Figure 1.
Galectin-1 blocks NiV-F and G mediated syncytia formation of endothelial and glial cells.
A, Quantification of galectin-1 inhibition. PK-13 (ephrinB2 negative) cells expressing NiV-F and NiV-G were added to monolayers of ephrinB2 positive cells, Vero (control), HUVEC, mVEC, and U87. Heterologous fusion in the absence and presence of 20µM galectin-1 (white and black bars respectively) were quantified as described in Experimental Procedures. Data are mean ± SD of triplicate samples from one of three replicate experiments. B, Representative images of cell fusion in the absence or presence of galectin-1. Left panels show multinucleated syncytia in the indicated cell type. Right panels are cells treated with galectin-1 (20×).
Figure 2.
Endogenous endothelial galectin-1 inhibits NiV-F and NiV-G mediated syncytia formation.
A, Activated HUVECs have increased cell surface galectin-1 compared to resting cells. Flow cytometric analysis of cell surface galectin-1 on resting (light grey) and activated (dark grey) HUVECs, and on resting HUVECs plus exogenous galectin-1 (black) Data are mean ± SEM of three independent experiments, each done in triplicate. B, Activated HUVECs are resistant to NiV-F and NiV-G mediated cell fusion. Heterologous cell fusion of resting (light grey) and activated (dark grey) HUVECs, and resting HUVECs plus exogenous galectin-1 (black). Data are mean ± SD of triplicate samples from one of three replicate experiments. C, Reduction of cell surface galectin-1 by siRNA. Flow cytometric analysis of cell surface galectin-1 on resting HUVECs (grey filled), siRNA treated HUVECs (grey line), and siRNA treated HUVECS with 20µM exogenous galectin-1 (black line). D, Reduction of cell surface galectin-1 in HUVECs increases susceptibility to NiV-F and G mediated cell fusion. Heterologous cell fusion of resting cells (light grey), cells with reduced galectin-1 (white), and cells with reduced galectin-1 plus exogenous galectin-1 (dark grey). Data are mean ± SD of triplicate samples from one of three replicate experiments.
Figure 3.
Galectin-1 interferes with lateral movement of NiV-F on the plasma membrane.
A, NiV-FGFP expression on the surface of two individual Vero cells. B, NiV-FGFP promotes cell fusion when transfected into Vero cells with NiV-G. (20×) C, Galectin-1 inhibits fusion mediated by NiV-FGFP. Fusion of Vero cells in the absence (−) or presence (+) of 20µM galectin-1 was measured as in Fig. 1. D, Galectin-1 inhibited fluorescence recovery after photobleaching. NiV-FGFP transfected Vero cells were treated with buffer control (black line with black squares) or 20µM galectin-1 (grey line with black triangles), and a portion of the membrane was bleached and measured for fluorescent recovery (y-axis) as a function of time in seconds (x-axis). Data are mean ± SD of six replicate measurements from one of two independent experiments.
Figure 4.
Galectin-1 inhibits NiV-F0 endocytosis and maturation.
A, Galectin-1 decreases internalization of NiV-F0 from the plasma membrane. Cells transfected with NiV-F were cell surface biotinylated, then incubated in the presence of 20µM galectin-1 (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized biotinylated NiV-F was quantified by ELISA. Percent internalization was determined as the amount of internalized biotinylated NiV-F compared to total biotinylated NiV-F at the initial timepoint. Data are mean ± SEM for seven replicate experiments. B, Galectin-1 inhibits NiV-F0 proteolytic processing. 293T cells expressing NiV-F were pulse-labeled with 35S-methionine, then chased for 4 or 6 hrs in the presence or absence of galectin-1. NiV-F was immunoprecipitated with anti-NiV-F polyclonal sera and proteolytic processing analyzed by immunoblotting. C, Graphic representation of data in B. Cleavage ratio was determined as the amount of processed NiV-F (F1+F2) compared to total NiV-F protein. Data are mean ± SEM of three replicate experiments.
Figure 5.
Galectin-1 inhibits function of mature NiV-F.
A, Chlorpromazine inhibits maturation of NiV-F0. PK-13 were transfected with NiV-F in the absence or presence of chlorpromazine (50µM). Cells were incubated overnight and NiV-F0 and NiV-F1 detected by immunoblotting. B, Galectin-1 inhibits heterologous cell fusion in the presence of chlorpromazine. BSRT7 ephrinB2 positive cells were added to a monolayer of PK13 cells transfected with NiV-F, NiV-G and a luciferase construct with a T7 dependent promoter in the presence or absence of chlorpromazine (50µM) and galectin-1 (20µM). * p = 0.0002, calculated using Student's t test. C, Fusion kinetics in the presence or absence of galectin-1. NiV-G and NiV-F were expressed in effector PK13 cells, and the relative rate of fusion assessed on target 293T cells loaded with CCF2 dye. Relative fusion is the ratio of blue to green fluorescence from NiV-G and NiV-F-transfected effector cells minus the ratio of background blue to green fluorescence from empty-vector (pcDNA3)-transfected cells. Each data point is the mean of three independent experiments. D, Galectin-1 inhibits the ability of NiV-F to be triggered for membrane fusion. CHO cells expressing NiV-F and NiV-G were mixed with CHO cells (negative control, grey shaded) or CHOB2 cells (ephrinB2 positive, black line) for 1.5 hr at 4°C. Cell mixtures were brought to 37°C or kept at 4°C for 1.5 hr with 1 µM biotinylated HR2 peptide, in the presence or absence of galectin-1GG; top, 4°C without and with galectin-1; bottom, 37°C without and with galectin-1. E, Inhibition of F triggering at 37°C; data are mean fluorescence intensity of triplicate determinations ± SEM. See also Supplementary Figure S1.
Figure 6.
The F3 N-glycan on NiV-F is a complex N-glycan containing putative binding sites for galectin-1.
A, MALDI-TOF mass spectrum of all permethylated N-glycans from NiV-F0. Annotated structures were deduced by taking into account theoretical compositions and knowledge of the biosynthetic pathways (for further information, refer to http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=glyco.TOC&depth=2). All molecular ions are [M+Na]+. Peaks labeled with * represent contaminating hexose polymers. Unlabelled peaks are non-carbohydrate contaminants or permethylation products. B, Glycan component of the F3 glycopeptide, GALEIYKNNTHDLVGDVR, and effect of sialidase S digestion. Top panel – NiV-F0 was digested with trypsin and the peptide/glycopeptide mixture was analysed by LC-ES-MS/MS; the summed MS data for the F3 glycopeptide are shown. Bottom panel – The LC-ES-MS/MS experiment was repeated after treatment of the tryptic digest of NiV-F0 with Sialidase S; summed MS data for the partially desialylated F3 glycopeptide are shown. Unannotated peaks correspond to peptides. Molecular ions attributable to glycopeptides are annotated with m/z values and subscripted charge states. Peaks labeled in bold correspond to molecular ions that have shifted on Sialidase S digestion; these peaks are also assigned a potential glycan structure. Symbol nomenclature is that used by the Consortium of Functional Glycomics (CFG) (see key below). See also Figure S2. Key: Galactose (yellow circle), Mannose (green circle), GlcNAc (blue square), Fucose (red triangle), NeuAc, (purple diamond).
Figure 7.
The F3 glycan is critical for galectin-1 inhibition of NiV-F maturation and function.
A, The F3 mutant (NiV-F missing the F3 glycan) is resistant to galectin-1 inhibition of syncytia formation in endothelial and glial cells, using the heterologous cell fusion assay described in Fig. 1. PK13 cells expressing NiV-G and either wildtype NiV-F (white) or NiV-F lacking the F3 glycan (black) were added to indicated cells in the presence of galectin-1 (HUVEC, 10µM; U87, 20µM). The y-axis shows percent inhibition of fusion. Data are mean ± SD of triplicate samples from one of three replicate experiments. * p = 0.0001, calculated using unpaired Student's t test. B, The F3 glycan on NiV-F is critical for galectin-1 inhibition of NiV-F0 internalization. Cells transfected with NiV-F3 (lacking the F3 glycan) were cell surface biotinylated and incubated in the presence of galectin-1 (20µM) (bold line), or buffer control (dashed line), for the indicated times to allow internalization. Internalized NiV-F3 was quantified as in Fig. 4. Data are mean ± SEM for seven replicate experiments.