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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.

SUBMITTER: Ramirez AS

PROVIDER: S-EPMC5421464 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs.

Ramírez Ana S AS Boilevin Jérémy J Biswas Rasomoy R Gan Bee Ha BH Janser Daniel D Aebi Markus M Darbre Tamis T Reymond Jean-Louis JL Locher Kaspar P KP

Glycobiology 20170601 6

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 ac ...[more]

PMID: 28204532

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Project description:Trypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N-glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs. Here, we show by blue-native gel electrophoresis and stable isotope labeling in cell culture proteomics that the TbSTT3A and TbSTT3B proteins associate with each other in large complexes that contain no other detectable protein subunits. We probed the peptide acceptor specificities of the OSTs in vivo using a transgenic glycoprotein reporter system and performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase H and peptide:N-glycosidase-F digestions. This allowed us to assess the relative occupancies of numerous N-glycosylation sites by endoglycosidase H-resistant N-glycans originating from Man5GlcNAc2-PP-dolichol transferred by TbSTT3A, and endoglycosidase H-sensitive N-glycans originating from Man9GlcNAc2-PP-dolichol transferred by TbSTT3B. Using machine learning, we assessed the features that best define TbSTT3A and TbSTT3B substrates in vivo and built an algorithm to predict the types of N-glycan most likely to predominate at all the putative N-glycosylation sites in the parasite proteome. Finally, molecular modeling was used to suggest why TbSTT3A has a distinct preference for sequons containing and/or flanked by acidic amino acid residues. Together, these studies provide insights into how a highly divergent eukaryote has re-wired protein N-glycosylation to provide protein sequence-specific N-glycan modifications. Data are available via ProteomeXchange with identifiers PXD007236, PXD007267, and PXD007268.

Dataset Information

Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs.

Publications

Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs.

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