Abstract
BackgroundPost-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.MethodsThe P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).ResultsWhile low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.ConclusionsThis study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.
Highlights
Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings
This nucleotide sugar is the substrate of O-GlcNAc transferase (OGT) and, it is crucial for O-GlcNAcylation processes
The UDPGlcNAc level is around tenfold lower when compared to human fibroblasts (MRC5 cell line was used as a control), the study showed that there is a pool of precursor for the O-GlcNAcylation processes managed by P. falciparum
Summary
Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. The estimated malaria death rate declined by over 50% during the last 15 years This has been the result of a considerable international effort to globally provide potent anti-malarial drugs, insecticide-impregnated bed nets and indoor residual spraying. In ensuing experiences it was shown that contrary to other known forms of glycosylation, O-GlcNAcylation is a dynamic modification [8] It cycles on and off proteins thanks to a pair of enzymes: the O-GlcNAc transferase (OGT) adds the monosaccharide [9] and the O-GlcNAcase (OGA) catalyzes its hydrolysis [10]. Given these features differences O-GlcNAcylation endows some functions that are distinct from complex glycosylations such as transcriptional processes, protein synthesis or proteasomal degradation. Deregulations in O-GlcNAcylation processes are found in different diseases including metabolic disorders [12], as well as neoplastic [13] and neurodegenerative diseases [14]
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