Figure 4: Effect of the single mutations on α-Gal aggregation and activity.

(a) Western blot of SEC fractions of WT α-Gal, selected aggregating mutants and single mutants in transiently transfected HeLa cells. WT α-Gal and single mutants eluted in later fractions (12.5–14.5 ml) than the aggregating mutants, corresponding to the active soluble form of the protein. (b) Quantification of the solubility of α-Gal mutants in transiently transfected HeLa cells. The band densities from western blot of SEC fractions from several experiments were quantified. Fractions from 6.5–10.5 ml elution were considered as aggregated, whereas from 12.5–14.5 ml as monomeric. WT α-Gal showed approximately 75% solubility, whereas aggregating mutants were highly insoluble. Single mutants reached ∼80–90% of total solubility (with the exception of the A368R mutant). Statistical significance was calculated as compared with WT α-Gal; ‘*’ P<0.05 and ‘****’ P<0.0001. (c) Quantification of the enzymatic activity of WT α-Gal and single mutations in transiently transfected HeLa cells. The activity of the WT and the mutants was similar, with a slight reduction in the case of the A368R mutant. Values are mean±s.e.m.