Identification of O-Glcnacylated Proteins in Trypanosoma cruzi.

Originally an anthropozoonosis in the Americas, Chagas disease has spread from its previous borders through migration. It is caused by the protozoan Trypanosoma cruzi. Differences in disease severity have been attributed to a natural pleomorphism in T. cruzi. Several post-translational modifications (PTMs) have been studied in T. cruzi, but to date no work has focused on O-GlcNAcylation, a highly conserved monosaccharide-PTM of serine and threonine residues mainly found in nucleus, cytoplasm, and mitochondrion proteins. O-GlcNAcylation is thought to regulate protein function analogously to protein phosphorylation; indeed, crosstalk between both PTMs allows the cell to regulate its functions in response to nutrient levels and stress. Herein, we demonstrate O-GlcNAcylation in T. cruzi epimastigotes by three methods: by using specific antibodies against the modification in lysates and whole parasites, by click chemistry labeling, and by proteomics. In total, 1,271 putative O-GlcNAcylated proteins and six modification sequences were identified by mass spectrometry (data available via ProteomeXchange, ID PXD010285). Most of these proteins have structural and metabolic functions that are essential for parasite survival and evolution. Furthermore, O-GlcNAcylation pattern variations were observed by antibody detection under glucose deprivation and heat stress conditions, supporting their possible role in the adaptive response. Given the numerous biological processes in which O-GlcNAcylated proteins participate, its identification in T. cruzi proteins opens a new research field in the biology of Trypanosomatids, improve our understanding of infection processes and may allow us to identify new therapeutic targets.

Apart From Rhoptries, Identification of Toxoplasma gondii's O-GlcNAcylated Proteins Reinforces the Universality of the O-GlcNAcome.

O-linked ß-N-acetylglucosaminylation or O-GlcNAcylation is a widespread post-translational modification that belongs to the large and heterogeneous group of glycosylations. The functions managed by O-GlcNAcylation are diverse and include regulation of transcription, replication, protein's fate, trafficking, and signaling. More and more evidences tend to show that deregulations in the homeostasis of O-GlcNAcylation are involved in the etiology of metabolic diseases, cancers and neuropathologies. O-GlcNAc transferase or OGT is the enzyme that transfers the N-acetylglucosamine residue onto target proteins confined within the cytosolic and nuclear compartments. A form of OGT was predicted for Toxoplasma and recently we were the first to show evidence of O-GlcNAcylation in the apicomplexans Toxoplasma gondii and Plasmodium falciparum. Numerous studies have explored the O-GlcNAcome in a wide variety of biological models but very few focus on protists. In the present work, we used enrichment on sWGA-beads and immunopurification to identify putative O-GlcNAcylated proteins in Toxoplasma gondii. Many of the proteins found to be O-GlcNAcylated were originally described in higher eukaryotes and participate in cell shape organization, response to stress, protein synthesis and metabolism. In a more original way, our proteomic analyses, confirmed by sWGA-enrichment and click-chemistry, revealed that rhoptries, proteins necessary for invasion, are glycosylated. Together, these data show that regardless of proteins strictly specific to organisms, O-GlcNAcylated proteins are rather similar among living beings.

Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

BACKGROUND: Post-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. METHODS: The 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). RESULTS: While 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. CONCLUSIONS: This 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.

OGT: a short overview of an enzyme standing out from usual glycosyltransferases.

O-GlcNAcylation is a highly dynamic post-translational modification whose level depends on nutrient status. Only two enzymes regulate O-GlcNAcylation cycling, the glycosyltransferase OGT (O-GlcNAc transferase) and the glycoside hydrolase OGA (O-GlcNAcase), that add and remove the GlcNAc moiety to and from acceptor proteins, respectively. During the last 30 years, OGT has emerged as a master regulator of cell life with O-GlcNAcylation being found in viruses, bacteria, insects, protists and metazoans. The study of OGT in different biological systems opens new perspectives for understanding this enzyme in many kingdoms of life. In this review, we summarize recent and older findings regarding the distribution of OGT in living organisms.

Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains.

Lipid microdomains (rafts) are cholesterol-enriched dynamic ordered lipid domains belonging to cell membranes involved in diverse cellular functions, including signal transduction, membrane trafficking, and infection. Many studies have reported relationships between insulin signaling and lipid rafts. Likewise, links between insulin signaling and O-GlcNAcylation have also been described. However, the potential connection between O-GlcNAc and raft dynamics remains unexplored. Here we show that O-GlcNAc and the enzyme that creates this modification, O-GlcNAc transferase (OGT), are localized in rafts. On insulin stimulation, we observe time-dependent increases in OGT expression and localization within rafts. We show that these processes depend on activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Inhibition of OGT does not significantly affect cholesterol synthesis and raft building but decreases insulin receptor expression and PI3K and mitogen-activated protein kinase pathway activation. Taken together, these findings indicate that O-GlcNAcylation, lipid rafts, and signaling pathways are spatiotemporally coordinated to enable fundamental cellular functions.