Identification of methylated GnTI‐dependent <i>N</i>‐glycans in <i>Botryococcus brauni</i>

Schulze, Stefan; Urzica, Eugen I.; Reijnders, Maarten J.M.F.; Geest, Henri van de; Warris, Sven; Bakker, Linda V.; Fufezan, Christian; Santos, Vítor A. P. Martins dos; Schaap, Peter J.; Peters, Sander; Hippler, Michael

doi:10.1111/nph.14713

Cited by 23 publications

(38 citation statements)

References 46 publications

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“…After data acquisition, peptide identification by common database search engines is carried out with HexNac and HexNAc(2) set as variable modifications. In addition, with help of the in-house developed python-based tool SugarPy (Schulze et al, 2017;Schulze et al, 2018), the N-glycopeptide fragment ion series observed in MS1 can be analyzed to reconstruct the N-glycan composition (examples of annotated spectra are given in Supplemental Figure 3). By gaining information on both, peptide and attached carbohydrate moiety, N-glycan compositions of specific N-glycosites found in two strains can be directly compared, i.e.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

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Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical b1,2-core xylose, a1,3-fucose residues, as well as plant atypical terminal b1,4-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 Nglycopeptides 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.

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Section: Resultsmentioning

confidence: 99%

“…The annotation of N-glycan compositions, employing the inhouse developed Python tool SugarPy can be found in Schulze et al, 2017 andSchulze et al, 2018. Therefore, the procedure is summarized here only briefly.…”

Section: Annotation Of N-glycan Compositionsmentioning

confidence: 99%

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

et al. 2020

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“…With regards to microalgae, studies have demonstrated that the Golgi maturation of N ‐glycans in microalgae like Volvox carteri , Porphyridium sp., Phaeodactylum tricornutum , Botryococcus braunii , Euglena gracilius , Chlorella vulgaris , also give rise to a large variety of oligosaccharides ranging from oligomannosidic to complex‐type N ‐glycans decorating with GlcNAc, xylose, fucose, aminoethylphosphonate moieties and/or O ‐methylated mannose residues (Balshüsemann and Jaenicke, ; Baïet et al , ; Levy‐Ontman et al , , ; O’Neill et al , ; Schulze et al , ; Mócsai et al , ). Among microalgae, Chlamydomonas reinhardtii is the best studied so far as it has been chosen as a model laboratory system for many biological processes ranging from photosynthesis over flagellar movement including acclimation to nutrient deficiency (Rochaix, ; Merchant et al , ; Marshall, ; Harris, ).…”

Section: Introductionmentioning

confidence: 99%

Multiple xylosyltransferases heterogeneously xylosylate protein N‐linked glycans in Chlamydomonas reinhardtii

et al. 2020

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Summary Nowadays, little information is available regarding the N‐glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N‐glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core β(1,2)‐xylose. Recently, N‐glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core β(1,2)‐xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N‐glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double‐mutant. The combination of mass spectrometry approaches allowed us to identify the major N‐glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core β(1,2)‐xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N‐glycan as well as in the partial addition of the core β(1,2)‐xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double‐mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N‐glycosylation pathway.

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“…After data acquisition, peptide identification by common database search engines is carried out with HexNac and HexNAc(2) set as variable modifications. In addition, with help of the in-house developed python-based tool SugarPy (Schulze et al, 2017, Schulze et al, 2018, the N-glycopeptide fragment ion series observed in MS1 can be analyzed to reconstruct the N-glycan composition (examples are given in Figure S3). By gaining information about both, peptide and attached carbohydrate moiety, N-glycan compositions of specific N-glycosites found in two strains can be directly compared, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…The annotation of N-glycan compositions, employing the in-house developed Python tool SugarPy can be found in Schulze et al, 2017 andSchulze et al, 2018. Therefore, the procedure is summarized here only briefly.…”

Section: Annotation Of N-glycan Compositionsmentioning

confidence: 99%

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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Identification of methylated GnTI‐dependent N‐glycans in Botryococcus brauni

Cited by 23 publications

References 46 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

Multiple xylosyltransferases heterogeneously xylosylate protein N‐linked glycans in Chlamydomonas reinhardtii

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

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