A Monoclonal Antibody that inhibits IgE binding to the High affinity IgE receptor recognizes a glycolipid (36.11)

Abstract

Abstract The mAb BD6 is a monoclonal IgM that binds to the surface of RBL-2H3 cells and inhibits the binding of IgE to FcεRI, without reacting with this receptor. By cloning, mAb BD6 binding was resulted by the expression of the alpha1,3-galactosyltransferase gene. In binding assays on RBL-2H3 and alpha1,3-galactosyltransferase transfected PEAK cells there was partial competition between mAb BD6 and IB4, a galactose binding lectin. Both galactose and melibiose decreased the binding of mAb BD6 in a dose dependent manner on RBL-2H3 cells and abolished its binding on alpha1,3-galactosyltransferase transfected PEAK cells. MAb BD6 recognized a low molecular weight band by immunoblotting suggesting that it may be binding a lipid. This was confirmed by blotting lipids from RBL-2H3 cells separated by thin layer chromatography. By sucrose gradient analysis mAb BD6 bound to the lipid rafts fractions. RBL-2H3 variants were isolated completely deficient in mAb BD6 binding; these cells were also deficient in the ganglioside GD1b and had low expression of ganglioside GM1. However, these cells deficient in mAb BD6 binding and GD1b ganglioside still had normal FceRI induced degranulation. These results demonstrate that mAb BD6 inhibits IgE binding and reacts with a glycolipid present in lipid rafts; furthermore the ganglioside GD1b is not essential for degranulation. Supported by the Intramural Research Program, NIDCR, NIH.