Abstract
The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.
Highlights
The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility that critically depends on actin filaments and myosin
MALDI-TOF-MS of N-Glycans Released from Detergent Extracts—To gain more insight into protein N-glycosylation by the parasite, we embarked on the determination of the major structures of N-glycans synthesized by T. gondii
No modification of terminal galactose and sialic acid was identified, suggesting that T. gondii may lack a part of the endoplasmic reticulum and Golgi trimming and maturation pathways that are highly conserved in other eukaryotes [10, 11]
Summary
Dolichol; ER, endoplasmic reticulum; PP, pyrophosphate; HFF, human foreskin fibroblast; DMEM, Dulbecco’s modified Eagle’s medium; ConA, concanavalin A; PSA, P. sativum agglutinin; GC, gas chromatography; SAG, surface antigen; MIC, micronemal protein; GRA, dense granule protein; ROP, rhoptry protein; PNGase, peptide-N4-(N-acetyl--glucosaminyl)asparagine amidase; PMAA, partially methylated alditol acetate; BLAST, Basic Local Alignment Search Tool; PFA, paraformaldehyde; mAb, monoclonal antibody; Hex, hexose; HexNAc, N-acetylhexosamine; Tg, T. gondii; MyoA, myosin A; MLC, myosin light chain; GAP, glidingassociated protein; RON, rhoptry neck protein; AMA, apical membrane antigen. This led to the elucidation of the major structures of protein N-linked glycans that suggests the presence of an almost complete early N-glycosylation biosynthetic pathway in T. gondii. We showed that several key proteins involved in gliding motility, moving junction, and other functions that are essential for host cell invasion by T. gondii are modified by N-glycosylation. Inhibition of this post-translational modification dramatically impaired the parasite’s motility, host cell invasion, and intracellular growth
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