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The Unfolded Protein Response (UPR) is a retrograde, ER-to-nucleus, signalling pathway which is conserved across kingdoms. In plants, it contributes to development, reproduction, immunity and tolerance to abiotic stress. This RNA sequencing (RNA-seq) dataset was produced from 14-day-old Arabidopsis thaliana seedlings challenged by tunicamycin (Tm), an antibiotic inhibiting Asn-linked glycosylation in the endoplasmic reticulum (ER), causing an ER stress and eventually activating the UPR. Wild-type (WT) and a double mutant deficient for two main actors of the UPR (INOSITOL-REQUIRING ENZYME 1A and INOSITOL-REQUIRING ENZYME 1B) were used as genetic backgrounds in our experimental setup, allowing to distinguish among differentially-expressed genes (DEGs) which ones are dependent on or independent on IRE1s. Also, shoots and roots were harvested separately to determine organ-specific transcriptomic responses to Tm. Library and sequencing were performed using DNBseq™ technology by the Beijing Genomics Institute. Reads were mapped and quantified against the Arabidopsis genome. Differentially-expressed genes were identified using Rflomics upon filtering and normalization by the Trimmed Mean of M-value (TMM) method. While the genotype effect was weak under mock conditions (with a total of 182 DEGs in shoots and 195 DEGs in roots), the tunicamycin effect on each genotype was characterized by several hundred of DEGs in both shoots and roots. Among these genes, 872 and 563 genes were statistically up- and down-regulated in the shoot tissues of the double mutant when compared to those of WT, respectively. In roots of Tm-challenged seedlings, 425 and 439 genes were significantly up- and down-regulated in mutants with respect to WT. We believe that our dataset could be reused for investigating any biological questions linked to ER homeostasis and its role in plant physiology.

The endoplasmic reticulum (ER) is equipped with multiple quality control systems (QCS) that are necessary for shaping the glycoproteome of eukaryotic cells. These systems facilitate the productive folding of glycoproteins, eliminate defective products, and function as effectors to evoke cellular signaling in response to various cellular stresses. These ER functions largely depend on glycans, which contain sugar‐based codes that, when needed, function to recruit carbohydrate‐binding proteins that determine the fate of glycoproteins. To ensure their functionality, the biosynthesis of such glycans is therefore strictly monitored by a system that selectively degrades structurally defective glycans before adding them to proteins. This system, which is referred to as the glycan QCS, serves as a mechanism to reduce the risk of abnormal glycosylation under conditions where glycan biosynthesis is genetically or metabolically stalled. On the other hand, glycan QCS increases the risk of global hypoglycosylation by limiting glycan availability, which can lead to protein misfolding and the activation of unfolded protein response to maintaining cell viability or to initiate cell death programs. This review summarizes the current state of our knowledge of the mechanisms underlying glycan QCS in mammals and its physiological and pathological roles in embryogenesis, tumor progression, and congenital disorders associated with abnormal glycosylation. [ABSTRACT FROM AUTHOR]

Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.

N-glycosylation is one of the most important protein modifications in eukaryotes. It has been well established that N-glycosylation plays multiple roles in regulating stress tolerance of plants. However, the effects and mechanism of N-glycosylation on photosynthesis have not been well understood. In the present study, an obvious decrease in photosynthetic capacity and dry mass were detected in alg3-3 and cgl1-1, two typical mutants in N-glycosylation process. The maximal photochemical efficiency of PSII decreased significantly in cgl1-1. The values of effective quantum yield of PSII photochemistry, rate of photosynthetic electron transport through PSII, and photochemical quenching coefficient, which reflected the photochemical efficiency of plants, decreased as well, while the values of quantum yield of nonregulated energy dissipation of PSII showed obvious enhancement, the similar tendency was also observed in alg3-3. Furthermore, we found that N-glycosylation was also required to maintain the stability of a chloroplast-located protein CAH1, which was closely related to photosynthesis. These results suggest that N-glycosylation plays crucial roles in maintaining photosynthetic efficiency.

Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana 'changed calcium elevation 1' (cce1) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole‐genome resequencing revealed a mutation in asparagine‐linked glycosylation 12 that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α‐1,6 linkage to the dolichol‐PP‐oligosaccharide N‐glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N‐glycosylation is required for proper functioning of client proteins. [ABSTRACT FROM AUTHOR]

N-glycosylation is one of the most important protein modifications in eukaryotes. It has been well established that N-glycosylation plays multiple roles in regulating stress tolerance of plants. However, the effects and mechanism of N-glycosylation on photosynthesis have not been well understood. In the present study, an obvious decrease in photosynthetic capacity and dry mass were detected in alg3-3 and cgl1-1, two typical mutants in N-glycosylation process. The maximal photochemical efficiency of PSII decreased significantly in cgl1-1. The values of effective quantum yield of PSII photochemistry, rate of photosynthetic electron transport through PSII, and photochemical quenching coefficient, which reflected the photochemical efficiency of plants, decreased as well, while the values of quantum yield of nonregulated energy dissipation of PSII showed obvious enhancement, the similar tendency was also observed in alg3-3. Furthermore, we found that N-glycosylation was also required to maintain the stability of a chloroplast-located protein CAH1, which was closely related to photosynthesis. These results suggest that N-glycosylation plays crucial roles in maintaining photosynthetic efficiency. [ABSTRACT FROM AUTHOR]

Given that Cav3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Cav3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Cav3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Cav3.2 in neuroendocrine-like differentiated prostate cancer cells.

The Unfolded Protein Response (UPR) is a retrograde, ER-to-nucleus, signalling pathway which is conserved across kingdoms. In plants, it contributes to development, reproduction, immunity and tolerance to abiotic stress. This RNA sequencing (RNA-seq) dataset was produced from 14-day-old Arabidopsis thaliana seedlings challenged by tunicamycin (Tm), an antibiotic inhibiting Asn-linked glycosylation in the endoplasmic reticulum (ER), causing an ER stress and eventually activating the UPR. Wild-type (WT) and a double mutant deficient for two main actors of the UPR ( INOSITOL-REQUIRING ENZYME 1A and INOSITOL-REQUIRING ENZYME 1B ) were used as genetic backgrounds in our experimental setup, allowing to distinguish among differentially-expressed genes (DEGs) which ones are dependent on or independent on IRE1s. Also, shoots and roots were harvested separately to determine organ-specific transcriptomic responses to Tm. Library and sequencing were performed using DNBseq™ technology by the Beijing Genomics Institute. Reads were mapped and quantified against the Arabidopsis genome. Differentially-expressed genes were identified using Rflomics upon filtering and normalization by the Trimmed Mean of M-value (TMM) method. While the genotype effect was weak under mock conditions (with a total of 182 DEGs in shoots and 195 DEGs in roots), the tunicamycin effect on each genotype was characterized by several hundred of DEGs in both shoots and roots. Among these genes, 872 and 563 genes were statistically up- and down-regulated in the shoot tissues of the double mutant when compared to those of WT, respectively. In roots of Tm-challenged seedlings, 425 and 439 genes were significantly up- and down-regulated in mutants with respect to WT. We believe that our dataset could be reused for investigating any biological questions linked to ER homeostasis and its role in plant physiology., (© 2024 The Author(s).)

A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc2Man9Glc3) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc2Man9 as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc2Man9Glc3 donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway. [ABSTRACT FROM AUTHOR]

Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked ( N -linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N -glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N -glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N -glycoproteins with advantageous biological and biophysical properties.

Asparagine-linked glycosylation of proteins by the oligosaccharyltransferase (OST) occurs when acceptor sites or sequons (N-x?P-T/S) on nascent polypeptides enter the lumen of the rough endoplasmic reticulum. Metazoan organisms assemble two isoforms of the OST that have different catalytic subunits (STT3A or STT3B) and partially non-overlapping cellular roles. Potential glycosylation sites move past the STT3A complex, which is associated with the translocation channel, at the protein synthesis elongation rate. Here, we investigated whether close spacing between acceptor sites in a nascent protein promotes site skipping by the STT3A complex. Biosynthetic analysis of four human glycoproteins revealed that closely spaced sites are efficiently glycosylated by an STT3Bindependent process unless the sequons contain non-optimal sequence features, including extreme close spacing between sequons (e.g. NxTNxT) or the presence of paired NxS sequons (e.g. NxSANxS). Many, but not all, glycosylation sites that are skipped by the STT3A complex can be glycosylated by the STT3B complex. Analysis of a murine glycoprotein database revealed that closely spaced sequons are surprisingly common, and are enriched for paired NxT sites when the gap between sequons is less than three residues. [ABSTRACT FROM AUTHOR]

Highlights: [•] A protocol for recombinant production of PglB, a bacterial oligosaccharyl transferase. [•] PglB was overexpressed in Escherichia coli using high-density autoinduction. [•] An average procedure gives 550-fold purification with 64% yield. [•] Pure enzyme retains full catalytic activity for over two months . [ABSTRACT FROM AUTHOR]

The article presents the findings of a research conducted on the complementation of a defect in the asparagine-linked glycosylation of a mouse FM3A mutant G258 cell line by spheroplast fusion of a human mega YAC cone 923f5. Under the research, the isolation of human genomic DNA transformants of G258 mutant, cell hybridization with X-ray were performed. It concludes that YAC clone 923f5 complemented both defects of the G258 mutant was found through spheroplast fusion.

Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel. [ABSTRACT FROM AUTHOR]

The biogenesis and functionality of pattern recognition receptors (PRRs) are critical for robust plant immune responses. Here, we present methods to determine the N-glycosylation state and ligand-induced activity of these receptors for comparative quantitative analysis. These techniques can be used to identify mutants and chemical inhibitors affecting PRR biogenesis and functionality. When combined, these techniques can provide useful insights on biological processes necessary to synthesize a properly membrane-localized and ligand-responsive PRR.

Given that Ca v 3.2 T-type Ca 2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Ca v 3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Ca v 3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Ca v 3.2 in neuroendocrine-like differentiated prostate cancer cells., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Prostate marker assays are widely used for detection of prostate cancer (PCa) but are associated with considerable sensitivity and specificity problems. Therefore, we investigated prostatic protein glycosylation profiles as a potential biomarker. We determined the urinary asparagine-linked glycan (N-glycan) profile of prostatic proteins of healthy volunteers (n = 25), patients with benign prostate hyperplasia (BPH; n = 62) and newly diagnosed PCa patients (n = 42) using DNA-sequencer-assisted fluorophore-assisted carbohydrate electrophoresis. Through squeezing of the prostate, a sufficient amount of prostatic proteins was obtained for direct structural analyses of N-glycan structures. N-glycans of PCa compared to BPH were characterized by a significant decrease in triantennary structures (p = 0.047) and overall fucosylation (p = 0.026). Prostate-specific antigen (PSA) and the urinary glycoprofile marker showed comparable overall receiver operating characteristic curve analysis as well as in the diagnostic gray zone with serum PSA values between 4 and 10 μg/L. However, when combining PSA and the urinary glycoprofile marker, the latter gave an additive diagnostic value to serum PSA (p ≤ 0.001). In conclusion, N-glycosylation profiling demonstrated differences between BPH and PCa. These changes could lead to the discovery of a new biomarker for PCa., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)