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We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.
We have recently identified the C-type lectin-like receptor, CLEC-2, as a novel activating receptor that is expressed on the surface of platelets and megakaryocytes . CLEC-2 can be activated by the snake venom toxin rhodocytin or by a specific antibody, triggering powerful platelet aggregation . CLEC-2 signals through a single YXXL motif that is present in its cytoplasmic domain . Crosslinking of CLEC-2 induces Src kinase-dependent tyrosine phosphorylation of the YXXL sequence, inducing activation of the tyrosine kinase Syk and initiation of a signalling pathway that culminates in activation of PLCγ2 . The CLEC-2 signalling pathway is similar to, but distinct from, that used by receptors that signal through an immunoreceptor tyrosine-based activation motif (ITAM), such as the major signalling receptor for collagen on platelets, the GPVI-FcR γ-chain complex. These two signalling pathways differ in that activation of PLCγ2 by ITAM receptors has an absolute requirement for two YXXL motifs and the SLP-76/Blnk family of adapter proteins , whereas CLEC-2 has a single YXXL sequence and is only partially dependent on this family of adapter proteins .
CLEC-2 is a member of the C-type lectin-like family of receptors, but its putative carbohydrate recognition domain lacks the key structural features that confer binding to carbohydrates, suggesting that its endogenous ligand may be a protein. We have recently crystallized CLEC-2 and shown that it has a compact C-type lectin-like domain with a flexible loop on its surface . A role for this loop in ligand binding is suggested by mutagenesis and surface plasmon resonance binding studies with rhodocytin .
CLEC-2 has recently been reported to facilitate capture of HIV-1 by CLEC-2-transfected cells and platelets . Importantly, CLEC-2 does not bind to the HIV-1 envelope protein suggesting that CLEC-2 binds to a membrane protein derived from the HIV-producing 293T cells, that is captured by the virus during viral budding. Thus, 293T cells must express an endogenous ligand for CLEC-2. Infection of renal cells and production of infective virus from these cells is well documented in HIV infection and renal disease is an important complication of HIV infection [6, 7]. Therefore, this CLEC-2 interaction could be a significant mechanism in viral dissemination. The aim of the present study was to identify this ligand and to investigate its ability to bind and activate CLEC-2.
For eukaryotic expression of glycosylated CLEC-2 protein, the extracellular domain of CLEC-2 was cloned into the plasmid pQCXIX (Clontech, CA) to encode a protein with an N-terminal 6-histidine tag followed by a BirA recognition sequence. The packaging cell line 293T-GP2 was transfected with the CLEC-2 expression construct, retrovirus harvested and used to infect 293T cells. The cell supernatant was centrifuged, filtered and protein purified by nickel affinity chromatography and size exclusion gel filtration using a Superdex 75 HR 26/60 column on an AKTA Purifier (GE Healthcare, Sweden).
For bacterial expression of unglycosylated protein, the extracellular domain of CLEC-2 was cloned into the plasmid pGMT7 to encode a protein with an N-terminal BirA recognition sequence. Recombinant CLEC-2 was expressed in E. coli strain BL21 DE3 pLysS as inclusion bodies as previously described [4, 8]. Briefly, inclusion bodies were isolated, washed in detergent, solubilized in guanidine, refolded in the presence of a redox couple and refolded protein purified by size exclusion chromatography.
Podoplanin was made as previously described . Briefly, podoplanin was cloned into the plasmid pCDM7Ig, which was transiently transfected into 293T cells to express podoplanin fused at its C-terminus to the human IgG1 Fc region. The fusion protein was purified by protein A affinity chromatography and size exclusion chromatography.
BirA enzyme was produced and used to biotinylate proteins with BirA recognition tags as previously described . A 4-fold molar excess of biotinylated protein was conjugated to phycoerythrin-labelled extravidin (Sigma, UK).
Cells were washed in cold PBS 0.01% azide, blocked with 1% bovine serum albumin for 10 min on ice, washed again and incubated with fluorochrome-labelled multimeric CLEC-2 protein (3.5μM) for 1 hour. Cells were then washed 3 times, fixed in PBS 1% formaldehyde and analyzed on a FACS Canto machine (Becton-Dickinson, UK) and subsequently with FlowJo software (Tristar, CA, USA). For competition studies, cells were pre-incubated with free unlabelled CLEC-2 protein, NKG2D, rat monoclonal anti-human podoplanin antibody NZ1 (AngioBio, CA, USA) or isotype control for 1 hour before the addition of multimeric fluorochrome-labelled CLEC-2. The monoclonal anti-human podoplanin antibody 18H5 (Santa Cruz Biotechnology, CA, USA) was also used for flow cytometry. For platelet analysis, whole blood was isolated in tri-sodium citrate, directly labelled, then diluted prior to flow cytometry.
Platelet rich plasma was isolated as previously described  and placed in a Born-lumiaggregometer with stirring at 1200 rpm at 37°C for 5 min before addition of 293T cells suspended in PBS. In some experiments, platelets were pre-incubated with the GPIIbIIIa antagonist, integrilin (9μM), or the Src kinase inhibitor, PD173952 (20μM; Pfizer, MI, USA), for 2 min prior to experimentation. Activation of CLEC-2 was monitored in DT-40 cells using an NFAT-luciferase reporter assay following transient transfection of CLEC-2 or the Y7F mutant of CLEC-2, as previously described . The DT-40-transfected cells were incubated with 293T cells for 6 hours before measurement of luciferase activity. In competition experiments, 293T cells were pre-incubated with 20μg/ml rat IgG or rat anti-human podoplanin antibody NZ-1 on ice for 20 min prior to experimentation.
Cells were resuspended in methionine-free medium and incubated for 1 hour. Cells were then incubated overnight in 35S-labelled methionine (GE Healthcare, UK), then washed and lysed on ice in 0.5% NP40, 0.25M NaCl, 10mM Tris pH 7.5 with protease inhibitors. Lysates were pre-cleared with protein G agarose beads (Sigma, UK) and incubated with or without 100μg recombinant biotinylated CLEC-2 for 1 hour on ice with gentle agitation. Anti-polyhistidine antibody (Sigma, UK) was added for 1h and then protein G agarose was added and the mixture rotated at 4°C for 3 hours. Beads were washed 5 times, and proteins were eluted in Laemmli reducing sample buffer and boiled for 5min. Proteins were separated on 12% SDS-PAGE gels, which were dried and exposed to photographic film. Western blots were performed in an equivalent manner without metabolic labelling. Gels were blotted onto nitrocellulose membranes, which were blocked in 5% milk, washed and incubated with 1μg/ml NZ1, washed and incubated with anti-rat immunoglobulin-HRP antibody (Dako, UK).
Surface plasmon resonance binding studies were conducted using a Biacore T100 machine (Biacore GE, Sweden) as described previously . The biological activity of recombinant CLEC-2 and podoplanin were established by demonstrating binding of each fluorescence-labelled protein to cells transfected with the other protein. Proteins were attached to the carboxymethylated dextran-coated surface of CM5 biosensor chips, using amine coupling chemistry and experiments performed in 10mM Hepes pH 7.4, 150mM NaCl, 3mM EDTA and 0.005% polysorbate 20 surfactant. Non-specific interactions were controlled for by subtraction of the signal from a reference flow cell coated with an irrelevant protein. All CLEC-2 protein used in binding studies was monomeric. To avoid avidity effects with podoplanin which was expressed as a dimeric Fc-fusion protein, experiments were only performed with podoplanin immobilized on the chip surface. Kd values were obtained by nonlinear curve fitting of the Langmuir isotherm to the data using the Levenberg-Marquardt algorithm as implemented in the program Origin (Microcal Software, MA, USA).
To confirm that 293T cells express a ligand for CLEC-2 and to provide a route to its identification, we expressed the extracellular domain of CLEC-2 in eukaryotic cells as a recombinant protein fused at its N-terminus to a BirA recognition sequence . This allowed site-specific enzymatic biotinylation at the membrane insertion point of the protein. Biotinylated protein was tethered to a fluorochrome-labelled avidin derivative with four biotin-binding sites to create a species that is tetrameric with respect to CLEC-2 and in which CLEC-2 retains the orientation that it would have on the cell surface, with the C-terminus of the protein available for interaction with a ligand. Flow cytometry studies using this fluorochrome-labelled CLEC-2 demonstrated binding of CLEC-2 to 293T cells, consistent with expression of an endogenous ligand for CLEC-2 on these cells (Figure 1A). No binding was seen with the fluorochrome-labelled extravidin alone. To confirm that the interaction of multimeric CLEC-2 protein was specific, binding was carried out in the presence of free CLEC-2. Increasing concentrations of free unlabelled CLEC-2 inhibited binding of the fluorescence-labelled protein in a concentration-dependent manner, consistent with CLEC-2 exhibiting a specific mode of binding (Figures 1A and 1B). By contrast, incubation with a range of concentrations of a free irrelevant protein (NKG2D) did not inhibit binding of fluorescent CLEC-2 (Figure 1B).
To confirm that the binding seen was not artefactual due to the multimeric nature of the CLEC-2 used, the experiment was repeated using monomeric CLEC-2. When monomeric biotinylated CLEC-2 was bound to cells, it could be detected by the addition of fluorochrome-labelled extravidin (Figure 1C). However, the binding was slightly less as predicted by the loss of avidity effects in the primary binding of monomeric CLEC-2 to the cells.
A range of other cell types were also screened for binding to the CLEC-2 tetramer. High levels of specific binding (i.e. binding that could be competed out with unlabelled CLEC-2), comparable to that seen on 293T cells, were observed on SuSa cells, which are derived from testis. Intermediate levels of specific binding were observed on HT1080 and U2OS cells, which are derived from connective tissue and osteocytes respectively, and low level binding was observed on the erythroid/megakaryocyte progenitor cell line, K562, and on ovarian SKOV3 cells. In contrast, there was no specific binding on T cells (Jurkat), B cells (Raji, Daudi, CIR), natural killer cells (NK92), mammary epithelial cells (T47D), monocytes (U937 and HL60), platelets or primary endothelial cells (HUVEC). This data is summarised in Table 1 and representative binding as measured by flow cytometry is shown in Figure 1D.
CLEC-2 is able to interact with a range of different cell types of diverse origin, suggesting that the ligand is widely expressed. Native CLEC-2 in platelets is glycosylated  and recombinant CLEC-2 has a molecular mass of ~33kDa compared to the predicted molecular mass of ~26kDa (Figure 2A). Treatment of this protein with PNGaseF (peptide N-glycosidase F), which removes N-linked glycans, reduced the molecular weight to its predicted value (Figure 2A), confirming that recombinant CLEC-2 is glycosylated. To establish whether the binding of CLEC-2 to its ligand was dependent on carbohydrate groups on CLEC-2, unglycosylated CLEC-2 was produced in E. coli. This protein was biotinylated and tethered to phycoerythrin-labelled extravidin and used for flow cytometry in the same manner as the CLEC-2 expressed from eukaryotic cells. Unglycosylated CLEC-2 binds to primary and cultured cells in a similar manner to glycosylated CLEC-2 as illustrated in Figure 2B and Table 1. Two other irrelevant C-type lectin-like molecules, made in a similar way in E. coli and refolded from inclusion bodies did not show any binding to the cells (data not shown). In addition, PNGaseF treated CLEC-2, which was made in eukaryotic cells, also bound to cells specifically, as demonstrated in Figure 2C. These observations demonstrate that sugar moieties on CLEC-2 are not necessary for CLEC-2 to bind to its ligand. Thus, recombinant CLEC-2 binds specifically to the surface of a number of cell types, including 293T cells, and this binding is independent of its glycosylation. These data strongly suggest that one, or possibly more than one, transmembrane protein functions as an endogenous ligand for CLEC-2.
The above results demonstrate the presence of a ligand for CLEC-2 on 293T cells and confirm our previous indirect evidence for this . Studies were therefore undertaken to investigate whether this putative ligand is able to activate platelets. Figure 3A demonstrates that 293T cells stimulate powerful aggregation of platelets, with the magnitude of response being determined by the number of 293T cells used. Importantly, this response was inhibited by the GPIIbIIIa integrin antagonist, integrilin, demonstrating that it is true integrin-dependent aggregation rather than agglutination (Figure 3B). Furthermore, activation by 293T cells was inhibited in the presence of the Src kinase inhibitor, PD173952 (Figure 3B). This is consistent with the observation that CLEC-2 signals through a Src kinase dependent pathway . Moreover, 293T cells also stimulated activation of FcR γ-chain deficient mouse platelets (data not shown) demonstrating that activation is independent of the platelet collagen receptor GPVI, the other major platelet activation receptor that induces aggregation via Src kinases.
To confirm that 293T cells are able to activate platelets via CLEC-2, we performed a series of studies on CLEC-2 transfected DT-40 cells, using an NFAT reporter assay . Activation of CLEC-2 in this model cell line leads to induction of NFAT activity and expression of luciferase, which can be measured by luminometry. Strikingly, exposure to 293T cells increases NFAT activity in CLEC-2 transfected, but not mock-transfected, DT-40 cells (Figure 3C), confirming that they express a ligand that is able to activate the C-type lectin-like receptor. Moreover, DT-40 cells transfected with a mutant of CLEC-2 in which the cytoplasmic motif YXXL was changed to FXXL (Figure 3C) were not activated by 293T cells. A similar result has previously been reported for activation of CLEC-2 by rhodocytin in DT-40 cells . Thus, these results confirm that 293T cells express a ligand for CLEC-2 that induces activation of the C-type lectin-like receptor through the same pathway as that used by the snake toxin, rhodocytin.
To characterize the surface proteins in 293T cells that bind to CLEC-2, pull down experiments were performed with recombinant CLEC-2 on cells that had been metabolically labelled with 35S-methionine. This approach identified several minor bands and a major band of 36 kDa that were not present in the control lane (Figure 4A). During the course of these studies, podoplanin, a 36 kDa protein that is expressed on certain tumours was shown to interact with CLEC-2 . Podoplanin derives its name from podocytes which express it [14, 15], and which are kidney epithelial cells and therefore closely related to the human embryonic kidney 293T cells.
Using a specific antibody against podoplanin, we have demonstrated that recombinant CLEC-2 precipitates podoplanin from 293T cells and that this migrates in the same region as the 36kDa protein identified in the metabolic labelling studies (Figure 4B). Further, we have confirmed expression of podoplanin on 293T cells, with strong staining using an anti-podoplanin antibody (Figure 5A), comparable to the staining seen with recombinant CLEC-2 (Figure 1). Moreover, binding of the podoplanin antibody paralleled that of binding of recombinant CLEC-2 on a range of cell lines as shown in Figure 5B and Table 1. Thus, these observations strongly suggest that podoplanin is the ligand that confers binding of 293T and a number of other cell lines to CLEC-2. Direct confirmation that this was the case was obtained using a specific antibody to podoplanin, NZ-1, that has been shown to block binding of podoplanin-expressing cells to platelets . As shown in Figure 5C, NZ-1 inhibited binding of recombinant CLEC-2 to 293T cells in a similar way to that observed with unlabelled CLEC-2 (Figure 1) confirming that binding was mediated by podoplanin. Moreover, NZ1 also blocked activation of CLEC-2 transfected DT-40 cells by 293T cells (Figure 3D), thereby confirming that 293T cells bind and activate CLEC-2 through podoplanin. In addition, recombinant podoplanin was able to activate CLEC-2 transfected DT-40 cells directly (Figure 5D), confirming that the interaction between podoplanin and CLEC-2 triggers signalling. Further, the signalling was dependent on the YXXL sequence of CLEC-2 confirming that it was mediated through the same mechanism as that used by rhodocytin (Figure 5D).
Thus, not only does this work confirm that podoplanin is a specific ligand for CLEC-2, it also confirms the biological activity of the recombinant CLEC-2 and the recombinant podoplanin that was used in these studies. In order to confirm a direct interaction between CLEC-2 and podoplanin and determine the affinity of the interaction, we have used the two recombinant proteins in surface plasmon resonance binding studies (Figure 6). For these studies, podoplanin was tethered to a biosensor surface and interacted with different concentrations of recombinant CLEC-2. A progressively higher signal was seen with increasing concentrations of CLEC-2 protein. No interaction was seen with control proteins. The data were consistent with a single site binding model and demonstrate that the interaction between the two proteins is direct with an affinity of 24.5 ± 3.7μM.
CLEC-2 is a newly characterized C-type lectin-like receptor, which has been shown to mediate platelet activation and aggregation upon binding to rhodocytin, a snake venom protein . CLEC-2 has also been shown to enhance infectivity of HIV-1 produced in 293T cells . This effect was not mediated by the viral envelope protein implying that a protein from the 293T cells was captured during viral budding and was responsible for the interaction of the virus with CLEC-2. These data suggested the presence of an endogenous, surface-bound ligand for CLEC-2 on 293T cells. We used recombinant CLEC-2 protein to confirm that 293T cells express a ligand for the receptor. This recombinant CLEC-2 was used to pull down potential ligand molecules from cell lysates and identified a candidate ligand of 36kDa that corresponds in size to podoplanin. We have shown that 293T and other CLEC-2 binding cells used in this study express podoplanin and that anti-podoplanin antibody inhibits both binding to and activation of CLEC-2 by 293T cells. A direct interaction between podoplanin and CLEC-2 was formally demonstrated by co-immunoprecipitation and surface plasmon resonance experiments, which determined the affinity to be 24.5μM.
These results are consistent with a report that certain cancer cells express podoplanin on their surface, which allows them to interact with CLEC-2 . The study by Suzuki-Inoue et al sought to identify a receptor for podoplanin and demonstrated that Src inhibitors blocked podoplanin-induced signalling through CLEC-2. Our study defines the signalling in CLEC-2 that is triggered by podoplanin and demonstrates that it is mediated through the YXXL motif in the cytoplasmic domain of CLEC-2. We have further demonstrated that glycosylation of CLEC-2 is not required for this interaction. There is good evidence that HIV infects renal cells, that these cells can produce infective HIV virions and that renal cells express podoplanin which may be incorporated into the budding virion [6, 14]. The present study has demonstrated that podoplanin binds directly to CLEC-2 with an affinity in the micromolar range, so inhibitors of this interaction may help prevent dissemination of HIV-1 by platelets.
Podoplanin was originally identified on a range of cells including airway epithelia, fibroblasts, keratinocytes, osteoblasts and renal tubular epithelial cells [17-20]. Podoplanin is also expressed at high levels on lymphatic endothelial cells  and on certain tumour cells, where it has been shown to activate platelets . In the renal glomerulus, podoplanin helps maintain the structure of podocytes, required for efficient glomerular filtration . Podocytes are not normally in contact with blood, but acute renal damage could expose them to blood, allowing interaction between CLEC-2 on platelets and podoplanin on podocytes. Physiologically, this would trigger platelet activation and formation of a haemostatic plug, thereby preventing further leakage into the collecting ducts, and possibly helping in renal repair through release of growth factors. However, this could also trigger intrarenal and especially intraglomerular thrombosis, which can be observed in renal diseases when there is renal epithelial damage, including during renal transplant rejection.
Inhibitors of the podoplanin-CLEC-2 interaction have potential use as novel anti-platelet agents. The significance of the interaction of podoplanin and CLEC-2 is unclear as the majority of cells that express podoplanin, with the exception of tumour cells undergoing metastasis, do not normally contact platelets. As with certain other C-type lectin-like receptors, it is possible that the function of CLEC-2 is primarily in defence, and specifically in this to prevent excessive blood loss and to respond to infection following contact with podoplanin. Further studies will provide information on the physiological and pathophysiological significance of the interaction of podoplanin and CLEC-2.
This work was supported by the Medical Research Council, the British Heart Foundation and the Wellcome Trust. We are grateful to Paul Harrison, Norma Masson, Neale Banerji, Alain Townsend, Hal Drakesmith, Lisa Schimanski, Emma Sweetland and Da Lin for helpful discussions.