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• Ammonium (NH₄⁺) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH₄⁺ toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. • Here, RNA-seq analysis was performed on Arabidopsis roots to screen for ammonium-responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH₄⁺, was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole-3-acetic acid (IAA)-conjugating genes by WRKY46 via yeast one-hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative real-time polymerase chain reaction (ChIP-qPCR). • Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH₄⁺-suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA-conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N-glycosylation, leading to an inhibition of NH₄⁺ efflux in the root elongation zone (EZ). • We identify TF involvement in the regulation of NH₄⁺ efflux in the EZ, and show that WRKY46 inhibits NH₄⁺ efflux by negative regulation of NUDX9 and IAA-conjugating genes.

The Campylobacter jejuni pgl locus encodes an N ‐linked protein glycosylation machinery that can be functionally transferred into Escherichia coli . In this system, we analyzed the elements in the C. jejuni N ‐glycoprotein AcrA required for accepting an N ‐glycan. We found that the eukaryotic primary consensus sequence for N ‐glycosylation is N terminally extended to D/E‐Y‐N‐X‐S/T (Y, X≠P) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N ‐glycosylated when they were either artificially introduced or when they were present in non‐ C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N ‐glycosylation sites and confirmed the requirement for a negatively charged side chain at position −2 in C. jejuni N ‐glycoproteins. N ‐glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the −2 position. Thus, bacterial N ‐glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.

Age‐related declines in oocyte quality and ovarian function are pivotal contributors to female subfertility in clinical settings. Yet, the mechanisms driving ovarian aging and oocyte senescence remain inadequately understood. The present study evaluated the alterations in N‐glycoproteins associated with ovarian aging and noted a pronounced elevation in N221 glycopeptides of cathepsin L (Ctsl) in the ovaries of reproductive‐aged mice (8–9 months and 11–12 months) compared to younger counterparts (6–8 weeks). Subsequent analysis examined the involvement of Ctsl in oocyte aging and demonstrated a significant elevation in Ctsl levels in aged oocytes. Further, it was revealed that the overexpression of Ctsl in young oocytes substantially diminished their quality, while oocytes expressing an N221‐glycosylation mutant of Ctsl did not suffer similar quality degradation. This finding implies that the N221 glycosylation of Ctsl is pivotal in modulating its effect on oocyte health. The introduction of a Ctsl inhibitor into the culture medium restored oocyte quality in aged oocytes by enhancing mitochondrial function, reducing accumulated reactive oxygen species (ROS), lowering apoptosis, and recovering lysosome capacity. Furthermore, the targeted downregulation of Ctsl using siRNA microinjection in aged oocytes enhanced fertilization capability and blastocyst formation, affirming the role of Ctsl knockdown in fostering oocyte quality and embryonic developmental potential. In conclusion, these findings underscore the detrimental effects of high expression of N‐glycosylated Ctsl on oocyte quality and its contribution to oocyte senescence, highlighting it as a potential therapeutic target to delay ovarian aging and enhance oocyte viability. Ctsl is a protein synthesized in the cytoplasm of oocytes, where it undergoes N‐glycosylation at the N221 site. In the process of oocyte aging, there is a notable accumulation of N‐glycosylated Ctsl, which adversely impacts oocyte quality by impairing mitochondrial function, increasing reactive oxygen species (ROS) production, promoting apoptosis, and compromising lysosomal capacity. These alterations ultimately have detrimental effects on meiotic maturation and embryonic development.

Aims/hypothesis We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes. Methods A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples. Results This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3 , MGAT5 and ST6GAL1 , encoding beta-1,4-mannosyl-glycoprotein 4-beta- N -acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta- N -acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3 ; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N -acetylglucosamine (GlcNAc) ( p discovery =7.65 × 10 −12 , p replication =8.33 × 10 −6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc ( p discovery =2.88 × 10 −10 , p replication =3.03 × 10 −3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3 . Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene ( C3 ) showed association with the oligomannose plasma protein N-glycan ( p discovery =2.43 × 10 −11 , p replication =8.66 × 10 −4 for the top associated SNP rs1047286). Conclusions/interpretation This study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility. Graphical abstract

The Golgi apparatus, a crucial organelle involved in protein processing, including glycosylation, exhibits complex sub-structures, i.e., cis-, medial, and trans-cisternae. This study investigated the distribution of glycosyltransferases within the Golgi apparatus of mammalian cells via 3D super-resolution imaging. Focusing on human glycosyltransferases involved in N-glycan modification, we found that even enzymes presumed to coexist in the same Golgi compartment exhibit nuanced variations in localization. By artificially making their N-terminal regions [composed of a cytoplasmic, transmembrane, and stem segment (CTS)] identical, it was possible to enhance the degree of their colocalization, suggesting the decisive role of this region in determining the sub-Golgi localization of enzymes. Ultimately, this study reveals the molecular codes within CTS regions as key determinants of glycosyltransferase localization, providing insights into precise control over the positioning of glycosyltransferases, and consequently, the interactions between glycosyltransferases and substrate glycoproteins as cargoes in the secretory pathway. This study advances our understanding of Golgi organization and opens avenues for programming the glycosylation of proteins for clinical applications.Key words: Golgi apparatus, glycosyltransferase, 3D super-resolution imaging, N-glycosylation

Sedentary life style is considered to be an independent risk factor for many disorders, including development of type 2 diabetes, obesity, immune dysfunction, asthma, and neurological or coronary heart disease. Irisin is released from myocytes during physical activity, and acts as a link between muscles and other tissues and organs. This myokine is produced as a result of proteolytic cleavage of FNDC5 protein present in the membrane of myocytes. Secretion of irisin is regulated by N-linked oligosaccharides attached to the protein molecule. The two N-glycan molecules, which constitute a significant part of the irisin glycoprotein, regulate the browning of adipocytes, which is the most important function of irisin. A receptor specific for irisin has still not been discovered. In some tissues irisin probably acts via integrins, which are widely expressed transmembrane receptors. Many studies have confirmed the multifunctional role of irisin and the beneficial effects of this molecule on body homeostasis. Irisin reduces systemic inflammation, maintains the balance between resorption and bone formation, and modulates metabolic processes and the functioning of the nervous system. It suppresses the expression and release of pro-inflammatory cytokines in obese individuals and attenuates inflammation in adipose tissue. The impact of irisin on cancer cell proliferation, migration, and invasion has also been demonstrated in numerous studies, which proves its role in carcinogenesis. Owing to these pleiotropic and beneficial properties, irisin may be a potential option to prevent and treat civilization-related diseases which are, nowadays, considered to be the major health problems in Western societies.

Summary N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.

Plant pathogens infect hosts through sophisticated molecular strategies, with N‐glycosylation serving as a critical post‐translational modification that regulates their virulence. This review comprehensively summarises and discusses the role of N‐glycosylation in regulating potential pathogenic mechanisms, including fungal growth and development, cell wall integrity, infection structures (e.g., appressoria, invasive hyphae) and the secretion of effector proteins. By ensuring proper protein folding, stability and interaction with host defences, N‐glycosylation enables pathogens to evade plant immune recognition, such as pathogen‐associated molecular pattern (PAMP)‐triggered immunity, and establish successful infections. Advances in glycomic techniques and proteomics offer tools to dissect these mechanisms with enhanced precision. Future research directions regarding N‐glycosylation in plant pathogens should emphasise the development of small‐molecule targeted drugs against pathogens and the creation of enzyme inhibitors to disrupt virulence factors. This work provides a perspective for further study regarding N‐glycosylation in the plant‐pathogenic mechanisms and effective disease control. The potential pathogenic mechanisms related to growth and development, infection structures, and the secretion of effector proteins influenced by N‐glycosylation modifications are described in detail.

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N -glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N -glycosylation profile determined at the released glycan level.

Chronic inflammation is the main feature of many long-term inflammatory diseases such as autoimmune diseases, metabolic disorders, and cancer. There is a growing number of studies in which alterations of N-glycosylation have been observed in many pathophysiological conditions, yet studies of the underlying mechanisms that precede N-glycome changes are still sparse. Proinflammatory cytokines have been shown to alter the substrate synthesis pathways as well as the expression of glycosyltransferases required for the biosynthesis of N-glycans. The resulting N-glycosylation changes can further contribute to disease pathogenesis through modulation of various aspects of immune cell processes, including those relevant to pathogen recognition and fine-tuning the inflammatory response. This review summarizes our current knowledge of inflammation-induced N-glycosylation changes, with a particular focus on specific subsets of immune cells of innate and adaptive immunity and how these changes affect their effector functions, cell interactions, and signal transduction.