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Simplified scheme of the N-linked glycosylation pathway in the endoplasmic reticulum and the Golgi apparatus. The N-linked glycosylation pathway is shown in a simplified scheme. The mRNA is being attached and translated at the membrane of the endoplasmic reticulum (I), and the glycan is attached by the oligosaccharyltransferase (OST) (II) and trimmed by α-glucosidase II (GC-II) (III). The protein enters the folding control (dashed box) (IV). Calreticulin and UDP-glucose glucosyltransferase (UGGT) are the main component of the system. Correctly folded proteins are further transported to the Golgi apparatus (V), and N-linked glycan structures are further processed by specific enzymes (VI). The following abbreviations are used: alpha-mannosidase (α-Man), N-acetylglucosaminyltransferase (GnTI), 1,3 fucosyltransferase ((1,3)-FucT), galactosyltransferase (GalTI). In grey are two enzymes as examples of missing proteins that are involved in glycan maturation in other organisms. Components of the glycan structure are indicated with blue being dolichol, yellow is phosphate, blue spheres are glucose residues, green spheres are mannose residues, and blue squares are N-acetylglucosamines.
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N-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Tha...
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... the outermost glucose onto the LLO (Fig. 1, highlighted in red). Our in-silico search revealed a putative flippase, potentially involved in turning the glycan structure from the outside of the endoplasmic reticulum to the inside 27 . The analysis also showed that T. oceanica possesses an OST to attach the glycan structure to the NXT/S motif (Fig. 2). We identified calreticulin in the T. oceanica proteome as a putative chaperone, mediating the protein folding control in the ER 28 (Table 1). Correctly folded proteins enter the Golgi apparatus, and the glycan structure is modified by a mannosidase (α-ManI) 29 , with further modifications are either GnT1-dependant or GnT1-independent ...
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... 46109 ( Mito undef 12 18 C. reinhardtii* 90 Proteins B) 16 and C. reinhardtii 15 . We analyzed the peptides from both studies with respect to the type of motif (NXT; NXS) and compared these findings to our data from T.oceanica. Shown are the total motifs found in each study and the percent of NXT and NXS motifs. www.nature.com/scientificreports/ (Fig. 2) 4 . Based on the presence of GnT1 in T. oceanica, both pathways are possible. In addition to GnT1, we identified a fucosyltransferase ((1,3)-FucT) and a galactosylase (β1,4-GalT) that can alter the glycan structure in the Golgi apparatus ...
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... In order to analyze changes in the secretory pathway, four transcripts for enzymes involved in N-linked glycosylation were analyzed. High-iron and low-iron reads of these transcripts were included in a previous publication on N-linked glycosylation in microalgae [33]. The transcripts included OST, calreticulin, Gnt1, and UGGT. ...
... The experimental design was followed as described elsewhere [33]. Briefly, Thalassiosira oceanica cultures were kept in exponential growth, and sampling was done in low-to mid-exponential phase. ...
In large areas of the ocean, iron concentrations are insufficient to promote phytoplankton growth. Numerous studies have been conducted to characterize the effect of iron on algae and how algae cope with fluctuating iron concentrations. Fertilization experiments in low-iron areas resulted primarily in diatom-dominated algal blooms, leading to laboratory studies on diatoms comparing low- and high-iron conditions. Here, we focus on the short-term temporal response following iron addition to an iron-starved open ocean diatom, Thalassiosira oceanica . We employed the NanoString platform and analyzed a high-resolution time series on 54 transcripts encoding proteins involved in photosynthesis, N-linked glycosylation, iron transport, as well as transcription factors. Nine transcripts were iron-responsive, with an immediate response to the addition of iron. The fastest response observed was the decrease in transcript levels of proteins involved in iron uptake, followed by an increase in transcript levels of iron-containing enzymes and a simultaneous decrease in the transcript levels of their iron-free replacement enzymes. The transcription inhibitor actinomycin D was used to understand the underlying mechanisms of the decrease of the iron-responsive transcripts and to determine their half-lives. Here, Mn-superoxide dismutase (Mn-SOD), plastocyanin (PETE), ferredoxin (PETF) and cellular repressor of EA1-stimulated genes (CREGx2) revealed longer than average half-lives. Four iron-responsive transcripts showed statistically significant differences in their decay rates between the iron-recovery samples and the actD treatment. These differences suggest regulatory mechanisms influencing gene transcription and mRNA stability. Overall, our study contributes towards a detailed understanding of diatom cell biology in the context of iron fertilization response and provides important observations to assess oceanic diatom responses following sudden changes in iron concentrations.
... Peptide-N-glycosidase F catalyzes the deglycosylation of most N-glycans and cleaves 9-highmannose, hybrid, and complex monosaccharide chains but has not been identified for O-glycan (80,81). Hence, N-linked glycan is preferred in several technologies of protein modification and biopharmaceutical functioning (82,83). Recent strategies of Nlinked glycan can be classified into site-directed mutagenesis and glycosylated linker. ...
Over the last 20 years, granulocyte colony-stimulating factors (G-CSFs) have become the major therapeutic option for the treatment of patients with neutropenia. Most of the current G-CSFs require daily injections, which are inconvenient and expensive for patients. Increased understanding of G-CSFs’ structure, expression, and mechanism of clearance has been very instrumental in the development of new generations of long-acting G-CSFs with improved efficacy. Several approaches to reducing G-CSF clearance via conjugation techniques have been investigated. PEGylation, glycosylation, polysialylation, or conjugation with immunoglobulins or albumins have successfully increased G-CSFs’ half-lives. Pegfilgrastim (Neulasta) has been successfully approved and marketed for the treatment of patients with neutropenia. The rapidly expanding market for G-CSFs has increased demand for G-CSF biosimilars. Therefore, the importance of this review is to highlight the principle, elimination’s route, half-life, clearance, safety, benefits, and limitations of different strategies and techniques used to increase the half-life of biotherapeutic G-CSFs. Understanding these strategies will allow for a new treatment with more competitive manufacturing and lower unit costs compared with that of Neulasta.
... In this study, N-glycopeptide enrichment combined with Q-Exactive identification technology was used for the first time to identify N-glycosylation sites and glycosylated proteins in mink testis to determine the effect of N-glycoprotein modification on mink spermatogenesis. To generate a universal tag for MS analysis, the enriched peptides were treated with PNGase F in H 18 2 O to remove N-glycans, convert asparagine (Asn) to heavy-oxygen aspartic acid (Asp) and cause a mass shift of + 2.9890 Da for glycosylation site identification by MS (18)(19)(20). This strategy is similar to previous proteolytic stable isotope labeling technique in which trypsin-digested peptides and individual proteins were labeled with heavy-oxygen water for the quantitative analysis of two proteome samples (19,21). ...
Spermatogenesis in the American mink is characterized by an annual cycle of transition involving completely inactive and fully activated stages. N-glycosylation of proteins has emerged as an important regulator as it affects protein folding, secretion, degradation, and activity. However, the function of protein N-glycosylation in seasonal spermatogenesis of the American mink remains unclear. In the present study, we established a proteome-wide stoichiometry of N-glycosylation in mink testes at various phases of spermatogenesis using N-linked glycosylated-peptide enrichment in combination with liquid chromatography-tandem mass spectrometry analysis. A total of 532 N-glycosylated sites matching the canonical Asn-X-Ser/Thr motif were identified in 357 testicular proteins. Both the number of glycoproteins and the sites of N-glycosylated proteins in mink testes were highly dynamic at different stages. Functional analyses showed that testicular proteins with different N-glycosylation might play a vital role in spermatogenesis by affecting their folding, distribution, stability, and activity. Overall, our data suggest that the dynamics of N-glycosylation of testicular proteins are involved in seasonal spermatogenesis in the American mink.
... This comparative study revealed that N-glycosylation pathway of proteins and N-glycan structures are species-dependent. To get more reliable data, high throughput N-glycoproteomics and N-glycomics analysis have been considered to study the N-glycoproteins and N-glycans in C. reinhardtii (Mathieurivet et al., 2013), B. braunii (Schulze et al., 2017), and Thalassiosira oceanica (Behnke et al., 2021). However, the information regarding the presence of total N-glycoproteins and N-glycans in the model diatom, i.e., P. tricornutum still needs to be gathered. ...
... This approach has been used in limited studies so far. So far, the N-glycoproteomic analysis was performed in three green algae C. reinhardtii (Mathieu-rivet et al., 2013), B. braunii (Schulze et al., 2017), and diatom T. oceanica (Behnke et al., 2021). Compared with the identified 135 N-glycopeptides in C. reinhardtii, total 517 unique N-glycosylated peptides in three strains of B. braunii and 118 N-linked glycosylated peptides in T. oceanica, 863 different N-glycopeptides corresponding to 639 proteins were identified in this study in of P. tricornutum. ...
... In C. reinhardtii and three B. braunii strains, the enrichments of N-glycopeptides were performed by using agarose-bound concanavalin A lectin, and the analysis methods of N-glycopeptides were PNGase F/ 18 O-method and in-source collision induced dissociation (IS-CID) methods (Mathieu-rivet et al., 2013) and LC-MS (Schulze et al., 2017), respectively. Furthermore, solid-phase extraction, hydrophilic-lipophilic balance (HLB) cartridge enrichment, LC-MS/MS analysis were carried out in the N-glycopeptide analysis of T. oceanica (Behnke et al., 2021). These enrichment methods are N-glycan-specific, especially the lectin enrichment, while HILIC microcolumn was considered in the current to get more enriched N-glycopeptides. ...
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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Plant development represents a continuous process in which the plant undergoes morphological, (epi)genetic and metabolic changes. Starting from pollination, seed maturation and germination, the plant continues to grow and develops specialized organs to survive, thrive and generate offspring. The development of plants and the interplay with its environment are highly linked to glycosylation of proteins and lipids as well as metabolism and signaling of sugars. Although the involvement of these protein modifications and sugars is well-studied, there is still a long road ahead to profoundly comprehend their nature, significance, importance for plant development and the interplay with stress responses. This review, approached from the plants’ perspective, aims to focus on some key findings highlighting the importance of glycosylation and sugar signaling for plant development.
The marine nitrogen cycle controls oceanic productivity through enzymatic processes mediated by microbes. Here, we report the construction, evaluation, and application of the OceansN CodeSet for the NanoString nCounter, which quantifies a suite of protein‐coding genes that are central to microbially mediated nitrogen cycle processes in the ocean. We also placed emphasis on quantifying a diverse set of marine diazotrophs within known nifH phylogenetic clades. The OceansN CodeSet provided direct hybridization‐based quantitation of 48 probes in a single sample, presenting advantages in terms of reduced sample handling, elimination of amplification bias, minimal DNA sample requirements, and the ability to assess targets ranging from relatively rare to abundant, with a reliable quantitation limit of ~ 1000 gene copies per target per sample. As such, our approach fills a unique methodological niche between the scale of high‐throughput amplicon sequencing (a compositional method) and quantitative polymerase chain reaction (qPCR) (a targeted method with generally lower throughput). When applied to North Atlantic environmental DNA samples, the OceansN CodeSet revealed temporal and spatial patterns in nitrogen assimilation, nitrification, and denitrification, as well as the abundance and distribution of various nitrogen‐fixing microorganisms (diazotrophs). Data from the nCounter was validated via internal and external controls, and by comparison to qPCR, nifH amplicon sequencing, and shotgun metagenomic sequencing.
N-glycosylation is a common post-translational modification (PTM) in plants that possesses important biological functions including protein folding, protein quality control, protein stability and protein-protein interactions. Nowadays, the interest in analysis of glycoproteins using mass spectrometry is increasing because of advances in instrumentation and automatic data analysis tools. Here, we describe the progresses in mass spectrometry-based N-glycomics and N-glycoproteomics in plants. We also provide a comprehensive summary of N-glycan structures in plants according to the biosynthetic rules and up-to-date reports, which serves as a theoretical database for all N-glycan-related search engines.
Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global human health. However, the effect of SARS-CoV-2 infection on the overall serum N-glycomic profile has been largely unexplored. Here, we extended our 96-well-plate-based high-throughput, high-sensitivity N-glycan profiling platform further with the aim of elucidating potential COVID-19-associated serum N-glycomic alterations. Use of this platform revealed both similarities and differences between the serum N-glycomic fingerprints of COVID-19 positive and control cohorts. Although there were no specific glycan peaks exclusively present or absent in COVID-19 positive cohort, this cohort showed significantly higher levels of glycans and variability. On the contrary, the overall N-glycomic profiles for healthy controls were well-contained within a narrow range. From the serum glycomic analysis, we were able to deduce changes in different glycan subclasses sharing certain structural features. Of significance was the hyperbranched and hypersialylated glycans and their derived glycan subclass traits. T-distributed stochastic neighbour embedding (tSNE) and hierarchical heatmap clustering analysis were performed to identify 13 serum glycomic variables that potentially distinguished the COVID-19 positive from healthy controls. Such serum N-glycomic changes described herein may indicate or correlate to the changes in serum glycoproteins upon COVID-19 infection. Furthermore, mapping the serum N-glycome following SARS-CoV-2 infection may help us better understand the disease and enable “Long-COVID” surveillance to capture the full spectrum of persistent symptoms.
