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.

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.

Impact of Pb on Chlamydomonas reinhardtii at Physiological and Transcriptional Levels.

Trace elements stress is one of the most damaging abiotic stresses in environment. Nevertheless, the defense mechanism in microalgae remains poorly understood. In this study, physiological and molecular methods were performed to analyze the defense responses in green alga Chlamydomonas reinhardtii. It was speculated that the defense responses might mainly be due to the regulation of hormone signaling, indicating its potential role in alleviating the Pb toxicity besides other physiological and molecular defense responses like decrease in growth rate, chlorophyll content and photosynthesis efficiency, intensification of antioxidative mechanisms, regulation of transcription factors, trace elements chelation, and sequestration into vacuole via trace elements transporters. The sole differentially expressed ATP-binding cassette (ABC) transporters indicated that ABC transporters might play a very important role in the transport and relocation of Pb in C. reinhardtii. Additionally, our data provide the required knowledge for future investigations regarding Pb toxicity and defense mechanisms in algae, and detection of trace elements pollution in environment.

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.

[Effects on the pharmacokinetics of furosemide after acute exposure to high altitude at 4010 meters in rats].

The paper is to report the pharmacokinetics of furosemide in rats living at plain area and high altitude. After intragastric administration of furosemide (2.87 mg x kg(-1)), serial blood samples (0.5 mL) were collected by retro-orbital puncture at 0, 20 min, 40 min, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h, samples were determined by LC-MS/MS, and plasma concentration-time data were analyzed by DAS 2.0 software to get the related pharmacokinetic parameters. The main pharmacokinetic parameters: area under curve (AUC), mean residence time (MRT), the biological half-life (t1/2) and the peak concentration (C(max)) of furosemide, were significantly increased at high altitude, the time to reach peak concentration (t(max)) and clearance (CL) was significantly decreased. This study found significant changes on the pharmacokinetics of furosemide under the special environment of high altitude. This finding may provide some references for clinical rational application of furosemide at high altitude.

[Synthesis and characterization of thermosensitive hydrogel based on PEG-PCL-PEG block copolymers].

In this paper, a series of low-molecular-weight PEG-PCL-PEG triblock copolymers were successfully synthesized by ring-opening polymerization method, and were characterized using 1H-NMR and FTIR. The aqueous solution displayed specific thermosensitive gel-sol transition when the concentration was above corresponding critical gel concentration (CGC). The gel-sol phase diagram was recorded using test tube-inverting method, which was depended on the hydrophilic/hydrophobic balance in macromolecular structure, as well as heating history. As a result, the gel-sol transition temperature range could be altered, which might be very useful for its application as injectable drug delivery system.