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

Ana S Ramírez,Rasomoy Biswas,Jérémy Boilevin,Daniel Janser,Tamis Darbre,Markus Aebi,Jean-Louis Reymond,Bee Ha Gan,Kaspar P Locher

doi:10.1093/glycob/cwx017

Ana S Ramírez, Rasomoy Biswas + Show 7 more

Open Access

PDF Available

https://doi.org/10.1093/glycob/cwx017

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

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.

Highlights

Protein N-glycosylation confers a multitude of functions to the acceptor macromolecules, facilitating diverse interactions and signaling pathways (Kelleher and Gilmore 2006; Breitling and Aebi 2013; Xu and Ng 2015; Cherepanova et al 2016)
Whereas TbSTT3B requires a c-branch in the linked oligosaccharide (LLO) and glycosylates sequons surrounded by neutral or basic residues, TbSTT3A preferentially transfer glycans from LLO donors lacking the c-branch such as Man5GlcNAc2-PP-Dol to sequons surrounded by acidic side chains (Izquierdo et al 2009, 2012)
The main difference between TbSTT3A and its orthologs is the length of its C-terminal domain, which is 23 amino acids shorter in TbSTT3A compared to TbSTT3B and TbSTT3C, this might influence the recombinant expression of the proteins

Summary

Introduction

Protein N-glycosylation confers a multitude of functions to the acceptor macromolecules, facilitating diverse interactions and signaling pathways (Kelleher and Gilmore 2006; Breitling and Aebi 2013; Xu and Ng 2015; Cherepanova et al 2016). The initial transfer of the glycan moiety from a lipid-linked oligosaccharide (LLO) donor to the. In vivo studies suggested that TbSTT3A and TbSTT3B have different preferences for the LLO donor as well as for acceptor sequon (Jones et al 2005; Manthri et al 2008). Whereas TbSTT3B requires a c-branch in the LLO (present in Man9GlcNAc2-PPDol) and glycosylates sequons surrounded by neutral or basic residues, TbSTT3A preferentially transfer glycans from LLO donors lacking the c-branch such as Man5GlcNAc2-PP-Dol to sequons surrounded by acidic side chains (Izquierdo et al 2009, 2012)

Methods

Results

Conclusion