卡波氏肉瘤相关疱疹病毒vGPCR具有巯基化酪氨酸的氨基端的表达和纯化

吴 慧，傅永明，肖 俊，周 曼，冯 浩

(湖南师范大学生命科学学院，蛋白质化学及发育生物学教育部重点实验室，中国 长沙 410081)

Kaposi’s sarcoma (KS) is an angioproliferative neoplasm consisting of characteristic spindle cells and infiltrating leukocytes and is a leading cause of morbidity and mortality among the AIDS population. The Kaposi’s Sarcoma-associated Herpesvirus (KSHV, also named Human Herpesvirus 8/HHV-8) is the etiologic agent for Kaposi’s Sarcoma, Primary Effusion Lymphoma (PEL), and Multicentric Castleman’s Disease (MCD)[1-6]. KSHV is a DNA virus and belongs to garmma-2 herpesvirus that is closely related to herpesvirus saimiri (HVS) and rhesus monkey rhadinovirus (RRV). The genome of KSHV is approximately 165～170 kb and contains more than eighty open reading frames[6-7].

vGPCR is encoded by KHSV open reading frame 74 (ORF74) and is a member of the family of CXC chemokine GPCRs. vGPCR is the homolog of human interleukin-8 receptor (IL-8R or CXCR2), however, unlike its cellular counterpart, it is constitutively activated without ligand association[8-9]. vGPCR triggers downstream signaling components including phopholipase C pathway, the PI3 kinase/Akt axis, and has broad signaling effects in tissue culture, activating NF-κB, NFAT and AP-1 family[10-18]. Emerging evidence supports that this KHSV lytic gene is an oncogene, vGPCR transformed NIH3T3 cells led to tumorigenesis in nude mice and vGPCR transgenic mice developed the human KS like disease[9, 19-20].

The constitutive activation of vGPCR makes the signaling molecule toxic to cells, including the KSHV latently infected PEL cell line[18, 21]. We have recently delineated that the KSHV encoded small membrane protein K7 retains vGPCR in the endoplasmic reticulum (ER) and thereby induces its rapid degradation by the proteasome. Both the signaling in tissue culture and the tumorigenesis in nude mice driven by vGPCR were greatly reduced when coexpressed with K7[22]. Correlating with the reports that vGPCR is translated under the biscistronic mechanism and regulated by viral macrophage inflammatory protein (vMIP) chemokines[23-24], our data suggest that KSHV has evolved distinct strategies to delicately regulate vGPCR protein expression and subsequent signal transduction.

vGPCR constitutively activates the downstream signaling even without ligand association, however, chemokines such as GRO-α and IP-10 bind vGPCR and modulate its signaling and tumorginecity. Accumulating evidence supports that the N-terminal of vGPCR is important for its chemokine association and tumorigenicity[25-26]. Understanding the molecular details of vGPCR association with chemokines will advance our knowledge of regulatory mechanisms that govern vGPCR signaling and tumorigenesis and will inform efforts to develop therapeutics that target the tumorigenic vGPCR for intervention. Our previous study showed that vGPCR contains the sulfated tyrosines at the N-terminal (residues at positions of 26 and 28, named Y26 and Y28 separately), which are necessary for GRO-α but not IP-10 binding. Sulfotyrosines of Y26 and Y28 are not critical for signaling downstream of vGPCR. However, they are crucial for vGPCR’s tumorigenicity in nude mice and GRO-α promotes vGPCR tumorigenesis in a sulfotyrosine dependent manner[27]. This striking feature of vGPCR offers us an opportunity to develop drug or antibody therapy for KS targeting vGPCR. In this study, the chimeras containing the N-terminal of vGPCR or its yydd counterpart (tyrosines at positions of 26 and 28 both had been alerted to aspartic acid, described previously) and mouse Fc were constructed (named wt-vG-N-mFc and yydd-vG-N-mFc separately). The fusion proteins were expressed in HEK293T cells and purified through affinity chromatography from the whole cell lysate and the media supernatant separately. The radioactive labeling test demonstrated that the wt-vG-N-mFc but not the yydd counterpart recruits the [35S]-sulfate. The purified wt-vG-N-mFc with the sulfated tyrosines has built a good foundation for its application in vGPCR tumorigenesis study in vivo.

1 Materials and Methods

1.1 Cells and Plasmids

HEK293T (293T) cells were obtained from American Type Cultrue Collection and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10%(V/V) fetal bovine serum, 5 mmol/L L-glutamine, 100 unit/mL penicillin, and 100 g/L streptomycin. pcDNA5/FRT/TO-vGPCR and pcDNA5/FRT/TO-yydd-vGPCR were kept in the lab as described previously[27]. DNA fragment coding for 1～49 amino acids of vGPCR (vG-N) or yydd-vGPCR (yydd-vG-N) was PCR amplified and cloned into pSecTag2A (Invitrogen) between Asc I and Xho I sites. Mouse Fc fragment with 6×His.tag tail (mFc) was PCR amplified based on the plasmid encoding LSF-mouse Fc hinge (a kind gift from Dr. Sally Ward, UTSW) and cloned into the above pSecTag2A constructs between Xho I and Apa Isites. The two recombinant plasmids are named pSecTag2A-wt-vG-N-mFc and pSecTag2A-yydd-vG-N-mFc accordingly. For lentiviral vector construction, pSecTag2A-wt-vG-N-mFc and pSecTag2A-yydd-vG-N-mFc were double-digested with Nhe I and Pme I, then the wt-vG-N-mFc or yydd-vG-N-mFc were cloned into pCDH-CMV-EF-Puro (System Bioscience) separately, and the plasmids are named CDH-wt-vG-N-mFc or CDH-yydd-vG-N-mFc accordingly.

1.2 Expression and purification of wt-vG-N-mFc and yydd variants

293T cells were transfected with plasmids expressing wt-vG-N-mFc or yydd-vG-N-mFc. Cells were harvested at 48 h post-transfection and lysed in Lysis Buffer-Tween (Tris-Hcl [PH7.4] 50 mmol/L,150 mmol/L NaCl, 10 g/L NP-40, 5 mmol/L EDTA,0.5 g/L Tween 20) containing the protease inhibitor cocktail (Roche). Both the cell lysates and the media supernatant were incubated with 50 g/L Ni-NTA Magnetic Agarose Bead suspension (QIAGEN) on ice for 1 h. The beads were washed and eluted according to the manufacture’s protocol. The eluate was isolated by 10% SDS-PAGE and applied for coomassie brilliant blue staining or western blot assay.

1.3 Immunoblotting

Immunoblot assay was performed as previously described[22]. Immunoblot detection of the wt-vG-N-mFc and yydd-vG-N-mFc was performed with anti-Penta.His antibody (1∶2 500, Sigma) or only with the secondary antibody (goat anti-mouse IgG peroxidase conjugate) separately.

1.4 Labeling and purification of wt-vG-N-mFc and YYDD variants

293T cells were transfected with pSecTag2A-wt-vG-N-mFc or pSecTag2A-yydd-vG-N-mFc separately. Cells were split at 10 h posttransfection. Approximately 36 h after transfection, cells were radiolabeled with [35S]-methionine/cysteine or [35S]-sulfate for 10 h individually. Labeled cells were lysed and applied for purification as above. The eluate was applied for autoradiography as before[27].

2 Results

2.1 The vG-N-mFc fusion expression vectors are constructed

The pSecTag2A-wt-vG-N-mFc and pSecTag2A-yydd-vG-N-mFc contain the chimera of vG-N-mFc, in which the Igκ leader sequence is at upstream of the DNA fragment coding for 1～49 amino acids of vGPCR or yydd-vGPCR (wt-vG-N or yydd-vG-N) and the mFc fragment with 6×His.tag tail is fused to the 3′ end of vG-N. The vG-N-mFc chimeras in the lentiviral vectors of CDH-wt-vG-N-mFc or CDH-yydd-vG-N-mFc are the same to their pSecTag2A counterpart except the restriction sites are different. The map and the amino acid sequence of the chimera are shown in figure 1 and figure 2 separately.

Pcmv: cmv promoter; Igκ leader: Igκ-chain leader sequence; BGH pA:BGH polyadenylation sequence; vG-N:wt-vGPCR or yydd-vGPCR extracellular N-terminal (1～49 amino acids); mFc: mouse Fc hinge.Figure 1 The map of the chimera of wt-vG-N-mFc or yydd-vG-N-mFc

2.2 The wt-vG-N-mFc and yydd-vG-N-mFc are purified and characterized

To obtain the wt-vG-N-mFc or yydd-vG-N-mFc fusion protein, HEK293T cells (ten plates of 293T cells for each sample) were transfected with either pSecTag-wt-vG-N-mFc or the yydd variant. Cells were harvested at 48 h posttransfection and cell lysate was applied for affinity chromatography. 100 μL eluate of each sample was obtained finally and 10 μL eluate of each was applied for coomassie brilliant blue staining. One exclusive band of each sample was found in the coomassie staining and the size was about 4×104～4.5×104, which matches the predicted size of wt-vG-N-mFc or the yydd variant (Figure 3A). The smear could be found in both lanes, which might be attributed to the glycosylation of wt-vG-N-mFc and yydd-vG-N-mFc. The band of wt-vG-N-mFc was weaker than that of yydd-vG-N-mFc, which implies the higher expression level of yydd-vG-N-mFc. Immunoblot (IB) assay with anti-Penta.This demonstrated that the purified proteins are unambiguous wt-vG-N-mFc and yydd-vG-N-mFc, which are both ended with 6×His.tag. The band for yydd-vG-N-mFc was thicker than that of wt-vG-N-mFc, which correlates with the commassie staining result (Figure 3B). To characterize the secretion of the wt-vG-N-mFc and yydd-vG-N-mFc, the HEK293T cells in six-well plate were transfected with pSecTag2A-wt-vG-N-mFc, pSecTag2A-yydd-vG-N-mFc, CDH-wt-vG-N-mFc or CDH-yydd-vG-N-mFc separately, and the media supernatant was applied for the affinity chromato-graphy. Eluate of 20 μL from each sample was obtained and applied for IB test, in which the secondary antibody (goat anti-mouse IgG peroxidase conjugate) was used only. The IB result demonstrated that both wt-vG-N-mFc and yydd-vG-N-mFc were secreted out into the media from the transfected cells. The thicker bands in the lanes of pSectag constructs suggests that the expression level of pSectag constructs is higher than that of CDH constructs (Figure 3B).

(A) (B) (A) HEK293T cells were transfected with pSecTag2A-wt-vG-N-mFc or pSecTag2A-yydd-vG-N-mFc separately and the cell lysates was applied for purification as described in methods. 10 μL eluate of each sample was isolated by 10%(V/V) SDS-PAGE and stained with coomassie brilliant blue; (B) Upper and middle: 2.5 μL eluate of each sample was applied for Penta-his IB and 5%(V/V) input for actin IB separately; Bottom: HEK293T cells were transfected with pSecTag2A or CDH-CMV-EF-puro constructs expressing wt-vG-N-mFc or yydd-vG-N-mFc separately and the media supernatant of the transfected cells was applied for the secreted fusion protein purification as described in methods. 20 μL of eluate for each sample was obtained and applied for IB, the goat anti-mouse IgG peroxidase conjugate was used for IB without the recruitment of primary antibody. M: Prestained protein marker; wt: wt-vG-N-mFc; yydd: yydd-vG-N-mFc; wcl: whole cell lysates; media: media supernatant from the transfected cells; cdh: CDH-CMV-EF-puro construct; sectag: pSectag2A construct. Figure 3 The expression and purification of wt-vG-N-mFc and yydd-vG-N-mFc

2.3 The wt-vG-N-mFc but not the yydd variant is modified by sulfate

HEK293T cells were transfected with pSecTag2A-wt-vG-N-mFc or pSecTag2A-yydd-vG-N-mFc separately. Cells were labeled with [35S]-methionine/cysteine (Met/Cys) or [35S]-sulfate (SO4) individually and lysed. Cell lysates were incubated with Ni-NTA beads as described in methods. Eluate was analyzed by SDS-PAGE and autoradiography. Left: [35S]-Met/Cys labeling; Right: [35S]-sulfate labeling. wt: wt-vG-N-mFc; yydd: yydd-vG-N-mFc. Figure 4 The wt-vG-N-mFc efficiently incorporates radiolabeled sulfate

To identify whether the vG-N-mFc fusion protein has the tyrosine sulfation, 293T cells (2 plates of 293T cells for each sample) were transfected with pSecTag2A-wt-vG-N-mFc or pSecTag2A-yydd-vG-N-mFc separately and radiolabeled with [35S]-methionine/cysteine or [35S]-sulfate individually. The [35S]-methionine/cysteine labeling result demonstrated that both the lanes of wt-vG-N-mFc and its yydd variant had the band of around 4×104～4.5×104, which correlates with the coomassie staining result(Fig. 4, left panel). However, in the [35S]-sulfate labeling test, the band of 4×104～4.5×104was found in the wt-vG-N-mFc lane but not yydd variant lane, which clearly demonstrated that only the wt-vG-N-mFc recruits [35S]-sulfate(Fig. 4, right panel). The smear in both lanes implies that the glycoprotein chains of the two fusion protein have the sulfation modification, which correlates with the coomassie staining result.

3 Discussion

vGPCR is a bona fide signaling molecular which is constitutively activated even without ligand association[9]. However, chemokine binding modulates its signaling and tumorigenicity in vitro and in vivo. For instance, IP-10 binding inhibits, while GRO-α association further promotes signaling downstream of vGPCR. Remarkably, the extracellular N-terminal of this viral protein is important for its ligand association[25-26]. Our previous study uncovered that GRO-α binds to vGPCR in a sulfotyrosine dependent manner and promotes vGPCR’s tumorigenesis in nude mice through autocrine activation[27].

This study is based on the striking feature of the extracellular N-terminal of vGPCR and its association with GRO-α. The chimera of vG-N-mFc is designed to mimic the N-terminus of vGPCR, in which the extracellular N-terminal (1～49 aa) of vGPCR is fused with mouse Fc fragment. Mouse Fc helps the fusion protein stable in vivo and makes it easily characterzed by anti-mouse IgG. Igκ-chain leader sequence at the upstream of the chimera is used to ensure the secretion of the fusion protein. The immunoblot test based on the media supernatant from the transfected cells demonstrated that the chimeras in both pSectag construct and CDH-CMV-EF-Puro construct were well translated and had been secreted out from the transfected cells. The secreted fusion proteins are stable in the media because that they had been purified by Ni-NTA beads which targeting the 6×His.tag tail of the chimera and characterized by the anti-mouse IgG which recognizing the mouse Fc part in the fusion protein.

In our previous study, we delineated that a sulfated peptide corresponding to residues 12 through 33 of the vGPCR N-terminus, but not an unsulfated equivalent, partially inhibits vGPCR association with GRO-α. The purified wt-vG-N-mFc and yydd-vG-N-mFc will be used for chemokine binding assay in place of the sulfated peptide, in which NIH3T3 stably expressing vGPCR will be used for the [125I] GRO-α association as previously described[27]. Our preliminary study uncovered that vGPCR recruits the glycoprotein chains and the asparagines at the position of 18, 22 and 31 are the potential N-glycosylation sites (data not shown). Both the coomassie staining and the radioactive labeling suggest that wt-vG-N-mFc is modified with glycosylation. What is more, this chimera contains the 1～49 amino acids of vGPCR, which compose the entire extracellular N terminus of the viral oncoprotein. All above support the idea that vG-N part of wt-vG-N-mFc will more likely have the similar composition of vGPCR extracellular N terminus compared with the synthesized sulfated peptide. Thus, it is predicted that the purified wt-vG-N-mFc with sulfotyrosines will inhibit vGPCR association with GRO-α more potently.

The secretion of vG-N-mFc from the 293T cells transfected with the lenti-constructs suggests it is feasible that the chimera will be used for the vGPCR tumorigenicity study in vivo. The lenti-virus containing wt-vG-N-mFc or yydd-vG-N-mFc have been made and used to infect the SVEC cells (simian virus 40, large T antigen-immortalized, murine endothelial cell) for stable cell line establishment. The cell lines stably expressing wt-vG-N-mFc or yydd-vG-N-mFc (already been established in the lab, data not shown) will be mixed with the vGPCR stably trasnfected cells and applied for the nude mice assay, in which the impact of vG-N-mFc on vGPCR tumorigenesis will be tested.

In this study, we demonstrated that the wt-vG-N-mFc containing entire extracellular N-terminal of vGPCR and its yydd variant were purified from the cell lystes and the media supernatant of the transfected HEK293T cells. The wt-vG-N-mFc but not the yydd-vG-N-mFc incorporates the [35S]-sulfate. The successful purification of wt-vG-N-mFc containing sulfotyrosines has established a good foundation for its application in the vGPCR tumorigenicity study in vivo and in the clinical therapy for KS targeting vGPCR.

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