Studies on O‐glycans of Plasmodium‐falciparum‐infected human erythrocytes Evidence for O‐GlcNAc and O‐GlcNAc‐transferase in malaria parasites

Abstract

) -EJB 930465/4 0-Glycosylation is the major form of protein glycosylation in human erythrocytes infected with the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum. This study compares aspects of 0-glycosylation in l? falciparum-infected and uninfected erythrocytes.Non-labeled and metabolically glucosamine-labeled 0-glycans were obtained from the protein fraction of infected or uninfected erythrocytes by p elimination. Additional label was introduced by reduction with sodium borohydride, or by the attachment of radioactive Gal to peripheral GlcNAc using galactosyltransferase. 2 -4-times more labeled 0-glycans were obtained from infected erythrocytes compared to the same number of uninfected ones, consistent with additional biosynthesis by the parasite. Our analysis of these 0-glycans showed no significant qualitative divergence between the 0-glycans of the infected and those of the uninfected red cell.According to preliminary alditol analyses, the 0-glycans of P. falciparum-infected red cells do not contain GalNAc at their reducing terminus. Moreover, GalNAc was not synthesized by P. falciparum from either Glc, Gal, GlcN or GalN. At least one 0-glycan found in P. faleiparurn-infected erythrocytes contains GlcNAc at its reducing terminus.Gel-filtration results had suggested the presence of 0-GlcNAc on proteins in the infected erythrocyte. Probing with a synthetic pentapeptide, we could show that P. falciparum expresses its own 0-GlcNAc transferase during intraerythrocytic development. Using this peptide, the enzyme was characterized to some degree. The localization and function of 0-GlcNAc in P. falciparum remains to be elucidated.Plasmodium falciparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, this disease still causes the death of several million people each year. 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. Little is known about the biological significance of oligosaccharides in I? falciparum, be they linked to lipids or to proteins.However, post-translational modifications of proteins by the covalent attachment of sugars are often very important for the function, localization and eventual antigenicity of proteins concerned (Rademacher et al., 1988 We have previously shown that glycoproteins of the asexual intraerythrocytic P. fulciparum bear mainly, if not exclusively, 0-glycans, whereas N-glycans seem to be lacking (Dieckmann-Schuppert et al., 1992a). The present study was undertaken in order to investigate the nature of the 0-glycans present in P. faleiparum-infected erythrocytes in general and the occurrence of 0-GlcNAc in particular.Despite the possibility of growing P. falciparum continuously in culture (Trager and Jensen, 1976), the biochemical analysis of this system is hampered by the facts t...

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

“…NAcT-The analysis was performed as described by Dieckmann-Schuppert et al (23). The saponin-released parasites were lysed in ice-cold water containing 1 g/ml leupeptin, 2 g/ml antipain, 1 g/ml pepstatin A, 1 g/ml chymostatin, 1 mM PMSF, and 5 mM iodoacetamide.…”

“…While one group reported that the parasite proteins also contain O-linked GalNAc (19,21), another group showed the absence of this sugar moiety and the lack of its biosynthesis (23). Kimura et al reported the presence of O-glycanase-releasable oligosaccharides as the major carbohydrates in the proteins of late trophozoite and schizont stage P. falciparum (28).…”

“…However, 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.Two laboratories have previously reported that O-glycosylation is the major carbohydrate modification in proteins of the intraerythrocytic asexual stage of P. falciparum (21,23) and that the O-linked carbohydrates are mainly single residues of GlcNAc with the remainder being oligosaccharides, some containing GlcNAc at the reducing end. While one group reported that the parasite proteins also contain O-linked GalNAc (19,21), another group showed the absence of this sugar moiety and the lack of its biosynthesis (23).…”

“…Two laboratories have previously reported that O-glycosylation is the major carbohydrate modification in proteins of the intraerythrocytic asexual stage of P. falciparum (21,23) and that the O-linked carbohydrates are mainly single residues of GlcNAc with the remainder being oligosaccharides, some containing GlcNAc at the reducing end. While one group reported that the parasite proteins also contain O-linked GalNAc (19,21), another group showed the absence of this sugar moiety and the lack of its biosynthesis (23).…”

“…Several proteins of erythrocytic stage P. falciparum are known to contain carbohydrates (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The deduced amino acid sequences of the parasite proteins have potential N-glycosylation sites (7, 24 -26).…”