Bandini G, Leon DR, Hoppe CM, Zhang Y, Agop-Nersesian C, Shears MJ, Mahal LK, Routier FH, Costtello CE, Samuelson J O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites Journal of Biological Chemistry294(6) (2019)
1967-1983
Glcp-(1-?)-Fucp-(1--/thrombospondin-type I repeats (TSR1, -3, -4, and -5) of microneme protein 2 (MIC2)/
The structure was elucidated in this paper NCBI PubMed ID:30538131 Publication DOI:10.1074/jbc.RA118.005179 Journal NLM ID:2985121R Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology Correspondence: jsamuelsbu.edu Institutions: Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118, Department of Clinical Biochemistry OE4340, Hannover Medical School, 30625 Hannover, Germany, Department of Chemistry, Biomedical Chemistry Institute, New York University, New York, New York 10003, Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the Δmic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum Δpofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.
Methods: ELISA, Western blotting, serological methods, genetic methods, nLC-MS/MS, immunofluorescence analysis, nUPLC-MS/MS Enzymes that release or process the structure: O-fucosyltransferase 2 (POFUT2) Comments, role: all four MIC2 TSRs with POFUT2 acceptor sites are modified by a dHexHex disaccharide, consistent with FucGlc, and TSR5 can also be modified by only a dHex.
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