N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonasreinhardtii

Schulze, Stefan; Oltmanns, Anne; Machnik, Nick; Gai, Liu; Xu, Nannan; Jarmatz, Niklas; Scholz, Martin; Sugimoto, Kazuhiko; Fufezan, Christian; Huang, Kaiyao; Hippler, Michael

doi:10.1104/pp.17.01450

Cited by 25 publications

(53 citation statements)

References 40 publications

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“…Additionally, data presented here confirm the previous hypothesis, that XylT1B cannot act on excessively trimmed N-glycans (Schulze et al, 2018), while it was furthermore shown, that its lack can be compensated by XylT1A as seen in IMXylT1B.…”

Section: Xylt1b Transfers β12-xylose Onto N-glycanssupporting

confidence: 90%

“…When looking at the anti-β1,2-xylose antibody recognition, low signal of IMXylT1A as well as high signal of IMMan1AxIMXylT1A and IMMan1AxIMXylT1AxIMFucT confirmed already published results (Schulze et al, 2018). When XylT1B is knocked down in IMMan1AxIMXylT1A, the antibody signal was reduced to a minimal background level indicating the loss of a core xylose, thereby confirming the loss of a core Pent as seen in mass spectrometric measurements ( Figure 3b).…”

Section: Immunoblotting Proves Knockout Of N-glycosylation Enzymessupporting

confidence: 86%

“…In general, both antibodies showed lower affinity towards N-glycoproteins secreted by WT than towards N-glycoproteins secreted by IMMan1AxIMXylT1A. As it was shown previously that WT N-glycans are highly methylated while N-glycans produced in IMMan1AxIMXylT1A are reduced in methylation, one possible explanation might be an altered accessibility to the Nglycan core epitopes (Schulze et al, 2018). While conformational changes have been shown to be of importance for antibody recognition (Kaulfurst-Soboll et al, 2011), methylation might change the overall conformation or reduce the antibody accessibility by direct shielding of the N-glycan core.…”

Section: Immunoblotting Proves Knockout Of N-glycosylation Enzymesmentioning

confidence: 52%

“…When assuming that the putative XylT1B can act as a core XylT, N-glycans found in IMXylT1A and IMXylT1AB should not differ as IMXylT1A is already depleted in core xylose (Schulze et al, 2018). Indeed, the overall N-glycan compositions did not differ and no reduction in the amount of Pent was observed when comparing the two mutant strains ( Figure S7).…”

Section: Xylt1b Cannot Act On Excessively Trimmed N-glycansmentioning

confidence: 96%

“…To confirm the hypothesis regarding substrate specificity of XylT1B towards WT length N-glycans (Schulze et al, 2018), N-glycans found in IMXylT1AxIMFucT and IMMan1AxIMXylT1AxIMFucT were compared. Notably, while both strains expressed XylT1B (Table 1) and were devoid of dHex to the same degree ( Figure S6d), N-glycans found in IMXylT1AxIMFucT were excessively trimmed and therefore significantly shorter than N-glycans from IMMan1AxIMXylT1AxIMFucT (Figure 3c).…”

Section: Xylt1b Cannot Act On Excessively Trimmed N-glycansmentioning

confidence: 99%

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Novel insights into N-glycan fucosylation and core xylosylation in C. reinhardtii

Oltmanns¹,

Hoepfner²,

Scholz³

et al. 2019

Preprint

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Chlamydomonas 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.Significance StatementOur data provide novel insights into the function of XylT1B and FucT in C. reinhardtii as N-glycan core modifying enzymes. In the course of our study, different mutants were created by genetic crosses showing either varying or a lack of N-glycan core modification, enabling comparative analyses in relation to single N-glycan core epitope and overall protein function in C. reinhardtii.

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Section: Xylt1b Transfers β12-xylose Onto N-glycanssupporting

confidence: 90%

Section: Immunoblotting Proves Knockout Of N-glycosylation Enzymessupporting

confidence: 86%

Section: Immunoblotting Proves Knockout Of N-glycosylation Enzymesmentioning

confidence: 52%

Section: Xylt1b Cannot Act On Excessively Trimmed N-glycansmentioning

confidence: 96%

Section: Xylt1b Cannot Act On Excessively Trimmed N-glycansmentioning

confidence: 99%

See 3 more Smart Citations

Novel insights into N-glycan fucosylation and core xylosylation in C. reinhardtii

Oltmanns¹,

Hoepfner²,

Scholz³

et al. 2019

Preprint

Self Cite

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show abstract

Maximizing Depth of PTM Coverage: Generating Robust MS Datasets for Computational Prediction Modeling

Iannetta

Hicks

2022

Methods in Molecular Biology

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Bioinformatic analysis and genetic engineering approaches for recombinant biopharmaceutical glycoproteins production in microalgae

Liu

Xie

et al. 2021

Algal Research

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N-Glycoproteomic Characterization of Mannosidase and Xylosyltransferase Mutant Strains of Chlamydomonasreinhardtii

Cited by 25 publications

References 40 publications

Novel insights into N-glycan fucosylation and core xylosylation in C. reinhardtii

Novel insights into N-glycan fucosylation and core xylosylation in C. reinhardtii

Maximizing Depth of PTM Coverage: Generating Robust MS Datasets for Computational Prediction Modeling

Bioinformatic analysis and genetic engineering approaches for recombinant biopharmaceutical glycoproteins production in microalgae

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