title: The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.
abstract: Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.
authors.affiliation: From the Departments of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, the Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Kazi
authors.initials: K
authors.lastname: Rahman
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, the Complex Carbohydrate Research Center, Athens, Georgia 30602, and.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: From the Departments of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Msano
authors.initials: M
authors.lastname: Mandalasi
authors.affiliation: From the Departments of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602.
authors.forename: Hanke
authors.initials: H
authors.lastname: van der Wel
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, the Complex Carbohydrate Research Center, Athens, Georgia 30602, and.
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.affiliation: the Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York 14214.
authors.forename: Ira J
authors.initials: IJ
authors.lastname: Blader
authors.affiliation: From the Departments of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, westcm@uga.edu.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D019143:Evolution, Molecular; D017353:Gene Deletion; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D006801:Humans; D006909:Hydroxyproline / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D014122:Toxoplasma / Q000201:enzymology / Q000254:growth & development / Q000502:physiology*
chemical_list: D006020:Glycopeptides; D009844:Oligosaccharides; D015800:Protozoan Proteins; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D016695:Glycosyltransferases; D006909:Hydroxyproline
keywords: E3 ubiquitin ligase; SCF-complex; Skp1; Toxoplasma gondii; cytoplasmic glycosylation; glycobiology; glycosyltransferase; parasitology