FIG 3.

Effect of A168T and F427I mutations on expression and cell-cell fusion activity of ectopically expressed GPC. (A and B) Metabolically labeled Candid#1 and MC2 GPCs were immunoprecipitated from transfected Vero-76 cells using the anti-GP1 MAb BF11 and resolved using NuPAGE. The mature GP1 and GP2 subunits comigrate as glycoproteins (A) and are resolved following deglycosylation by treatment with PNGase F (B). The uncleaved GP1GP2 precursor is labeled, and deglycosylated polypeptides are indicated as pp. GPC expression was quantitated by using a Fuji FLA3000G phosphorimager and ImageGauge software. Photostimulated luminescence (PSL) values in the respective GP1GP2 precursor bands in panel A are 427,881, 174,626, 360,045, 68,087, 339,735, and 53,852. This pattern of expression was confirmed in three replicate analyses. (C) Cell-cell fusion activity of the ectopically expressed MC2 (red) and Candid#1 (blue) GPCs was triggered by exposure to medium adjusted to pH 5.0 and determined using a chemiluminescent β-galactosidase fusion reporter. Relative light units are plotted. Error bars represent the standard errors of the means from replicate fusion reactions. Complete and partial replicates of this experiment (n > 4) yielded concordant results. A pairwise statistical comparison using a 2-tailed Mann-Whitney analysis, as implemented in GraphPad Prism software, demonstrated that Can GPC is significantly more fusogenic than MC2 GPC (P = 0.0002).