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O-Fucosylation of Thrombospondin-Like Repeats Is Required for Processing of MIC2 and for Efficient Host Cell Invasion by Toxoplasma Gondii Tachyzoites

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O-Fucosylation of Thrombospondin-Like Repeats Is Required for Processing of MIC2 and for Efficient Host Cell Invasion by Toxoplasma Gondii Tachyzoites bioRxiv preprint doi: https://doi.org/10.1101/382515; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. O-fucosylation of thrombospondin-like repeats is required for processing of MIC2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites Giulia Bandini‡, Deborah R. Leon§, Carolin M. Hoppe¶, Yue Zhang∫, Carolina Agop-Nersesian‡, Melanie J. Shears◊, Lara K. Mahal∫, Françoise H. Routier¶, Catherine E. Costello§, and John Samuelson‡,1 Running title: O-fucosylation of MIC2 TSRs in T. gondii From the ‡ Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA, §Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA 02118, USA, ¶ Department of Clinical Biochemistry OE4340, Hannover Medical School, 30625 Hannover, Germany, ∫ Department of Chemistry, Biomedical Chemistry Institute, New York University, New York, NY 10003, USA, and ◊Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA 1 To whom correspondence may be addressed: Dept. of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 72 East Concord Street, Boston, MA 02118, USA. E-mail: [email protected] Key words: O-fucosylation; Toxoplasma gondii; thrombospondin-like repeats; MIC2; invasion. Abstract O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Toxoplasma gondii is an intracellular Trp residues within three TSRs are also modified parasite that causes disseminated infections which with C-mannose. Disruption of genes encoding can lead to neurological damage in fetuses and either pofut2 or nucleotide sugar transporter 2 immunocompromised individuals. Microneme 2 (nst2), the putative GDP-fucose transporter, results protein 2 (MIC2) , a member of the in loss of MIC2 O-fucosylation, as detected by an thrombospondin-related anonymous protein antibody against the GlcFuc disaccharide, and (TRAP) family, is a secreted protein important for markedly reduced cellular levels of MIC2. motility, host cell attachment, invasion, and Furthermore, in 10-15% of the Δpofut2 or Δnst2 egress. MIC2 contains six thrombospondin type I vacuoles, MIC2 accumulates earlier in the repeats (TSRs) that are modified by C-mannose secretory pathway rather than localizing to and O-fucose in Plasmodium spp. and mammals. micronemes. Dissemination of tachyzoites in Here we used mass spectrometry to show human foreskin fibroblasts is reduced in these that the four TSRs in T. gondii MIC2 with protein knockouts, which both show defects in attachment to and invasion of host cells comparable to the 2 The abbreviations used are: MIC2, microneme phenotype observed in the Δmic2. protein 2; TSR, thrombospondin repeat; POFUT2, These results, which show O-fucosylation protein O-fucosyltransferase 2; NST2, nucleotide of TSRs is required for efficient processing of sugar transporter 2; M2AP, MIC2 associated MIC2 and for normal parasite invasion, are protein; mGFP, green fluorescent protein; mRFP, consistent with the recent demonstration that P. red fluorescent protein; AAL, Aleuria aurantia falciparum Δpofut2 has decreased virulence and lectin; KLH, keyhole limpet hemocyanin; dHex, support a conserved role for this glycosylation deoxyhexose; Hex, hexose; Fuc, fucose; Glc, pathway in quality control of TSR-containing glucose; Man, mannose; HCD, higher collision proteins in eukaryotes. energy dissociation; ETD, electron transfer dissociation; PV, parasitophorous vacuole. bioRxiv preprint doi: https://doi.org/10.1101/382515; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. O-fucosylation of MIC2 TSRs in T. gondii Introduction M2AP/MIC2 complex to the micronemes is important for its function. Knockout of m2ap Toxoplasma gondii is a eukaryotic parasite results in inefficient localization of MIC2 to the that belongs to the Apicomplexa phylum, together micronemes and defects in the parasite’s ability to with other medically relevant parasites such as glide and invade (14). Cleavage-resistant Plasmodium spp. and Cryptosporidium parvum. T. mutations of m2ap, which affect protein gondii, which has the ability to invade all warm maturation and consequently localization of the blooded animals, causes transient, disseminated M2AP/MIC2 complex, also result in inefficient infections that can lead to neurological damage in gliding and attachment (15, 16). immunocompromised individuals and Thromobospondin-like repeats, which are developmental defects in fetuses (1, 2). T. gondii each about 60 amino acids long, are present in replicates asexually in all of its hosts, with the many extracellular proteins. TSRs have a exception of felids (cats), where the parasite also conserved structure characterized by a tryptophan undergoes a sexual cycle, which concludes in ladder composed of stacked Trp and arginine production and shedding of infectious oocysts in residues and a set of conserved cysteines that are the feces (3). involved in disulfide bridges (17). In metazoans, Because T. gondii is an obligate, TSRs are glycosylated via two pathways: O- intracellular pathogen, its virulence is dependent fucosylation by action of the protein O- on the ability to invade host cells. The parasite has fucosyltransferase 2 (POFUT2) and C- specialized secretory organelles, which localize at mannosylation by the C-mannosyltransferase the apical end of asexual stages and whose content DPY-19 (18, 19). C-Man is transferred via an is sequentially secreted during the invasion unusual carbon-carbon bond to the Trp on a process (4). Secretion of microneme proteins is WXXWXXC1 sequence, where C1 is the first dependent upon elevation of parasite calcium conserved cysteine in the TSR (20). P. falciparum levels and is the first event upon parasite and T. gondii each encode for a DPY-19 attachment (4, 5). orthologue that can transfer C-Man to the Trp Microneme protein 2 (MIC2) is a member residues in the conserved motif, both in vitro and of the thrombospondin-related anonymous protein in vivo (21). (TRAP) family (6). MIC2 is a type 1 membrane POFUT2 transfers α-fucose to the protein composed of an A/I von Willebrand hydroxyl group on Ser/Thr residues in the domain, six thrombospondin-like repeats, and a 1 2 1 consensus sequence C X2-3(S/T)C X2G, where C short cytoplasmic tail (7). The A/I domain has and C2 are the first two conserved cysteines and X been shown to be involved in recognition of host is any amino acid (22). In metazoans, fucose is cells, possibly through binding to ICAM-1, transferred on already folded TSR domains (23) heparin, and sulfated glycosaminoglycans (8-10). and can be elongated by a β1,3-glucose by action MIC2 forms a hexameric complex with the MIC2- of the glucosyltransferase B3GLCT (24). Mass associated protein (M2AP) in the endoplasmic spectrometry analyses of P. falciparum TRAP and reticulum; together they traffic to the micronemes circumsporozoite protein (CSP) indicate that a and, upon secretion, to the apical cell surface (8, hexose (Hex) residue elongates the fucose on 11). As host cell penetration concludes, the Apicomplexa TSRs; this sugar may be glucose, MIC2/M2AP complex relocates to the posterior but has not yet been unequivocally identified (21, end of the parasite and is released into the 25). environment by cleavage of the C-terminal TSRs glycosylation is carried out in the transmembrane helix that is catalyzed by the endoplasmic reticulum (22, 26) and, in studies MPP1 protease (7, 12). performed in mammalian cell and C. elegans, Although knockout of the mic2 gene defects in both C-mannosylation and O- strongly affects motility and invasion, MIC2 is not fucosylation have been shown to affect protein essential for parasite viability. Tachyzoites lacking stabilization and secretion, albeit with protein-to- this protein still glide, attach to, and invade protein variability (19, 23, 27-31). A knockout of fibroblasts, albeit inefficiently (13). Numerous pofut2 is embryonically lethal in mice (31). The studies have shown that correct trafficking of the human recessive disorder Peter Plus syndrome is 2 bioRxiv preprint doi: https://doi.org/10.1101/382515; this version posted August 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. O-fucosylation of MIC2 TSRs in T. gondii caused by mutations in the B3GLCT MIC2 has six contiguous TSRs. All six glycosyltransferase (23). Recently, a knockout of contain C-mannosylation sites, whereas only four P. falciparum pofut2 showed O-fucosylation of (TSR1, 3, 4, and 5) have the consensus O- 1 2 TSRs is required for efficient mosquito infection fucosylation motif (C X2-3(S/T)C X2G) (Fig. 1A). by ookinetes and for hepatocyte invasion by The targeted HCD and ETD experiments allowed sporozoites (32). These phenotypes are due to localization of a dHexHex disaccharide on the defective folding/stabilization of the parasite TSR- predicted POFUT2 acceptor residues on MIC2 containing proteins, such as TRAP and CSP, TSR1 and TSR4 and a dHex +/-Hex on TSR5 consistent with the observations made in (25). TSR3 is also modified with a dHexHex, metazoans and a potential role for O-fucosylation although the modification site could not be in efficient folding and/or stabilization of TSRs assigned (Table 1, Figs. 1, 2, 3 and S3). TSR6 (23, 32). lacks a consensus site for POFUT2 and is not In this paper we use mass spectrometry to modified by C-Man; TSR2 lacks an O- show that four of six TSRs of MIC2 are modified fucosylation consensus site and was not sampled.
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