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

Moyira Osny Aquino-Gil,Eduardo Pérez Campos,Stéphan Hardivillé,Frédéric Krzewinski,Ralph T Schwarz,Marlène Mortuaire,Jean-François Dubremetz,Nao Yamakawa,Fabrice Bray,Yobana Perez-Cervera,Christian Rolando,Tony Lefebvre,Jörg Schmidt,Hosam Shams-Eldin,Edgar Zenteno,Mattis Kupferschmid

doi:10.3389/fendo.2018.00450

Abstract

O-linked β-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.

Highlights

Most of the proteins are subjected to covalent chemical modifications that occur co- or post-translationally
The absence of contamination of parasite preparations by host cells was unequivocally attested by microscopy examination and anti-αtubulin staining (Figure 1A). α-tubulin is expressed in Vero cells, HFF and T. gondii but the difference of molecular weight observed between the two cell types and the parasite asserts that there was no contamination during T. gondii purification
Distinct approaches based on antibodies, click-chemistry and lectin-beads enrichment were used to visualize the occurrence of O-GlcNAcylation in T. gondii

Summary

Introduction

Most of the proteins are subjected to covalent chemical modifications that occur co- or post-translationally. Posttranslational modifications (PTMs) are very diverse and consist in the addition—usually enzymatically—of simple or complex (e.g., peptides, proteins, glycosylphosphatidylinositol) groups, or in the proteolytic cleavage to enlarge the complexity of the proteome [1]. O-GlcNAcylation cycles on and off targeted proteins, and the addition and hydrolysis of the GlcNAc moiety is respectively controlled by OGT (O-GlcNAc transferase) and OGA (OGlcNAcase) [2, 3]. It was found that beyond protein O-GlcNAcylation, OGT proteolytically processes the cell cycle regulator HCF-1 (Host cell factor-1) into a mature form through a glutamate glycosylation dependent manner [8, 9]. Deregulations in O-GlcNAcylation cycling were reported in different human pathologies: cancers, type-2 diabetes, cardiovascular and neuronal disorders [10]

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