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

2021 ◽  
Vol 12 ◽  
Author(s):  
Xihui Xie ◽  
Hong Du ◽  
Jichen Chen ◽  
Muhammad Aslam ◽  
Wanna Wang ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Metabolic Pathways ◽  
Phaeodactylum Tricornutum ◽  
Glucose Residue ◽  
Clinical Therapeutics ◽  
Glcnac Residue ◽  
Hybrid Type ◽  
High Mannose ◽  
Glycosylation Pathway

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.

scholarly journals Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum

Marine Drugs ◽  
2018 ◽  
Vol 16 (8) ◽  
pp. 272 ◽  
Author(s):  
Zhiqian Yi ◽  
Yixi Su ◽  
Maonian Xu ◽  
Andreas Bergmann ◽  
Saevar Ingthorsson ◽  
...  
Keyword(s):  
High Throughput ◽  
Metabolic Pathways ◽  
Phaeodactylum Tricornutum ◽  
Mass Spectrometry Data ◽  
Marine Diatom ◽  
Chemical Mutagenesis ◽  
Carotenoid Content ◽  
Enzymatic Reactions ◽  
Wild Type ◽  
A Genome

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

scholarly journals In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

2004 ◽  
Vol 70 (5) ◽  
pp. 2639-2646 ◽  
Author(s):  
Wouter Vervecken ◽  
Vladimir Kaigorodov ◽  
Nico Callewaert ◽  
Steven Geysens ◽  
Kristof De Vusser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Golgi Apparatus ◽  
Downstream Processing ◽  
Human Cells ◽  
Oligosaccharide Structure ◽  
Hybrid Type ◽  
Glycosylation Pathway ◽  
Expression Strain ◽  
Terminal Mannose

ABSTRACT The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.

scholarly journals Structural Features ofN-Glycans Linked to Royal Jelly Glycoproteins: Structures of high-mannose type, hybrid type, and biantennary type glycans

2000 ◽  
Vol 64 (10) ◽  
pp. 2109-2120 ◽  
Author(s):  
Yoshinobu KIMURA ◽  
Chiyoko MIYAGI ◽  
Mariko KIMURA ◽  
Teruhiko NITODA ◽  
Nobuyuki KAWAI ◽  
...  
Keyword(s):  
Royal Jelly ◽  
Structural Features ◽  
Hybrid Type ◽  
High Mannose

Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin

1981 ◽  
Vol 669 (2) ◽  
pp. 216-221 ◽  
Author(s):  
Hideko Ishihara ◽  
Noriko Takahashi ◽  
Jinichi Ito ◽  
Eriko Takeuchi ◽  
Setsuzo Tejima
Keyword(s):  
Hybrid Type ◽  
High Mannose ◽  
Asparagine Residue

scholarly journals Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride CotransporterSlc12a2

2015 ◽  
Vol 2015 ◽  
pp. 1-20 ◽  
Author(s):  
Richa Singh ◽  
Mohammed Mashari Almutairi ◽  
Romario Pacheco-Andrade ◽  
Mohamed Y. Mahmoud Almiahuob ◽  
Mauricio Di Fulvio
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Basolateral Membrane ◽  
Transport Function ◽  
Ion Regulation ◽  
Hybrid Type ◽  
Membrane Complex ◽  
Polarized Cells ◽  
Intracellular Compartments ◽  
High Mannose

The Na+K+2Cl−cotransporter-1 (Slc12a2, NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates in the plasma membrane of all cells studied, particularly in the basolateral membrane of most polarized cells. Although the mechanisms involved in plasma membrane sorting of NKCC1 are poorly understood, it is assumed that N-glycosylation is necessary. Here, we characterize expression, N-glycosylation, and distribution of NKCC1 in COS7 cells. We show that ~25% of NKCC1 is complex N-glycosylated whereas the rest of it corresponds to core/high-mannose and hybrid-type N-glycosylated forms. Further, ~10% of NKCC1 reaches the plasma membrane, mostly as core/high-mannose type, whereas ~90% of NKCC1 is distributed in defined intracellular compartments. In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function. Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function. Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

scholarly journals N-glycosylation in sugarcane

2001 ◽  
Vol 24 (1-4) ◽  
pp. 231-234 ◽  
Author(s):  
Ivan G. Maia ◽  
Adilson Leite
Keyword(s):  
Posttranslational Modification ◽  
Protein Function ◽  
Biosynthetic Pathway ◽  
Expressed Sequence Tag ◽  
Sequence Similarity ◽  
Genetic Regulation ◽  
Gene Products ◽  
Significant Sequence Similarity ◽  
Glycosylation Pathway ◽  
Expressed Sequence

The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST) project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST) clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

scholarly journals Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat α1-proteinase inhibitor and α1-acid glycoprotein

1986 ◽  
Vol 236 (3) ◽  
pp. 853-860 ◽  
Author(s):  
V Gross ◽  
T A Tran-Thi ◽  
R T Schwarz ◽  
A D Elbein ◽  
K Decker ◽  
...  
Keyword(s):  
Proteinase Inhibitor ◽  
De Novo ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Complex Type ◽  
Glucose Residue ◽  
Acid Glycoprotein ◽  
Glucosidase Inhibitors ◽  
Oligosaccharide Processing ◽  
High Mannose

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.

scholarly journals AglJ Adds the First Sugar of the N-Linked Pentasaccharide Decorating the Haloferax volcanii S-Layer Glycoprotein

2010 ◽  
Vol 192 (21) ◽  
pp. 5572-5579 ◽  
Author(s):  
Lina Kaminski ◽  
Mehtap Abu-Qarn ◽  
Ziqiang Guan ◽  
Shai Naparstek ◽  
Valeria V. Ventura ◽  
...  
Keyword(s):  
Surface Layer ◽  
Posttranslational Modification ◽  
Model System ◽  
Mass Spectrometric ◽  
Haloferax Volcanii ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
System A ◽  
Genes Encoding ◽  
Glycosylation Pathway

ABSTRACT Like the Eukarya and Bacteria, the Archaea also perform N glycosylation. Using the haloarchaeon Haloferax volcanii as a model system, a series of Agl proteins involved in the archaeal version of this posttranslational modification has been identified. In the present study, the participation of HVO_1517 in N glycosylation was considered, given its homology to a known component of the eukaryal N-glycosylation pathway and because of the genomic proximity of HVO_1517 to agl genes encoding known elements of the H. volcanii N-glycosylation process. By combining the deletion of HVO_1517 with mass spectrometric analysis of both dolichol phosphate monosaccharide-charged carriers and the S-layer glycoprotein, evidence was obtained showing the participation of HVO_1517, renamed AglJ, in adding the first hexose of the N-linked pentasaccharide decorating this reporter glycoprotein. The deletion of aglJ, however, did not fully prevent the attachment of a hexose residue to the S-layer glycoprotein. Moreover, in the absence of AglJ, the level of only one of the three monosaccharide-charged dolichol phosphate carriers detected in the cell was reduced. Nonetheless, in cells lacking AglJ, no further sugar subunits were added to the remaining monosaccharide-charged dolichol phosphate carriers or to the monosaccharide-modified S-layer glycoprotein, pointing to the importance of the sugar added through the actions of AglJ for proper N glycosylation. Finally, while aglJ can be deleted, H. volcanii surface layer integrity is compromised in the absence of the encoded protein.

Remodeling of the glycosylation pathway in the methylotrophic yeast Hansenula polymorpha to produce human hybrid-type N-glycans

2012 ◽  
Vol 50 (2) ◽  
pp. 341-348 ◽  
Author(s):  
Seon Ah Cheon ◽  
Hyunah Kim ◽  
Doo-Byoung Oh ◽  
Ohsuk Kwon ◽  
Hyun Ah Kang
Keyword(s):  
Hansenula Polymorpha ◽  
Methylotrophic Yeast ◽  
Hybrid Type ◽  
Glycosylation Pathway

scholarly journals Histone lysine methacrylation is a dynamic post-translational modification regulated by HAT1 and SIRT2

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Kyle Delaney ◽  
Minjia Tan ◽  
Zhesi Zhu ◽  
Jinjun Gao ◽  
Lunzhi Dai ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Metabolic Pathways ◽  
Histone Mark ◽  
Structural Isomer ◽  
Post Translational Modification ◽  
Site Specific ◽  
Affinity Enrichment ◽  
Histone Lysine ◽  
Biochemical Studies ◽  
New Type

AbstractHistone lysine crotonylation is a posttranslational modification with demonstrated functions in transcriptional regulation. Here we report the discovery of a new type of histone posttranslational modification, lysine methacrylation (Kmea), corresponding to a structural isomer of crotonyllysine. We validate the identity of this modification using diverse chemical approaches and further confirm the occurrence of this type of histone mark by pan specific and site-specific anti-methacryllysine antibodies. In total, we identify 27 Kmea modified histone sites in HeLa cells using affinity enrichment with a pan Kmea antibody and mass spectrometry. Subsequent biochemical studies show that histone Kmea is a dynamic mark, which is controlled by HAT1 as a methacryltransferase and SIRT2 as a de-methacrylase. Altogether, these investigations uncover a new type of enzyme-catalyzed histone modification and suggest that methacrylyl-CoA generating metabolism is part of a growing number of epigenome-associated metabolic pathways.

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