Novel Insights Into N-Glycan Fucosylation and Core Xylosylation in C. reinhardtii.

Anne Oltmanns,Michael Hippler,Karen Zinzius,Martin Scholz,Lara Hoepfner,Stefan Schulze

doi:10.3389/fpls.2019.01686

Anne Oltmanns, Michael Hippler + Show 4 more

Open Access

https://doi.org/10.3389/fpls.2019.01686

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Abstract

Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical β1,2-core xylose, α1,3-fucose residues, as well as plant atypical terminal β1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact N-glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.

Highlights

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes. It starts in the endoplasmic reticulum (ER) with the co-translational transfer of glucose3mannose9Nacetylglucosamine2 from a lipid-linked oligosaccharide precursor onto the asparagine of the consensus sequence N-X-S/T by the oligosaccharyltransferase complex (OST)
To analyze the role of the putative XylT1B and FucT, insertional mutants were obtained from the Chlamydomonas Library Project (CLiP) (Li et al, 2016)
Strains derived from single cell colonies were checked by PCR for insertional cassettes (Supplemental Figure 1) and Parallel Reaction Monitoring (PRM) measurements were employed to quantify relative protein abundances of Man1A, XylT1A and XylT1B (Table 2; Supplemental Figure 2A)

Summary

Introduction

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes. It starts in the endoplasmic reticulum (ER) with the co-translational transfer of glucose3mannose9Nacetylglucosamine from a lipid-linked oligosaccharide precursor onto the asparagine of the consensus sequence N-X-S/T (where X may be any amino acid except proline) by the oligosaccharyltransferase complex (OST). The high diversity of so-called complex type or paucimannosidic N-glycans in plants is a result of the manifold Golgi enzyme repertoire among different species. Typical N-glycan modifications absent in mammals but found in vascular plants include b1,2-xylose and a1,3-fucose. N-glycans in plants can be terminally capped by b1,3-galactose and a1,4-fucose, a structure referred to as Lewisa epitope

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