Delineation of three pathways of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana. Precursors from different pathways are assembled on distinct pools of phosphatidylinositol and undergo fatty acid remodeling.

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 abundant lipophosphoglycan as well as being the major glycolipids (GIPLs) in the plasma membrane. In this study we have identified putative precursors for the protein and lipophosphoglycan anchors and delineated the complete pathway for GIPL biosynthesis in Leishmania mexicana promastigotes. Based on the structural analyses of these GPI intermediates and their kinetics of labeling in vivo and in cell-free systems, we provide evidence that the GIPLs are the products of an independent biosynthetic pathway rather than being excess precursors of the anchor pathways. 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 and 1-O-(C24:0/C26:0)-2-stearoyl-PI, respectively. These PI species account for 20 and 1% of the total PI pool, respectively, indicating a remarkable specificity in their selection. Second, analysis of the flux of intermediates through these pathways in vivo and in a cell-free system suggests that the GIPL and anchor pathways are independently regulated. We also show that GIPL biosynthesis requires fatty acid remodeling, in which the sn-2 stearoyl chains are replaced with myristoyl or lauroyl chains. Fatty acid remodeling is dependent on CoA and ATP and occurs on pre-existing but not on de novo synthesized GIPLs. We suggest that the compartmentalization of different GPI pathways may be important in regulating the species and stage-specific expression of different GPI structures in these parasites.