Differential Reactivities of Fucosyl GM1 and GM1 Gangliosides on Rat Erythrocyte Membrane Revealed by Analysis with Anti-Fucosyl GM1 and GM1 Antisera1

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.

Altered expression of gangliosides in erythrocytes of paroxysmal nocturnal hemoglobinuria.

“…4, lane 2) as well as the control IV6NeuAc-nLc4Cer from human meconium (Fig. 4, lane 1) (21). Differences in mobility on TLC plates may be due to the structural difference in their ceramide moieties.…”

Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera

“…As to GM1 on RRBC, we previously found that its reactivity was confined to small ligands on flow cytometric analysis, that is, cholera toxin B-subunit (56 kDa in molecular weight) had the ability to bind with GM1 readily bound to RRBC, but anti-GM1 antibodies (150 to 970 kDa in molecular weight) could not bind to RRBC at all [16,27]. Thus, one of the reasons why anti-GM1 antibodies were not produced on immunization with RRBC was supposed to be the masking of GM1 by coexisting molecules on RRBC, and the conformation of GM1 probably differs from those of Gg 4 Cer, the unknown glycolipid (IV 2 Fucα, IV 3 Galα-Gg 4 Cer), and FGM1, all of which effectively produced antibodies to themselves in anti-RRBC antisera.…”

“…), and fractionated into neutral and acidic lipids on a DEAE-Sephadex column (A-25, acetate form; GE Healthcare). Then, the neutral glycolipids were separated from the unabsorbed neutral lipid fraction by acetylation, separation of the acetylated derivatives, deacetylation and desalting, whereas the gangliosides were prepared from the absorbed acidic lipid fraction by cleavage of the ester-containing lipids, followed by dialysis [16,17]. The gangliosides and neutral glycolipids thus obtained were examined by TLC as above.…”

“…5, Forssman glycolipid on SRBC was revealed to react well with the antibodies on flow cytometry, hemagglutination and hemolysis, like that on liposomes on liposome aggregation, liposome lysis and CF. The antibody titers with several procedures were determined to be 1:2 17 for flow cytometry, 1:2 16 for ELISA, 1:2 14 by CF, 1:2 13 for liposome lysis, 1:2 12 for hemolysis, 1:2 9 for liposome aggregation, and 1:2 7 for hemagglutination, and thus flow cytometry and ELISA allowed the detection of antibodies in the most highly diluted antisera among the procedures applied.…”

Selective terminal α2–3 and α2–6 sialylation of glycosphingolipids with lacto‐series type 1 and 2 chains in human meconium

“…Extraction of total lipids from human meconium (A and O donors), followed by fractionation into neutral and acidic lipids by DEAE-Sephadex (A-25, acetate form) column chromatography, was carried out as described previously [1,2]. The acidic glycosphingolipids were prepared from the acidic fraction by saponification and dialysis, and then purified by ganglioside-mapping [3] and high-performance liquid chromatography (HPLC) on a column (2 cm internal diameter x 25 cm) packed with Iatrobeads (6RS8010).…”