Identification of N-glycan oligomannoside isomers in the diatom Phaeodactylum tricornutum.

Microalgae are emerging production systems for recombinant proteins like monoclonal antibodies. In this context, the characterization of the host cell N-glycosylation machinery and of the microalgae-made biopharmaceuticals, which are mainly glycoprotein-based products, requires efficient analytical methodologies dedicated to the profiling of the N-glycans. Herein, in order to gain knowledge regarding its N-glycosylation pathway, we profile the protein N-linked oligosaccharides isolated from the diatom Phaeodactylum tricornutum that has been used successfully to produce functional monoclonal antibodies. The combination of ion mobility spectrometry-mass Spectrometry and electrospray ionization-multistage tandem mass spectrometry allows us to decipher the detailed structure of the oligomannoside isomers and to demonstrate that the processing of the oligomannosides N-linked to proteins occurs in this diatom as reported in mammals. Therefore, P. tricornutum synthesizes human-like oligomannosides in contrast to other microalgae species. This represent an advantage as an alternative ecofriendly expression system to produce biopharmaceuticals used for human therapy.

Dumontier R ，Loutelier-Bourhis C ，Walet-Balieu ML ，Burel C ，Mareck A ，Afonso C ，Lerouge P ，Bardor M ... -  《-》

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

Vanier G ，Hempel F ，Chan P ，Rodamer M ，Vaudry D ，Maier UG ，Lerouge P ，Bardor M ... -  《PLoS One》

User-friendly extraction and multistage tandem mass spectrometry based analysis of lipid-linked oligosaccharides in microalgae.

Lucas PL ，Dumontier R ，Loutelier-Bourhis C ，Mareck A ，Afonso C ，Lerouge P ，Mati-Baouche N ，Bardor M ... -  《Plant Methods》

Golgi fucosyltransferase 1 reveals its important role in α-1,4-fucose modification of N-glycan in CRISPR/Cas9 diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.

Xie X ，Yang J ，Du H ，Chen J ，Sanganyado E ，Gong Y ，Du H ，Chen W ，Liu Z ，Liu X ... -  《Microbial Cell Factories》

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

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.

Xie X ，Du H ，Chen J ，Aslam M ，Wang W ，Chen W ，Li P ，Du H ，Liu X ... -  《Frontiers in Plant Science》