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Use of RhD Fusion Protein Expressed on K562 Cell Surface in the Study of Molecular Basis for D Antigenic Epitopes

1999; Elsevier BV; Volume: 274; Issue: 9 Linguagem: Inglês

10.1074/jbc.274.9.5731

1083-351X

Alex Zhu, Stephanie Haller, Hua Li, Asok Chaudhuri, Antoine Blancher, Kimita Suyama,

Monoclonal and Polyclonal Antibodies Research

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323–331(A) and 350–354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes. The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323–331(A) and 350–354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes. polymerase chain reaction phosphate-buffered saline polyacrylamide gel electrophoresis mean fluorescence intensity 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. 1Blancher A. Socha W.W. Blancher A. Klein J. Socha W.W. The Rhesus System in Molecular Biology and Evolution of Blood Group & MHC Antigens in Primates. Springer-Verlag, Berlin1997: 147-218Google Scholar and 2Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (93) Google Scholar). Hartel-Schenk and Agre (3Hartel-Schenk S. Agre P. J. Cell Biol. 1992; 267: 5569-5574Google Scholar) first demonstrated that the Rh polypeptides migrated through sucrose gradients as a complex with an apparentM r of 170,000. Based on proteolytic digestion and immunoblotting, Eyers et al. (4Eyers S. Ridgwell K. Mawby W.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6417-6423Abstract Full Text PDF PubMed Google Scholar) 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 (5Cherif-Zahar B. Mattei G. LeVan K.C. Bailly P. Cartron J.-P. Colin Y. Hum. Genet. 1991; 86: 398-400Crossref PubMed Scopus (137) Google Scholar), whereas RH50gene is at chromosome 6p11–21 (6Ridgwell K. Spurr N.K. Laguda B. Maggeoch C. Avent N.D. Tanner M.J.A. Biochem. J. 1992; 287: 223-228Crossref PubMed Scopus (114) Google Scholar). 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) (2Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (93) Google Scholar). 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 (7Lomas C. McColl K. Tippett P. Transfus. Med. 1993; 3: 67-69Crossref PubMed Scopus (72) Google Scholar). 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 (2Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (93) Google Scholar). Thus, DVIphenotype, characterized as lack of epD1, 2, 5, 6, 7, and 8, contains rearranged RHD gene where its exons 4–6 are replaced withRHCE equivalents (8Mouro I. Le Van K.C. Rouillac C. van Rhenen D.J. LePennec P.Y. Bailly P. Cartron J.-P. Blood. 1994; 83: 1129-1135Crossref PubMed Google Scholar). 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 (9Rouillac C. Colin Y. Hughes-Jones N.C. Boelet M. D'Ambrosio A.M. Cartron J.-P. Le Van Kim C. Blood. 1995; 85: 2937-2944Crossref PubMed Google Scholar). 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 (10Chang T.Y. Siegel D.L. Blood. 1998; 91: 3066-3078Crossref PubMed Google Scholar). With the expression of Rh30 on the surface of K562 cells (11Smythe J.S. Avent N.D. Judson P.A. Parson S.F. Martin P.G. Anstee D.J. Blood. 1996; 87: 2968-2973Crossref PubMed Google Scholar), it is now possible to elucidate the molecular basis for the D epitopes using the approach of molecular biology. However, it has been reported (12Suyama K. Lunn R. Haller S. Goldstein J. Blood. 1994; 84: 1975-1981Crossref PubMed Google Scholar) 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 (13Hermand P. Mouro I. Huet M. Bloy C. Suyama K. Goldstein J. Cartron J.P. Bailly P. Blood. 1993; 82: 669-676Crossref PubMed Google Scholar, 14Suyama K. Roy S. Lunn R. Goldstein J. Blood. 1993; 82: 1006-1009Crossref PubMed Google Scholar). 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 (15Apoil P.A. Reid M.E. Halverson G. Mouro I. Colin Y. Roubinet F. Cartron J.-P. Blancher A. Br. J. Haematol. 1997; 98: 365-374Crossref PubMed Scopus (33) Google Scholar). 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 andNotI. 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 TableI) 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 (16Zhu A. Wang Z.-K. Eur. J. Biochem. 1996; 235: 332-337Crossref PubMed Scopus (12) Google Scholar). 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. 2legend, the plasmid pR-DD1 was constructed via a three-fragment ligation at three restriction sites, AseI, BbsI, and BglII. The sequence between BbsI andBglII 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.Table IOligonucleotides used in the construction of plasmidsOligonucleotides used in construction of plasmids for RhD fusion proteinsP-15′-ACC CTT GGA AGC AAA TCC↓AGC TCT AAG TAC CCG CGG-3′333 338 2 7P-25′-TGG CAG CTC TGC CCT GGC↓AGC TCT AAG TAC CCG CGG-3′92 97 2 7P-35′-GGA AGC GTC ATA GTG GGT↓ACT AGT↓CAG TGC AGA GTC ATC CAG-3′59 64 SpeI 32 37Oligonucleotides used in construction of RhD mutantsP-A5′-C ATC TCC GTC ATG CAC T CC ATC TTC AGC TTG-3′325 334P-Ac5′-A GTG CAT GAC GGA GAT GTG GTG AAT CCC CAG-3′P-B5′-T CAT ACT GTC TGG AAC GGC AAT GGC A-3′349 356P-Bc5′-C GTT CCA GAC AGT ATG AAG CAC CAG C-3′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. Open table in a new tab Figure 2Construction 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) withBbsI and BglII. Finally, the ligation of these three resulted in the plasmid pR-DD1.View Large Image Figure ViewerDownload (PPT) 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. 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. 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. 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. 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 (17Rajasekaran A.K. Humphrey J.S. Wagner M. Miesenbock G. Le Bivic A. Bonifacino J.S. Rodriguez-Boulan E. Mol. Biol. Cell. 1994; 5: 1093-1103Crossref PubMed Scopus (51) Google Scholar). Photographs were taken with 2100 ASA Ektachrome film. 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 (14Suyama K. Roy S. Lunn R. Goldstein J. Blood. 1993; 82: 1006-1009Crossref PubMed Google Scholar). 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. 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.4 A, 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.4 A. Although no endogenous Fy antigen was detectable under the applied conditions (the first graph in Fig. 4 B), the same reasoning and procedure were applied in constructing Fig.4 B. 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.Table IIMean fluorescence intensity and relative fluorescence derived from the flow cytometry analysis shown in Fig. 5LOR15C9Anti-Fy6PercentageMFIPercentageMFIControl25.03 (0.77)26.29 (7.44)0.649.39RhD92.77143.610.326.25Fy35.1825.8837.4272.68DD144.2233.094.4414.17DD283.9770.0236.9532.25DD374.3658.1125.2728.67 Open table in a new tab As shown in Fig. 5, the presence of endogenous RhD antigen in K562 cells was demonstrated by comparing panels 1and 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. 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 (markeda), 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. A second approach involved a preparation of membrane fractions from transfected cells following the procedure of Chaudhuri et al. (18Chaudhuri A. Zbrzezna V. Polyakova J. Pogo A.O. Hesselgesser J. Horuk R. J. Biol. Chem. 1994; 269: 7835-7838Abstract Full Text PDF PubMed Google Scholar). 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 inlanes 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. 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 (19Roubinet F. Apoil P.A. Blancher A. Transfus. Clin. Biol. 1996; 3: 247-255Crossref PubMed Scopus (15) Google Scholar) (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.Table IIIAntigenic profile of RhD on the K562 cell surfaceMonoclonal reagentsSpecificity (eptD)MFIBackgroundK562DD2RBCLORIFA6, 74.718.259.1HLOR15C93410.537.5HSAL20/S16, 74.7932.3HGAN-4B56, 7416.568.2HNOI6, 73.812.269.2HLOR11–12E223.83.94.9HLOR17–6C743.84.96.1HMab1–12324.17.541.7HH2G11G6G4.112.243.6HNine 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). Open table in a new tab 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). 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 (4Eyers S. Ridgwell K. Mawby W.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6417-6423Abstract Full Text PDF PubMed Google Scholar) than to the DD2-expressing cells (3Hartel-Schenk S. Agre P. J. Cell Biol. 1992; 267: 5569-5574Google Scholar). More strikingly, mutation in region B (5Cherif-Zahar B. Mattei G. LeVan K.C. Bailly P. Cartron J.-P. Colin Y. Hum. Genet. 1991; 86: 398-400Crossref PubMed Scopus (137) Google Scholar) resulted in severe reduction in antibody binding. 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 (18Chaudhuri A. Zbrzezna V. Polyakova J. Pogo A.O. Hesselgesser J. Horuk R. J. Biol. Chem. 1994; 269: 7835-7838Abstract Full Text PDF PubMed Google Scholar, 20Chaudhuri A. Polyakova J. Zbrzezna V. Williams K. Gulati S. Pogo A.O. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10793-10797Crossref PubMed Scopus (216) Google Scholar). 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 (21Chitnis C.E. Chaudhuri A. Horuk R. Pogo A.O. Miller L.H. J. Exp. Med. 1996; 184: 1531-1536Crossref PubMed Scopus (155) Google Scholar). 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. (22Nunoki K. Ishii K. Okada H. Yamagishi T. Murakoshi H. Taira N. J. Biol. Chem. 1994; 269: 24138-24142Abstract Full Text PDF PubMed Google Scholar) 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 (23Avent N.D. Liu W. Warner K.M. Mawby W.J. Jones J.W. Ridgwell K. Tanner M.J. J. Biol. Chem. 1996; 271: 14233-14239Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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. 2A. Zhu and K. Suyama, unpublished data. 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. (11Smythe J.S. Avent N.D. Judson P.A. Parson S.F. Martin P.G. Anstee D.J. Blood. 1996; 87: 2968-2973Crossref PubMed Google Scholar) 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.(15Apoil P.A. Reid M.E. Halverson G. Mouro I. Colin Y. Roubinet F. Cartron J.-P. Blancher A. Br. J. Haematol. 1997; 98: 365-374Crossref PubMed Scopus (33) Google Scholar). 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 (24Scott M. Transfus. Clin. Biol. 1996; 3: 333-337Crossref PubMed Scopus (53) Google Scholar). 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. We thank Dr. Jack Goldstein for support of the project and critical reading of the manuscript. We are also grateful to Dr. Ayyappen K. Rajasekaran at Cornell University Medical College for helpful discussion regarding the confocal microscope analysis and Ruth Croson-Lowney at the New York Blood Center for excellent technical assistance in flow cytometric analysis.