Two glycoforms are present in the GPI-membrane anchor of the surface antigen 1 (P30) of Toxoplasma gondii

SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite , the life cycle stage associated with the acute phase of infection. The pr otein is inserted into the parasite's plasma membrane by a glycosyl-phospha tidylinositol anchor, a modification that is present on all T. gondii surfa ce proteins characterized so far. Here we describe a detailed structural an alysis of this anchor. GPI anchor peptides were isolated from [H-3]glucosam ine labeled purified P30 by protease digestion and phase partitioning. Neut ral glycans were prepared from this material by dephosphorylation and deami nation. Two glycoforms were characterized by gel filtration and high perfor mance ion exchange chromatography in combination with exoglycosidase treatm ent. Both forms were shown to carry an N-acetylgalactosamine bound to the f irst mannose of the conserved three-mannosyl core. Glycan B carries an addi tional terminal hexose linked to GaINAc. To identify the nature of this hex ose, bulk anchor peptide was prepared and glycans were purified by aminopro pyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-alpha1,2-Man-a lpha1,6-Man-[GalNAc-beta1,4-]-alpha1,4-GlcN-PI and Man-alpha1,2-Man-alpha1, 6-Man [Glc-alpha1,4-GalNAc-beta1,4-]-alpha ,4--GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein. (C) 2001 Published by Elsevier Science B.V.