Glycosylphosphatidylinositol Anchors Represent the Major Carbohydrate Modification in Proteins of Intraerythrocytic Stage Plasmodium falciparum

Abstract

The nature and extent of carbohydrate modification in intraerythrocytic stage Plasmodium falciparum proteins have been controversial. This study describes the characterization of the carbohydrates in intraerythrocytic P. falciparum proteins and provides an overall picture of the nature of carbohydrate modification in the parasite proteins. P. falciparum strains were metabolically labeled with radioactive sugar precursors and ethanolamine at different developmental stages. The individual parasite proteins separated on SDS-polyacrylamide gels and whole parasite cell lysates were analyzed for the carbohydrate moieties. The results established the following: 1) glycosylphosphatidylinositol (GPI) anchors represent the major carbohydrate modification in the intraerythrocytic stage P. falciparum proteins; 2) in contrast to previous reports, O-linked carbohydrates are either absent or present only at very low levels in the parasite; and 3) P. falciparum contains low levels of N-glycosylation capability. The amount of N-linked carbohydrates in whole parasite proteins is ϳ6% compared with the GPI anchors attached to proteins based on radioactive GlcN incorporated into the proteins.The glycan cores of multiple parasite protein GPI anchors are all similar, consisting of protein-ethanolamine-phosphate-(Man␣1-2)6Man␣1-2Man␣1-6Man␣1-4GlcN. The fourth Man residues distal to GlcN of the GPI anchor glycan cores contain unidentified substituents that are susceptible to conditions of nitrous acid deamination. This unusual structural feature may contribute to the reported pathogenic properties of the P. falciparum GPI anchors.Malaria, a life-threatening disease caused by parasitic protozoa of the genus Plasmodium, is a major health problem throughout the tropical and subtropical regions of the world. Among the four species that infect humans, Plasmodium falciparum is the most virulent. New approaches such as vaccine development and novel therapeutic agents are urgently needed due to the emergence of parasite strains resistant to chloroquine and other commonly used drugs (1).Vaccines based on antigens of the blood stage parasite are under intensive study. A major focus has been on synthetic peptides or recombinant proteins of cell surface antigens (2-6). However, this approach has not been highly effective, although immunization with native cell surface proteins purified from the erythrocytic stage parasite is known to confer significant protective immunity (7-10). It is plausible that post-translational modifications play an important role in antigenicity. Accordingly, a basic understanding of such modifications may assist in the development of effective vaccines.Glycosylation is an often extensive post-translational modification of eukaryotic proteins. Carbohydrate moieties of glycoproteins perform a variety of functions including modulation of immunological properties, receptor-ligand interactions, sorting and localization of proteins, cell adhesion, and cell-cell communication (11). In addition, they contribute to protein conform...

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

“…The P. falciparum GPIs contain a conserved core structure, ethanolamine-phosphate-6Man␣1-2Man␣1-6Man␣1-4GlcN, that is ␣-(1-6)-linked to the inositol residue of PI (8,13,14). 2 The glycan core has an additional Man residue linked to the third Man at O-2.…”

“…More than 15 proteins of the intraerythrocytic stage P. falciparum including functionally important proteins such as merozoite surface protein-1, -2, and -4, a 71-kDa heat shock family protein, a 102-kDa transferrin receptor, and a 75-kDa serine protease are modified with GPI anchors (8, 10, 11). These observations suggest that GPI biosynthesis is vital to the parasite.The P. falciparum GPIs contain a conserved core structure, ethanolamine-phosphate-6Man␣1-2Man␣1-6Man␣1-4GlcN, that is ␣-(1-6)-linked to the inositol residue of PI (8,13,14). 2 The glycan core has an additional Man residue linked to the third Man at O-2.…”

The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases

“…Second, Tetrahymena pyriformis, which is a free-living ciliate, lacks all of the mannosylating activity in the ER lumen and makes dolichol-PP-GlcNAc 2 Man 5 Glc 3 (19). Third, it has been difficult to identify N-glycans from either Giardia lamblia (cause of diarrhea) or Plasmodium falicparum (cause of severe malaria) (20)(21)(22).…”

Biosynthesis ofO -N -Acetylglucosamine-linked Glycans inTrypanosoma cruzi

“…NAc is either absent or present only at very low levels in P. falciparum (45) and that glucose rather than N-acetylglucosamine is the O-linked sugar in G. lamblia (46). Because the O-GlcNAc-transferase activity of T. cruzi is associated with the microsomal fraction, and because its known natural substrates are GPI-anchored N-linked surface glycoproteins (10), it seems likely that it is associated with some compartment of the secretory pathway, although confirmation of this must await completion of detailed localization studies.…”