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

Ramachandra S Naik,Eugene A Davidson,D Channe Gowda

doi:10.1074/jbc.m002151200

Ramachandra S Naik, Eugene A Davidson + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m002151200

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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 Manalpha1-6Manalpha1-4GlcN-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.

Highlights

Glycosylphosphatidylinositol (GPI)1 anchors represent a distinct class of glycolipids found covalently attached to proteins in almost all eukaryotic cells [1, 2]; they are abun
Effect of ManN on the Survival of P. falciparum at Different Stages of the Intraerythrocytic Development—We investigated the developmental stage-specific biosynthesis of GPIs in intraerythrocytic P. falciparum and the effect of inhibition of the GPI biosynthetic pathway on the survival of the parasite
Four important findings emerge from this study: 1) intraerythrocytic P. falciparum synthesizes GPI anchors in a developmental stage-specific manner exclusively at the trophozoite stage; 2) GPI biosynthesis is critical to the differentiation of trophozoites into schizonts, and the inhibition of GPI biosynthesis causes parasite growth arrest at the trophozoite stage; 3) in P. falciparum, ManN inhibits GPI biosynthesis by interfering with the attachment of first Man to the inositolacylated GlcN-PI intermediate, a mechanism that is distinctively different from those in other organisms, including man; and 4) The matured GPI, EtN-P-Man4-GlcN-PI, is formed by two different reaction sequences: transfer of a mannose residue to EtN-P-Man3-GlcN-PI and to Man3-GlcN-PI; Man4-GlcN-PI is substituted with ethanolamine phosphate

Summary

EXPERIMENTAL PROCEDURES

Materials—RPMI 1640 culture medium and HEPES were purchased from Life Technologies, Inc. Treatment with Mannosidase—The [3H]GlcN-labeled GPIs (25,000 – 50,000 cpm) were treated with jack bean ␣-mannosidase (30 units/ml) in 40 ␮l of 100 mM NaOAc, 2 mM Zn2ϩ, pH 5.0, containing 0.1% sodium taurodeoxycholate at room temperature for 2 h and at 37 °C for 22 h [27]. Thin Layer Chromatography—The [3H]GlcN-labeled GPIs were applied onto HPTLC plates, developed with chloroform/methanol/water (10:10:2.4, v/v/v), and dried. The glycan cores obtained by treatment of GPIs with HF and nitrous acid/NaBH4 and their mannosidase digestion products were analyzed by HPTLC using 1-propanol/acetone/water (10: 6:4, v/v/v). SDS-PAGE and Fluorography—The [3H]GlcN- or [35S]Met-labeled parasites were dissolved in nonreducing SDS-PAGE sample buffer, and the lysates were heated in a boiling water bath for 5 min and electrophoresed under nonreducing conditions on 6 –20% SDS-polyacrylamide gradient gels [28]. The gels were fixed, washed with water, soaked in 1 M sodium salicylate in water for 30 min, dried, and exposed to x-ray films at Ϫ80 °C

RESULTS

Culture timea and parasite development stage

Glycan core identified

DISCUSSION