Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum

Abstract

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

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

“…2 Therefore, it is likely that the low content of N-linked carbohydrates is due to very low N-glycosylation capacity of the parasite. This conclusion is consistent with the previously reported undetectable levels of GDP-Man, dolichol pyrophosphate-oligosaccharide intermediates, and peptide N-glycosidic oligosaccharyltransferase activity (27).…”

“…This explains why the presence of N-glycosylation was evident only on examination of whole cell lysates and was not readily detectable in individual parasite proteins. The low content of N-linked carbohydrates in P. falciparum is also in agreement with the previously reported undetectable levels of dolichol pyrophosphate-oligosaccharide intermediates, and peptide N-glycosidic oligosaccharyltransferase activity in the parasite (27).…”

“…The deduced amino acid sequences of the parasite proteins have potential N-glycosylation sites (7, 24 -26). However, Dieckmann-Schuppert et al (27) have reported that P. falciparum has no N-glycosylation capability based on their finding that dolichol phosphate-linked oligosaccharides, the obligatory biosynthetic intermediates, and dolichol phosphate-oligosaccharyltransferase activity were not detectable in the parasite. However, while this work was in review, Kimura et al (28) reported the existence of N-linked glycans in P. falciparum.…”

N-linked glycosylation of proteins in the protozoan parasite Toxoplasma gondii

“…There have been conflicting reports on the presence of Nlinked oligosaccharides on Plasmodium proteins. Dieckmann-Schnittler et al could not detect any evidence for the presence of ALG7 activity in P. falciparum lysates or oligosaccharide transferase activity, nor could they detect any evidence for sensitivity to tunicamycin [16]. Although this would appear to be in conflict with the analysis of Samuelson et al, it is important to remember that biochemical analyses of Plasmodium extracts is complicated by the small yield and by the extensive contamination by host cell material.…”

“…Despite the importance of N-glycosylation of proteins in other eukaryotic genera, several studies have suggested that this post-translational modification is a rare event in apicomplexan parasites [14][15][16][17]. In spite of this, the apicomplexan genomes do appear to contain genes encoding at least a number of the core enzymes required for synthesis of the dolichol-linked precursor oligosaccharide as well as the Rft1 flippase and members of the OST complex [18].…”