Differential reactivities of fucosyl GM1 and GM1 gangliosides on rat erythrocyte membrane revealed by analysis with anti-fucosyl GM1 and GM1 antisera

Abstract

Rat erythrocytes contained ganglio-series gangliosides, GM1, fucosyl GM1, and GD1a, in a high concentration. The concentrations of GM1, fucosyl GM1, and GD1a in rat erythrocyte ghosts were 889.0 nmol, 470.6 nmol, and 462.0 nmol per g dry weight, respectively, and the molar ratio of lipid-bound sialic acid, cholesterol and lipid-bound phosphorus was 3.1:73.9:100.0. The reactions of fucosyl GM1 and GM1 on rat erythrocytes with rabbit anti-fucosyl GM1 and anti-GM1 antisera were measured by means of haemolysis in the presence of complement and a binding assay of antibodies with a FACS after staining erythrocytes by the indirect membrane immunofluorescence technique. When measured by ELISA, anti-fucosyl GM1 antiserum was found to react almost exclusively with fucosyl GM1 with a slight cross-reaction with GM1, but anti-GM1 antiserum cross-reacted to a significant extent with asialo GM1. Rat erythrocytes were haemolyzed specifically with anti-fucosyl GM1 antiserum, but not with antisera to GM1, asialo GM1, asialo GM2, Forssman and globoside, and the haemolysis was proved to be definitely caused by the specific recognition of fucosyl GM1 on rat erythrocytes by anti-fucosyl GM1 antibody according to the haemolysis inhibition reaction using various glycosphingolipid-containing liposomes as inhibitors. In addition, although the binding of anti-fucosyl GM1 antibody on rat erythrocytes was clearly demonstrated with a FACS, anti-GM1 antibody did not bind. The observations that the haemolysis of rat erythrocytes and the binding of antibody to rat erythrocytes were found only with anti-fucosyl GM1 antiserum, and not with anti-GM1 antiserum, but that nevertheless the titer of anti-GM1 antiserum was higher than that of anti-fucosyl GM1 antiserum and GM1 on rat erythrocytes was more abundant in concentration than fucosyl GM1, seem to be a matter of great importance in assessing the specificity of anti-ganglioside antibody and the surface distribution of gangliosides on the cell.