flow cytometry analysis but also the
denatured proteins by immunoblotting. Our observations that both
mutations (A and B) decreased but did not abolish the binding of
LOR15C9 as determined by flow cytometry further support the notion that the binding of LOR15C9 depends on conformation as well as sequence of
the antigen within RhD protein.
Our analysis of the RhD antigenic profile of K562 cells suggests subtle
changes in the conformation or microenvironment of RhD (both endogenous
and exogenous) on the K562 cell surface in comparison with its
counterpart on red blood cells. Specifically, the epD2 and 4 recognized
by LOR11-12E2 and LOR17-6C7, respectively, may have been somehow
affected by K562 cells. The lack of activity for LOR-12E2 was further
confirmed by a related antibody, LOR-12E2-1C6 (data not shown).
Although Mag 1-123 may possibly have an overlapping epD specificity
with LOR11-12E2, there is evidence suggesting that these two reagents
are serologically distinctive (24). In addition, we tested four
different monoclonal reagents with the same specificity (epD6 and 7).
As expected, they all reacted positively with expressed DD2, as well as
with endogenous RhD on the K562 cell surface. The information generated
from such antigenic profile analysis will be important in designing
approaches and interpreting data to determine the molecular basis for
RhD antigenic epitopes using K562 cells as an expression host.