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Pregnancy |
Institute of Biological Chemistry,3 Academia Sinica, Nankang, Taipei 115, Taiwan
Graduate Institute of Biochemical Sciences,4 National Taiwan University, Taipei 106, Taiwan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cell fusion, placenta, pregnancy, syncytin, syncytiotrophoblast, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, a fusogenic membrane protein called syncytin was identified and shown to be specifically expressed in the syncytiotrophoblast layer of human placenta [2]. Syncytin is encoded by an envelope (Env) gene of the human endogenous retrovirus family W (HERV-W). It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit by analogy to other retroviral Env proteins. Syncytin has been demonstrated to mediate cell fusion in a variety of mammalian cell lines, including BeWo, COS, and 293 cells [2, 3]. As in the fusion process of retroviral Env protein, syncytin binds its cognate receptor to initiate the fusion process. Currently, two human sodium-dependent amino acid transporters, ASCT1 and 2, have been reported as the putative syncytin receptors [4]. Murine cells expressing these transporters were able to form syncytia when the syncytin expression plasmid was also introduced into these cells. However, a direct interaction between syncytin and these transporters has not been demonstrated. Furthermore, it is not known if any auxiliary receptor is involved in the interaction between syncytin and these transporters.
A maturation process is required during the biosynthesis of fusogenic retroviral Env glycoproteins [5]. It is believed that protease(s) in the trans-Golgi compartment cleaves the Env protein precursor to release the SU and TM subunits, which then associate together and traffic to the cell surface. The fusion peptide located at the amino terminus of TM interacts with the target cell membrane during the interaction between SU and retroviral receptor. However, currently, neither the biosynthetic pathway of syncytin nor the structure-function relationship of syncytin is clear. Both issues are important for understanding the molecular mechanism underlying syncytin-mediated cell fusion. In this article, we approached these issues by functional characterization of syncytin protein using linker-scanning and deletion mutagenesis. Twenty two linker-inserted and 17 deletion mutants were generated and assessed for their fusogenic activities in 293T and HeLa cells, which do not normally express syncytin. The cytoplasmic domain of syncytin is not essential for syncytin-mediated cell fusion but may play a regulatory role in the process and an intact N-terminal region of syncytin is required for fusion, with the exception of three flexible sites (S51, V139, and E156). In addition, two intervening heptad repeat regions (HRA and HRB) were identified in the TM subunit. The involvement of the heptad repeat regions in syncytin-mediated cell fusion is further discussed in this study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Syncytin cDNA was subcloned into the pIRESDsRed plasmid (BD Biosciences Clontech, Palo Alto, CA), which harbors a red fluorescent protein (RFP) downstream of an IRES (internal ribosomal entry site) element. The resultant plasmid containing syncytin-IRES-DsRed, was further cleaved and subcloned into pTM1 plasmid (kindly provided by Dr. Bernard Moss, National Institutes of Health) to generate pSynIRESRFP. Linker-scanning mutagenesis was performed using a commercial kit based on the in vitro Tn7-based transposition system (NEB, Beverly, MA) (Fig. 1A). In brief, a plasmid containing the syncytin cDNA fragment was used as the target template for in vitro transposition, to which a donor plasmid containing a kanamycin selection marker flanked by two attachment sites (Tn7L and R) for transposase was added. Transposition was initiated after addition of transposase and the reaction mixture was incubated at 25°C for 30 min. The reaction mixture was then transformed into HB101, and kanamycin-resistant colonies were selected. Plasmids prepared from kanamycin-resistant colonies were digested with Pme I to cleave off the selection marker. The resultant linearized syncytin plasmid was self-ligated, resulting in a 15-base pair (bp) insertion in syncytin cDNA. Sequence for the 15-bp insertion is TGTTTAAACA(N)5, where (N)5 are the duplicated nucleic acid residues at the insertion site. After sequencing, mutant syncytin cDNA fragments were further cleaved and substituted for the wild-type syncytin fragment in pSynIRESRFP.
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Cell Culture and Transfection
All cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA). 293T or HeLa cells were maintained at 37°C in minimal essential medium alpha medium, 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. For syncytin expression, 293T or HeLa cells in six-well culture plates were infected with vTF7–3 [6], a recombinant vaccinia virus expressing T7 RNA polymerase, at a multiplicity of infection of 10 viral particles per cell for 90 min. After infection, cells were transfected with 1 µg of expression plasmid using the calcium phosphate coprecipitation method for 293T cells or the Geneporter system (Gene Therapy Systems, San Diego, CA) for HeLa cells.
Fusion Assay of Syncytin Protein
We began to monitor syncytin-mediated cell fusion 12 h after transfection by tracing RFP-expressing cells, which coexpress syncytin. In addition, red fluorescence in transfected cells was monitored to estimate transfection efficiency. As fusion proceeded, patches of red fluorescence were observed under the microscope and scored as fusion events (Fig. 1B). Fusion events were also scored under bright-field microscopy as the formation of multinucleated syncytia. Photographs were taken with an Olympus microscope (Tokyo, Japan) equipped with a cooled charge-coupled device camera (DP50). Images were prepared for presentation using Adobe Photoshop 6.0. The fusogenic activity of syncytin was calculated as a fusion index of (N – S)/T, where N is the number of nuclei in the syncytia, S is the number of syncytia, and T is the total number of nuclei counted, to measure the number of fusion events [7]. The Student t-test was used to determine statistical significance for differences between means. A P value of less than 0.05 was considered significant.
Peptides were synthesized by 9-fluorenylmethoxycarbonyl chemistry [8], purified by HPLC on a C18 column, and verified by mass spectrometry. All peptides were acetylated at the N-terminus and amidated at the C-terminus to enhance biological half-life. For inhibition assays, transfected 293T cells in 24-well plates were treated with peptides at the indicated concentration for 24 h. Multinucleated syncytia were counted by microscopic examination at 40x magnification, which allowed visualization of the entire well in a single field.
Metabolic Labeling, Immunoprecipitation, and Cell Surface Biotinylation
Cells were infected with vTF7-3 and transfected with the indicated expression plasmid as described above. Twelve hours after transfection, cells were starved with DMEM deficient in methionine and cysteine (DMEM-met/-cys) for 45 min and then pulse-labeled with 100 µCi of 35S-methionine and -cysteine in DMEM-met/-cys for 45 min. After labeling, cells were washed once with complete DMEM and then chased by incubation in complete DMEM plus 5 µg/ml of methionine and cysteine for the indicated amount of time. For surface biotinylation, chased cells were washed three times with PBS and biotinylated at 4°C for 30 min with 0.5 mg of NHS-SS-biotin (Pierce, Rockford, IL)/ml in PBS. Cells were lysed in RIPA (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) and incubated with guinea pig antiserum against the syncytin TM subunit at 4°C overnight. Protein A-agarose beads (Oncogene, Boston, MA) were added to precipitate the antigen-antibody complex. After extensive washing, the precipitated complex was boiled in 10% SDS for 5 min, diluted with RIPA, and incubated with streptavidin-magnetic beads (Polysciences, Warrington, PA) at 4°C overnight. After extensive washing, proteins were analyzed by SDS-PAGE and fluorography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For functional characterization of syncytin, we established a fusion assay for syncytin using the vaccine virus-T7 expression system [6]. To monitor fusion of syncytin-expressing cells, an expression plasmid named pSynIRESRFP was constructed to coexpress the DsRed red fluorescent protein (RFP) and syncytin in 293T cells (Fig. 1, A and B). Twelve hours after transfection, we were able to observe red fluorescence in cultured cells. As shown in Figure 1B, spots of red fluorescence were suggestive of transfected cells expressing syncytin and RFP. Moreover, patches of red fluorescence were observed, indicating fusion between syncytin-expressing cells and neighboring cells. However, different intensities of red fluorescence were noticed in different patches, suggesting that cell fusion did not synchronously proceed throughout the cell population.
Functional Characterization of Syncytin Mutants
To investigate the structure-function relationship of syncytin, we characterized the functional domains of syncytin using a series of syncytin mutants generated by linker-scanning and deletion mutagenesis. As shown in Table 1, 22 linker-scanning and 17 deletion mutants in total were tested in the fusion assay. Of the 22 linker-scanning mutants, only InS(51), InV(139), and InE(156) with linker sequence inserted in the syncytin SU subunit and InS(493), InA(506), and InL(529) with linker sequence inserted in the syncytin TM subunit were fusogenic (Fig. 2A). Therefore, regions around amino acid residues S51, V139, and E156 in the amino terminus of syncytin and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation to tolerate insertion of an extra five amino acids. In contrast, mutant InI(337) carrying an extra five amino acids adjacent to the C-terminus of the fusion peptide, without changing the helical structure of fusion peptide, still lost its fusogenicity. In the deletion mutagenesis study, mutants were fusogenic as long as the deletion was limited to the cytoplasmic domain. Of note, the deletion mutant DelI(480), which contains only four amino acid residues of the cytoplasmic domain, was also fusogenic (Fig. 2A). The loss of fusogenic activities in those null mutants was not due to low transfection efficiencies or a low level of transcript because the number of red fluorescent cells and the detection of Northern signals were similar in all mutants tested (data not shown).
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Cytoplasmic Domain of Syncytin Regulates its Fusion Activity
While characterizing the deletion mutants, we observed a conspicuous enhancement of fusion in 293T or HeLa cells transfected with the mutant DelI(480) in comparison with the wild-type syncytin. As shown in Figure 3, A and B, fusion occurred to a higher degree in the DelI(480)-transfected cells when compared with the wild-type syncytin-transfected cells. The fusion index of the mutant DelI(480) was almost threefold higher than that of wild-type sycnytin in 293T cells (Fig. 3C). Similar results were observed in HeLa cells (data not shown). To further understand the differential fusogenic activity between wild-type and mutant DelI(480), we analyzed the biosynthesis of both proteins on the cell membranes of transfected HeLa cells. HeLa cells transfected with wild-type or mutant DelI(480) syncytin were pulsed with 35S-methionine and -cysteine and chased for 30, 60, 120, and 240 min. Surface-bound wild-type and mutant syncytin proteins were sequentially biotinylated and precipitated with an antiserum against the TM subunit of syncytin and streptavidin-magnetic beads. As shown in Figure 3D, wild-type and DelI(480) syncytin precursor proteins were detected on cell surfaces 30 min after chasing (lanes 1 and 5). As the length of the chase period increased, more precursor proteins were detected on cell surfaces. Cleaved TM subunits from wild-type and DelI(480) syncytin precursor proteins were detected 120 min after the chase (Fig. 3D, lanes 3 and 7). Considering the number of methionine residues in the wild type and DelI(480) TM subunits, the signals of both TM subunits were not significantly different. Therefore, the differential fusogenic activity between wild-type and DelI(480) syncytin proteins is unlikely due to a differential proteolytic cleavage of the precursor proteins during their biosyntheses. Based on these observations, the cytoplasmic domain of syncytin may have an inhibitory effect on its cell-fusion process.
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Peptide Inhibition of Syncytin-Mediated Fusion
One of the structural features of retroviral envelope proteins lies in the ectodomain of the TM subunit, whose primary sequence is in part predicted to be extended amphipathic 
-helices like the leucine zipper motif [5, 9, 10]. Synthetic peptides derived from these regions have been used to study the relationship between the secondary structure and the fusogenic activity of envelope proteins [11, 12]. Therefore, we analyzed the primary sequence of syncytin using the Lupas algorithm [13, 14] for predicting secondary structure of leucine zipper-like motif. As shown in Figure 2B, two heptad repeat regions (HRA and HRB) were identified in the TM subunit of syncytin. To investigate the functional relationship between these two heptad repeat regions and the fusogenicity of syncytin, three peptides, HRA1, HRA2, and HRB1, located in the HRA and B regions were synthesized (Fig. 4A). As negative controls, two unrelated peptides, H32 and DP178, were also synthesized (Fig. 4A). The former contained three copies of the hemagglutinin tag, whereas the latter was derived from the TM subunit (gp41) of HIV-1 and is a potent inhibitor of HIV-1-mediated cell-cell fusion [12]. We tested if these peptides could block syncytin-mediated cell-cell fusion in 293T cells transiently expressing syncytin. As shown in Figure 4B H32, DP178, and HRA1 did not block cell fusion; however, HRB1 was a very potent inhibitor that completely blocked cell fusion at 1.6 µg/ml. Compared with HRB1, HRA2 was a less potent inhibitor that, even at a relatively high concentration of 5.2 µg/ml, only inhibited about 35% cell fusion relative to the mock control (Fig. 4B). Together with the findings from the linker-scanning mutagenesis study that mutants InM(358), InM(358)Alt, InK(419), and InQ(426), harboring linker sequences in HRA and B in frame with the polypeptide backbone were not fusogenic (Table 1 and Fig. 2), these results suggested that HRB1 is a specific peptide inhibitor for syncytin-mediated cell-cell fusion and the heptad repeat regions are involved in the fusion process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inhibitory effect of syncytin CTM on cell fusion is reminiscent of the inhibitory R-peptide in the CTM of the envelope protein of murine leukemia virus (MLV). Deletion of this R-peptide in the MLV envelope protein causes extensive fusion with the formation of large syncytia, indicating that R-peptide inhibits membrane fusion [17, 18]. Indeed, the MLV R-peptide is a fragment of 16 amino acids, which is cleaved by the viral protease during virion maturation [19]. Apart from MLV, R-peptide cleavage has been demonstrated in other viruses such as Mason-Pfizer monkey virus and spleen necrosis virus, which are type D and C retroviruses, respectively [20, 21]. Although it is not known whether a similar R-peptide cleavage is required for the maturation of syncytin, the fusogenicity of DelI(480) was enhanced in the absence of most of CTM, suggesting this region may function in a similar manner to R-peptides. Recently, Lavillette et al. [4] demonstrated that an HERV-W Env mutant (termed HERV-Wcyt16), containing a truncated CTM of 16 amino acids, enhances the infectivity of HIV/HERV-Wcyt16 Env pseudotyped viruses. In their study, this enhancement effect was attributed to the interference of the long CTM in the maturation process of the Env protein precursor. Nevertheless, we did not observe a significant difference between wild-type and DelI(480) syncytin in the maturation process in HeLa (Fig. 3D) and 293T (data not shown) cells. This discrepancy may be due to fact that different assay systems (pseudotyped viruses vs. cultured cells) were tested in these two studies. Here we would like to point out that syncytin is a placental membrane protein; therefore, the cultured cell system used in this study is more likely to mimic the physiological condition during the maturation process of syncytin.
Heptad repeat sequences are conserved in the fusion proteins of retroviruses, paramyxoviruses, influenza viruses, and coronaviruses [9]. All of these heptad repeat sequences, when displayed on an -helical wheel, demonstrate an extensive region containing seven amino acid residue repeats of amino acids in a sequence of a, b, c, d, e, f, g, with nonpolar residues in all a positions and in most d positions. These sequences are predicted to form long amphipathic -helices. For HIV-1 Env protein, two heptad repeat sequences (HR1 and 2) have been identified in the ectodomain of its TM subunit, gp41 [9–12]. Peptides DP107 and DP178, respectively, derived from HR1 and 2 of HIV-1 Env protein, are potent inhibitors of HIV-1-Env-mediated cell fusion [11, 12]. Furthermore, the stable -helical structure of the DP107 peptide can be disrupted in the presence of DP178 based on CD studies, suggesting an interaction between the two peptides [12]. The predicted HRA and B regions in the ectodomain of the syncytin TM subunit are highly likely to interact with each other during the cell-cell fusion process because the HRB1 peptide may specifically interact with HRA to block cell fusion. Moreover, insertion of linkers into the HRA region (InM[358] and InM[358]Alt) and the HRB region (InK[419] and InQ[426]) also completely abolished the fusion event, possibly by impeding the interaction between the two regions. Several possibilities may explain the observations in this study that peptide HRA1 did not block fusion and peptide HRA2 had a low efficiency in blocking fusion. First, both peptides may not interact well with HRB. Second, there may be an intrinsic property causing peptides derived from the HRA region of syncytin to be inefficient inhibitors. As in the HIV-1-Env-mediated cell fusion, the HR1-derived DP107 peptide is also a much less efficient inhibitor compared with the HR2-derived DP178 peptide [12]. Third, HRB may be conformationally inaccessible to HRA peptides.
Finally, this study suggests that amino acids S51, V139, and E156 in the SU subunit of syncytin are not critically involved in receptor recognition, binding, and the subsequent conformational rearrangement important for formation of the active TM subunit. Nevertheless, identification of these three amino acid residues may provide a basis for engineering a syncytin polypeptide for therapeutic purposes. Recently, it has been shown that, when expressed in tumor cells, fusogenic envelope protein was able to cause syncytia formation and subsequent cell death [22, 23]. Therefore, targeted expression of the syncytin gene in tumor cells is a feasible gene therapy for tumor growth. To this end, it would be possible to insert into the syncytin polypeptide backbone an epitope or motif, which could specifically interact with surface protein in tumor cells. For example, many tumor cells express a higher level of EGF receptor (EGFR) [24]. Insertion of an EGF motif into syncytin may direct a specific interaction between tumor cells expressing a chimeric protein and EGFR. In this study, three potential motif insertion sites (S51, V139, and E156) were identified in the syncytin SU subunit since insertion of linker sequence in these sites did not affect the fusogenic activity of syncytin. Moreover, because DelI(480) has a higher fusogenicity, engineering or insertion of therapeutic motifs could use the backbone of a DelI(480) polypeptide.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 011 886 2 27889759; hwchen{at}gate.sinica.edu.tw
Received: 18 June 2004.
First decision: 9 July 2004.
Accepted: 21 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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