title: Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography.
abstract: Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, 90095-1489, United States of America.
authors.forename: Kevin
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, 90095-1489, United States of America.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Peng
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, 90095-1489, United States of America.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Huang
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Dong
authors.initials: D
authors.lastname: Xia
authors.affiliation: Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Vermont
authors.affiliation: UMR 5235 CNRS, Université de Montpellier 1 and 2, 34095, Montpellier, France.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Lentini
authors.affiliation: UMR 5235 CNRS, Université de Montpellier 1 and 2, 34095, Montpellier, France.
authors.forename: Maryse
authors.initials: M
authors.lastname: Lebrun
authors.affiliation: Faculty of Natural Sciences, University of Keele, Staffordshire, United Kingdom.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Wastling
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, 90095-1489, United States of America.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Bradley
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D006031:Glycosylation; D037121:Plant Lectins / Q000378:metabolism*; D059748:Proteolysis; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D014122:Toxoplasma / Q000166:cytology; D014617:Vacuoles / Q000378:metabolism
chemical_list: D037121:Plant Lectins; D015800:Protozoan Proteins; C042242:Vicia lectins; D000116:Acetylgalactosamine