DELINEATION OF 3 PATHWAYS OF GLYCOSYLPHOSPHATIDYLINOSITOL BIOSYNTHESIS IN LEISHMANIA-MEXICANA - PRECURSORS FROM DIFFERENT PATHWAYS ARE ASSEMBLED ON DISTINCT POOLS OF PHOSPHATIDYLINOSITOL AND UNDERGO FATTY-ACIDREMODELING

Glycosylphosphatidylinositol (GPI) glycolipids are major cell surface constituents in the Leishmania parasites. Distinct classes of GPI are present as membrane anchors for several surface glycoproteins and an a bundant lipophosphoglycan as well as being the major glycolipids (GIPL s) in the plasma membrane. In this study we have identified putative p recursors for the protein and lipophosphoglycan anchors and delineated the complete pathway for GIPL biosynthesis in Leishmania mexicana pro mastigotes. Based on the structural analyses of these GPI intermediate s and their kinetics of labeling in vivo and in cell-free systems, we provide evidence that the GIPLs are the products of an independent bio synthetic pathway rather than being excess precursors of the anchor pa thways, First, we show that the similar glycan head groups of the GIPL and protein/lipophosphoglycan anchor precursors are assembled on two distinct pools of PI corresponding to 1-O-(C18:0)alkyl-2-stearoyl-PI a nd 1-O-(C24:0/C26:0)-2-stearoyl-PI, respectively, These PI species acc ount for 20 and 1% of the total PI pool, respectively, indicating a re markable specificity in their selection, Second, analysis of the flux of intermediates through these pathways in vivo and in a cell-free sys tem suggests that the GIPL and anchor pathways are independently regul ated. We also show that GIPL biosynthesis requires fatty acid remodeli ng, in which the sn-2 stearoyl chains are replaced with myristoyl or l auroyl chains, Fatty acid remodeling is dependent on CoA and ATP and o ccurs on pre-existing but not on de novo synthesized GIPLs. We suggest that the compartmentalization of different GPI pathways may be import ant in regulating the species and stage-specific expression of differe nt GPI structures in these parasites.