Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum

Zhang, Peiqing; Burel, Carole; Plasson, Carole; Kiefer‐Meyer, Marie‐Christine; Ovide, Clément; Gügi, Bruno; Wan, Corrine; Teo, Gavin; Mak, Amelia; Song, Zhiwei; Driouich, Azeddine; Lerouge, Patrice; Bardor, Muriel

doi:10.3389/fpls.2019.00610

Cited by 15 publications

(23 citation statements)

References 95 publications

(155 reference statements)

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“…Chlamydomonas reinhardtii protein extracts were separated on a NUPAGE 4–12% gel electrophoresis and were electrotransferred onto nitrocellulose membrane as previously described (Zhang et al , ). Immunodetection with anti‐β(1,2)‐xylose‐specific antibodies (AS07 267; Agrisera, Vannas, Sweden) was performed according to Fitchette et al ().…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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“…B. braunii shows native N-glycans similar to human "hybrid" structures, and P. tricornutum presents paucimannosidic glycans, an important pattern found in a specific class of biopharmaceuticals (as explained in Section 5.1.2) [79,112]. Nonetheless, α(1,3)-fucose residues in P. tricornutum [113] and methylation of mannoses in B. braunii [110] are both N-glycan characteristics absent in humans.…”

Section: N-glycosylationmentioning

confidence: 96%

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Barolo

Abbriano

Commault

et al. 2020

Cells

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Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

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“…For examples, the heterologous expression of P. tricornutum GnTI restored the complex type N-glycans maturation in the Chinese hamster ovary (CHO) mutant, indicating its importance on the synthesis of complex type N-glycans ( Baïet et al, 2011 ). Subsequently, it was observed that the GDP-fucose transporter and FucT1 were involved in the fucose modification of proteins in P. tricornutum ( Zhang et al, 2019 ). In addition to P. tricornutum , the functions of genes, such as xylosyltransferase A (XylTA), xylosyltransferase B (XylTB), fucosyltransferase (FucT), and mannosidase 1A (Man1A) were studied during the protein N-glycosylation pathway of C. reinhardtii ( Mathieu-Rivet et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

Xie

Chen

et al. 2021

Front. Plant Sci.

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N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

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Characterization of a GDP-Fucose Transporter and a Fucosyltransferase Involved in the Fucosylation of Glycoproteins in the Diatom Phaeodactylum tricornutum

Cited by 15 publications

References 95 publications

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

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

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

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