Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 μg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

Chen J ，Du H ，Liu Z ，Li T ，Du H ，Wang W ，Aslam M ，Chen W ，Li P ，Luo H ，Fang H ，Liu X ... -  《Microbial Cell Factories》

Endoplasmic Reticulum-Mediated Protein Quality Control and Endoplasmic Reticulum-Associated Degradation Pathway Explain the Reduction of N-glycoprotein Level Under the Lead Stress.

Different anthropogenic activities result in the continuous increase of metal lead (Pb) in the environment and adversely affect living organisms. Therefore, it is important to investigate the tolerance mechanism in a model organism. Chlamydomonas reinhardtii is an important green eukaryotic model microalga for studying different kinds of biological questions. In this study, the responses of C. reinhardtii were revealed via a comprehensive approach, including physiological, genomic, transcriptomic, glycomic, and bioinformatic techniques. Physiological results showed that the growth rate and soluble protein content were significantly reduced under the high lead stress. Also, the results obtained from the genomic and transcriptomic analyses presented that the endoplasmic reticulum-mediated protein quality control (ERQC) system and endoplasmic reticulum-associated degradation (ERAD) pathway were activated under the third day of high lead stress. The unique upregulated protein disulfide isomerase genes on the ERQC system were proposed to be important for the protein level and protein quality control. The accumulation of specific N-glycans indicated that specific N-glycosylation of proteins might alter the biological functions of proteins to alleviate the Pb stress in alga and/or lead to the degradation of incomplete/misfolded proteins. At the same time, it was observed that genes involved in each process of ERAD were upregulated, suggesting that the ERAD pathway was activated to assist the degradation of incomplete/misfolded proteins. Therefore, it is reasonable to speculate that the reduction of protein level under the high lead stress was related to the activated ERQC system and QRAD pathway. Our findings will provide a solid and reliable foundation and a proposed ERAD working model for further in-depth study of the ERQC system and ERAD pathway under the Pb stress and even other biotic and abiotic stresses.

Du H ，Zheng C ，Aslam M ，Xie X ，Wang W ，Yang Y ，Liu X ... -  《Frontiers in Plant Science》

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》

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme.

Baïet B ，Burel C ，Saint-Jean B ，Louvet R ，Menu-Bouaouiche L ，Kiefer-Meyer MC ，Mathieu-Rivet E ，Lefebvre T ，Castel H ，Carlier A ，Cadoret JP ，Lerouge P ，Bardor M ... -  《-》

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》