Figure 1.
Comparison of CD9 and CD81 sequences and structures.
Fig. 1A: sequences for the large extracellular domains (EC2) of human CD9 and CD81 and mouse CD9, aligned using ClustalW [49] in JalView [50]. Conserved residues are coloured according to physicochemical properties (http://www.ebi.ac.uk/Tools/msa/clustalw2/help/faq.html#24). Asterisks show residues that were mutated and the gray/black line indicates regions that were exchanged to form chimeric EC2 fusion proteins. Fig. 1B, C: Structures of CD9 (modelled on CD81 EC2 (PDB IV5) using I-TASSER [51]) and CD81 (from PDB file 1V5 [18]) and, showing regions exchanged in the production of the chimeras in alternating black and gray, as in Fig. 1A. Structures visualised using the UCSF Chimera package, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, funded by grants from the National Institutes of Health National Center for Research Resources (2P41RR001081) and National Institute of General Medical Sciences (9P41GM103311) [52].
Figure 2.
Effects of 500 nM EC2 domains on multinucleate giant cell formation.
Fig. 2 A, B shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A alone, or with Con A and 200 nM lipopolysaccharide, 500 nM GST or the indicated recombinant tetraspanin EC2 GST fusion protein, except for the data indicated where monocytes were treated with Con A and 250 nM each of the respective EC2 protein. Data are the means of at least 6 experiments ± SEM. Significance was calculated using one way ANOVA with Bonferroni post-test; p values ***:<0.0001; **: <0.01; *: <0.05. Unless otherwise indicated, the significance of the difference from the Con A control (clear bar) is shown.
Figure 3.
Effects of 500 nM CD9/CD81 chimeric EC2 domains on multinucleate giant cell formation.
Fig. 3 A, B shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A and 500 nM GST or 500 nM of the indicated recombinant chimeric EC2 GST fusion protein, in which different CD81 sequences were used to replace the relevant CD9 sequence. Fig. 3 C, D shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A and 500 nM GST or 500 nM of the indicated recombinant chimeric EC2 GST fusion protein, in which different CD9 sequences were used to replace the relevant CD81 sequence. Data are the means of 6 experiments ± SEM. Significance was calculated using one way ANOVA with Bonferroni post-test; p values ***:<0.0001; **: <0.01; *: <0.05. Unless otherwise indicated, the significance of the difference from the GST only control (clear bar) is shown.
Figure 4.
Effects of 5 nM CD9/CD81 chimeric EC2 domains on multinucleate giant cell formation.
Fig. 4 A, B shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively, of increasing concentrations of human CD9 EC2 GST fusion protein. Data are the means of 2 experiments ± SEM. Fig. 4 C, D shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A and 5 nM GST or 5 nM of the indicated recombinant chimeric EC2 GST fusion protein, in which different CD81 sequences were used to replace the relevant CD9 sequence. Fig. 4 E, F shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A and 5 nM GST or 5 nM of the indicated recombinant chimeric EC2 GST fusion protein, in which different CD9 sequences were used to replace the relevant CD81 sequence. Data in Fig. 4C–F are the means of 6 experiments ± SEM. Significance was calculated using one way ANOVA with Bonferroni post-test; p values ***:<0.0001; **: <0.01; *: <0.05. Unless otherwise indicated, the significance of the difference from the GST only control (clear bar) is shown.
Figure 5.
Effects of point mutations on the ability of CD9 EC2 to inhibit multinucleate giant cell formation.
Fig. 5 A, B shows the effects on fusion index and average number of nuclei per giant cell (>5 nuclei per cell), respectively. Monocytes were treated with Con A and 500 nM GST or 500 nM of the indicated recombinant mutant CD9 EC2 GST fusion protein. Data are the means of at least 6 experiments ± SEM. Significance was calculated using one way ANOVA with Bonferroni post-test; p values ***:<0.0001; **: <0.01; *: <0.05. Unless otherwise indicated, the significance of the difference from the GST only control (clear bar) is shown. Fig. 5C shows the positions of selected mutations on the model of CD9 EC2 protein, with the important T175-V178 region highlighted in red.
Figure 6.
Depiction of the regions of CD9 EC2 involved in the inhibition of multinucleate giant cell formation.
Structure of CD9 (modelled on CD81 EC2 (PDB IV5) using I-TASSER [51]), with surface polarity depicted from low (red) to high (blue). Distances between residues involved in the inhibitory effect (F186, Y148) or at the N-terminal end of the scond (B) helix (D135) are shown in Å, measured from the backbone amide N atom. Structure visualised using the UCSF Chimera package, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, funded by grants from the National Institutes of Health National Center for Research Resources (2P41RR001081) and National Institute of General Medical Sciences (9P41GM103311) [52].