Glycosphingolipid antigens of Leishmania (Leishmania) amazonensis amastigotes identified by use of a monoclonal antibody

Abstract

Monoclonal antibodies directed against Leishmania (Leishmania) amazonensis amastigotes were produced. One monoclonal antibody (1C3) selected by indirect immunofluorescence reacted with both amastigotes and promastigotes of L. (L.) amazonensis. Glycolipid extraction from L. (L.) amazonensis amastigotes and separation by high-performance thin-layer chromatography followed by immunoblotting demonstrated that 1C3 reacts with two glycosphingolipids which migrate chromatographically similarly to ceramide-N-acetylneuraminic acid (GM1) and ceramide-N-tetrose-di-acetylneuraminic acid (GD1a). The antibody did not react with glycosphingolipids from L. (L.) amazonensis promastigotes. Immunoprecipitation of 125I- and 35S-methionine-labeled promastigotes demonstrated that 1C3 recognizes gp63 from L. (L.) amazonensis promastigotes. Biosynthetic incorporation of labeled lipids by L. (L.) amazonensis amastigotes indicated that the glycosphingolipids reactive with 1C3 contain oleic acid in their structures. Surface labeling with galactose oxidase and sodium boro[3H]hydride indicated that galactose is present in 1C3-reactive antigens, strongly suggesting that these glycosphingolipids are localized on the surface of L. (L.) amazonensis amastigotes. Inhibition experiments of macrophage infection implicated the 1C3-reactive glycosphingolipids from L. (L.) amazonensis amastigotes in Leishmania invasion. The role of gp63 in promastigote-macrophage attachment was also demonstrated by inhibition experiments performed with 1C3, consistent with data from the literature.