MOLECULAR-STRUCTURE OF THE LOW-MOLECULAR-WEIGHT ANTIGEN OF TOXOPLASMA-GONDII - A GLUCOSE ALPHA-1-4 N-ACETYLGALACTOSAMINE MAKES FREE GLYCOSYL-PHOSPHATIDYLINOSITOLS HIGHLY IMMUNOGENIC

Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenit al infection and severe encephalitis in the course of the acquired imm unodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii ha ve been shown to be identical with the low molecular weight antigen wh ich elicits an early immunoglobulin M immune response in humans. A det ailed study of the structures of these glycolipid antigens was perform ed. Radiolabelled glycolipids were extensively analysed by chemical an d exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk prep arations of unlabelled glycolipids by high performance liquid chromato graphy were subjected to two-dimensional H-1 nuclear magnetic resonanc e spectroscopy, fast-atom bombardment-mass spectrometry, and methylati on linkage analysis in order to elucidate the structure of T. gondii G PIs. The following structures were identified: (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha-inosito l-PO4-lipid and the novel structure (ethanolamine-PO4)-Man alpha 1-2Ma n alpha 1-6(Glc alpha 1-4GalNAc beta 1-4)Man alpha 1-4GlcN alpha-inosi tol-PO4-lipid both with and without terminal ethanolamine phosphate. E vidence is provided, that only T. gondii GPIs bearing the unique gluco se-N-acetylgalactosamine side branch are immunogenic in humans and tha t this structure is widely distributed among T. gondii isolates. Monoc lonal antibodies have been characterized to recognize structures with different degrees of side-chain modification. We suggest that these re agents in combination with recently devised techniques for insertional mutagenisis in T. gondii should greatly facilitate the cloning of gen es essential for GPI side-chain modification. (C) 1997 Academic Press Limited.