Developmental Stage-specific Biosynthesis of Glycosylphosphatidylinositol Anchors in IntraerythrocyticPlasmodium falciparum and Its Inhibition in a Novel Manner by Mannosamine

Abstract

Glycosylphosphatidylinositols (GPIs) are the major glycoconjugates in intraerythrocytic stage Plasmodium falciparum. Several functional proteins including merozoite surface protein 1 are anchored to the cell surface by GPI modification, and GPIs are vital to the parasite. Here, we studied the developmental stage-specific biosynthesis of GPIs by intraerythrocytic P. falciparum. The parasite synthesizes GPIs exclusively during the maturation of early trophozoites to late trophozoites but not during the development of rings to early trophozoites or late trophozoites to schizonts and merozoites. Mannosamine, an inhibitor of GPI biosynthesis, inhibits the growth of the parasite specifically at the trophozoite stage, preventing further development to schizonts and causing death. Mannosamine has no effect on the development of either rings to early trophozoites or late trophozoites to schizonts and merozoites. The analysis of GPIs and proteins synthesized by the parasite in the presence of mannosamine demonstrates that the effect is because of the inhibition of GPI biosynthesis. The data also show that mannosamine inhibits GPI biosynthesis by interfering with the addition of mannose to an inositol-acylated GlcN-phosphatidylinositol (PI) intermediate, which is distinctively different from the pattern seen in other organisms. In other systems, mannosamine inhibits GPI biosynthesis by interfering with either the transfer of a mannose residue to the Man␣1-6Man␣1-4GlcN-PI intermediate or the formation of ManN-Man-GlcN-PI, an aberrant GPI intermediate, which cannot be a substrate for further addition of mannose. Thus, the parasite GPI biosynthetic pathway could be a specific target for antimalarial drug development. Glycosylphosphatidylinositol (GPI)1 anchors represent a distinct class of glycolipids found covalently attached to proteins in almost all eukaryotic cells (1, 2); they are particularly abundant in parasites, in which they occur as free lipids or linked to proteins (3,4). Although the primary function of GPIs is to anchor proteins to cell surfaces, they have been implicated in a number of biological responses including transmembrane signaling, apical targeting of proteins in polarized cells, insulin mimetic activity, and endocytic mechanisms (1,5,6).Plasmodium falciparum, the most virulent parasite among the four species of plasmodial apicomplexan protozoa that infect man, causes millions of deaths each year in tropical and subtropical countries (7). In the past decades, chloroquine and other drugs have been very useful in reducing mortality due to malaria. Although these conventional drugs are still widely used for the treatment of parasite infection, the parasite is rapidly becoming drug-resistant, raising the death toll from malaria (7). Therefore, malaria is once again a global concern, and novel antimalarial drugs are urgently needed.In P. falciparum, GPIs are the major carbohydrate moieties, whereas N-and O-linked carbohydrates or other glycolipids are either absent or present only at very low levels (8, 9)....

Plasmodium falciparumGlycosylphosphatidylinositol-induced TNF-α Secretion by Macrophages Is Mediated without Membrane Insertion or Endocytosis

“…The compound that lacked all three acyl substituents (EtN-P-Man 4 -GlcN-IP-G) was prepared by hydrolysis with ammonia in aqueous methanol. The identity of the above products was established by determining mannose content, by identifying the glycan core by specific degradative cleavage, and by analysis of products by HPTLC and mass spectrometry (16,17,24).…”

“…Metabolic Labeling of GPIs-Metabolic labeling of parasites with [ ]myristic acid (50 Ci/ml) was performed as described (17).…”

“…Cell signaling is mediated by a previously unrecognized mode of outside-in signal transduction without inserting into cell membranes or internalization of GPIs. P. falciparum Cell Culture-Culturing of intraerythrocytic P. falciparum (FCR-3 strain), synchronization, and mycoplasma testing were performed as described (17)(18)(19).…”

Plasmodium falciparum GPI mannosyltransferase-III has novel signature sequence and is functional

“…These parasite proteins have been studied as invasion-blocking vaccine candidates [6]. Further, as in other parasites, P. falciparum expresses GPIs in large excess than the requirement for protein anchoring [7], leading to GPI-dependent recognition by the host immune system [8]. The parasite GPIs are thought to be the major factors responsible for malaria pathogenesis [4].…”

“…falciparum (3D7 strain) was cultured in RPMI 1640 using human O-positive blood and 10% O-positive human serum. Parasites were synchronized with 5% sorbitol [7]. …”