title: Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs.
abstract: The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyltransferase (OST), which is generally a multisubunit membrane protein complex in the endoplasmic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesizing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25) and with different double bond stereochemistry. We found that in addition to proline, charged residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the -2 position significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST. While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction, although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs.
authors.affiliation: Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich, Switzerland.
authors.forename: Ana S
authors.initials: AS
authors.lastname: Ramírez
authors.affiliation: Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Boilevin
authors.affiliation: Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
authors.forename: Rasomoy
authors.initials: R
authors.lastname: Biswas
authors.affiliation: Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
authors.forename: Bee Ha
authors.initials: BH
authors.lastname: Gan
authors.affiliation: Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Janser
authors.affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.affiliation: Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
authors.forename: Tamis
authors.initials: T
authors.lastname: Darbre
authors.affiliation: Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Reymond
authors.affiliation: Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), CH-8093 Zürich, Switzerland.
authors.forename: Kaspar P
authors.initials: KP
authors.lastname: Locher
mesh_terms: D004187:Disaccharides / Q000737:chemistry; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D004187:Disaccharides; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D010455:Peptides; D015800:Protozoan Proteins; C023023:lipid-linked oligosaccharides; C032438:chitobiose; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: N-glycosylation; enzyme kinetics; lipid-linked oligosaccharide; oligosaccharyltransferase