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Epithelial-to-mesenchymal transition (EMT) gives rise to cells with properties similar to cancer stem cells (CSCs). Targeting the EMT program to selectively eliminate CSCs is a promising way to improve cancer therapy. Salinomycin (Sal), a K+/H+ ionophore, was identified as highly selective towards CSC-like cells, but its mechanism of action and selectivity remains elusive. Here, we show that Sal, similar to monensin and nigericin, disturbs the function of the Golgi. Sal alters the expression of Golgi-related genes and leads to marked changes in Golgi morphology, particularly in cells that have undergone EMT. Moreover, Golgi-disturbing agents severely affect post-translational modifications of proteins, including protein processing, glycosylation and secretion. We discover that the alterations induced by Golgidisturbing agents specifically affect the viability of EMT cells. Collectively, our work reveals a novel vulnerability related to the EMT, suggesting an important role for the Golgi in the EMT and that targeting the Golgi could represent a novel therapeutic approach against CSCs. [ABSTRACT FROM AUTHOR]

Summary: Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N‐glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non‐human residues, is usually not controlled. The presence and composition of N‐glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow‐2 (BY‐2) cell lines through knock out and ectopic expression of genes involved in the N‐glycosylation pathway. Here, we report on the generation of BY‐2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co‐expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY‐2 cell lines producing only high mannose N‐glycans. Endoglycosidase T cleaves high mannose N‐glycans to generate single, asparagine‐linked, N‐acetylglucosamine residues. The N‐glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N‐glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N‐glycosylation sites, was observed. N‐glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N‐glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY‐2 cells expands the set of glycoengineered BY‐2 cell lines. [ABSTRACT FROM AUTHOR]

BACKGROUND: The pathological mechanism of chronic thromboembolic pulmonary hypertension (CTEPH) is not fully understood, and inflammation has been reported to be one of its etiological factors. IgG regulates systemic inflammatory homeostasis, primarily through its N-glycans. Little is known about IgG N-glycosylation in CTEPH. We aimed to map the IgG N-glycome of CTEPH to provide new insights into its pathogenesis and discover novel markers and therapies. METHODS: We characterized the plasma IgG N-glycome of patients with CTEPH in a discovery cohort and validated our results in an independent validation cohort using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Thereafter, we correlated IgG N-glycans with clinical parameters and circulating inflammatory cytokines in patients with CTEPH. Furthermore, we determined IgG N-glycan quantitative trait loci in CTEPH to reveal partial mechanisms underlying glycan changes. RESULTS: Decreased IgG galactosylation representing a proinflammatory phenotype was found in CTEPH. The distribution of IgG galactosylation showed a strong association with NT-proBNP (N-terminal pro-B-type natriuretic peptide) in CTEPH. In line with the glycomic findings, IgG pro-/anti-inflammatory N-glycans correlated well with a series of inflammatory markers and gene loci that have been reported to be involved in the regulation of these glycans or inflammatory immune responses. CONCLUSIONS: This is the first study to reveal the full signature of the IgG N-glycome of a proinflammatory phenotype and the genes involved in its regulation in CTEPH. Plasma IgG galactosylation may be useful for evaluating the inflammatory state in patients with CTEPH; however, this requires further validation. This study improves our understanding of the mechanisms underlying CTEPH inflammation from the perspective of glycomics. [ABSTRACT FROM AUTHOR]

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)-1 or 7.5 ± 0.4 mg L-1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.

The Dolichol kinase (DOLK) gene encodes the polytopic DOLK protein associated with the endoplasmic reticulum (ER) N-glycosylation pathway catalyzing the final step in the biosynthesis of dolichol phosphate. Dolichol phosphate is an oligosaccharide carrier required for N-glycosylation of DOLK protein, with its deficiency leading to a severe hypo glycosylation phenotype in humans which can cause congenital disorders of glycosylation and death in early infancy. The aim of the present study is to identify the phylogenetic relationship between human and ortholog species based on their conserved sequences in DOLK gene. Sequence alignment of DOLK was carried out in this study and the evolutionarily conserved regulatory sequences were identified using bioinformatics. Promoter sequence of human DOLK was compared with orthologous sequences from different organisms. Conserved non-coding sequences (CNS) and motifs in promoter regions were found by analyzing upstream promoter sequences of Homo sapiens DOLK and its orthologous genes in other organisms. Conserved sequences were predicted in the promoter regions in CNS1 and CNS2. Conserved protein sequences were also identified by alignment of the orthologous sequences. Organisms with similar gene sequences are assumed to be closely related and the ER N-glycosylation pathway is conserved in them.

Abstract Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute complex signaling hubs that are crucial for the development and homeostasis of the human body. Most of FGFs are released by cells using the conventional secretory pathway and are N-glycosylated, yet the role of FGFs glycosylation is largely unknown. Here, we identify N-glycans of FGFs as binding sites for a specific set of extracellular lectins, galectins − 1, -3, -7 and − 8. We demonstrate that galectins attract N-glycosylated FGF4 to the cell surface, forming a reservoir of the growth factor in the extracellular matrix. Furthermore, we show that distinct galectins differentially modulate FGF4 signaling and FGF4-dependent cellular processes. Using engineered variants of galectins with altered valency we demonstrate that multivalency of galectins is critical for the adjustment of FGF4 activity. Summarizing, our data reveal a novel regulatory module within FGF signaling, in which the glyco-code in FGFs provides previously unanticipated information differentially deciphered by multivalent galectins, affecting signal transduction and cell physiology. Video Abstract

Abstract Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future. Graphical Abstract

Abstract Background SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect this wide spectrum of disease presentations. Methods Our pilot cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins (APPs) by nephelometry, and full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). Results When compared to controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, α1-acid glycoprotein (AGP), α1-antitrypsin (AAT), ceruloplasmin (CP), haptoglobin (HP), and high-sensitivity C-reactive protein (hs-CRP). The concentrations of α1-antichymotrypsin (ACT), α2-macroglobulin (A2MG) and serum amyloid A (SAA) proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose- and sialylated di-antennary glycans and decreased non-sialylated di-antennary glycan A2G2 in COVID-19 patients compared to controls. Conclusions COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight complexity of inflammatory process and grant further investigations.

Abstract Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application. Graphical Abstract

The scavenger receptor cysteine‐rich (SRCR) domain is a key constituent in diverse proteins. N‐glycosylation is important in protein expression and function. In the SRCR domain of different proteins, N‐glycosylation sites and functionality vary substantially. In this study, we examined the importance of N‐glycosylation site positions in the SRCR domain of hepsin, a type II transmembrane serine protease involved in many pathophysiological processes. We analysed hepsin mutants with alternative N‐glycosylation sites in the SRCR and protease domains using three‐dimensional modelling, site‐directed mutagenesis, HepG2 cell expression, immunostaining, and western blotting. We found that the N‐glycan function in the SRCR domain in promoting hepsin expression and activation on the cell surface cannot be replaced by alternatively created N‐glycans in the protease domain. Within the SRCR domain, the presence of an N‐glycan in a confined surface area was essential for calnexin‐assisted protein folding, endoplasmic reticulum (ER) exiting, and zymogen activation of hepsin on the cell surface. Hepsin mutants with alternative N‐glycosylation sites on the opposite side of the SRCR domain were trapped by ER chaperones, resulting in the activation of the unfolded protein response in HepG2 cells. These results indicate that the spatial N‐glycan positioning in the SRCR domain is a key determinant in the interaction with calnexin and subsequent cell surface expression of hepsin. These findings may help to understand the conservation and functionality of N‐glycosylation sites in the SRCR domains of different proteins. [ABSTRACT FROM AUTHOR]

The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of immune cell receptors. Although knowledge in the field of glycoimmunology is still limited, numerous studies point to the key role of glycosylation in maintaining homeostasis, but also in reflecting its disruption. Changes in oligosaccharide patterns can lead to impairment of both innate and acquired immune responses, with important implications in the pathogenesis of diseases, including autoimmunity. B cells appear to be unique within the immune system, since they exhibit both innate and adaptive immune activity. B cell surface is rich in glycosylated proteins and lectins which recognise glycosylated ligands on other cells. Glycans are important in the development, selection, and maturation of B cells. Changes in sialylation and fucosylation of cell surface proteins affect B cell signal transduction through BCRs, CD22 inhibitory coreceptor and Siglec-G. Plasmocytes, as the final stage of B cell differentiation, produce and secrete immunoglobulins (Igs), of which IgGs are the most abundant N-glycosylated proteins in human serum with the conserved Nglycosylation site at Asn297. N-oligosaccharide composition of the IgG Fc region affects its secretion, structure, half-life and effector functions (ADCC, CDC). IgG N-glycosylation undergoes little change during homeostasis, and may gradually be modified with age and during ongoing inflammatory processes. Hyperactivated B lymphocytes secrete autoreactive antibodies responsible for the development of autoimmunity. The altered profile of IgG N-glycans contributes to disease progression and remission and is sensitive to the application of therapeutic substances and immunosuppressive agents. In this review, we focus on the role of N-glycans in B-cell biology and IgG activity, the rearrangement of IgG oligosaccharides in aging, autoimmunity and immunosuppressive therapy. [ABSTRACT FROM AUTHOR]

T cell‐mediated antitumor immunity is modulated, in part, by N‐glycosylation. However, the interplay between N‐glycosylation and the loss of effector function in exhausted T cells has not yet been fully investigated. Here, we delineated the impact of N‐glycosylation on the exhaustion of tumor‐infiltrating lymphocytes in a murine colon adenocarcinoma model, focusing on the IFN‐γ‐mediated immune response. We found that exhausted CD8+ T cells downregulated the oligosaccharyltransferase complex, which is indispensable for N‐glycan transfer. Concordant N‐glycosylation deficiency in tumor‐infiltrating lymphocytes leads to loss of antitumor immunity. Complementing the oligosaccharyltransferase complex restored IFN‐γ production and alleviated CD8+ T cell exhaustion, resulting in reduced tumor growth. Thus, aberrant glycosylation induced in the tumor microenvironment incapacitates effector CD8+ T cells. Our findings provide insights into CD8+ T cell exhaustion by incorporating N‐glycosylation to understand the characteristic loss of IFN‐γ, opening new opportunities to amend the glycosylation status in cancer immunotherapies. [ABSTRACT FROM AUTHOR]

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics. [ABSTRACT FROM AUTHOR]

In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy. [ABSTRACT FROM AUTHOR]

Degeneration of the intervertebral disc is an age‐related condition. It also accompanies the disappearance of the notochordal cells, which are remnants of the developmental stages of the nucleus pulposus (NP). Molecular changes such as extracellular matrix catabolism, cellular phenotype, and glycosaminoglycan loss in the NP have been extensively studied. However, as one of the most significant co‐ and posttranslational modifications, glycosylation has been overlooked in cells in degeneration. Here, we aim to characterize the N‐glycome of young and mature NP and identify patterns related to aging. Accordingly, we isolated N‐glycans from notochordal cell‐rich NP from porcine discs, characterized them using a combined approach of exoglycosidase digestions and analysis with hydrophilic interaction ultra‐performance liquid chromatography and mass spectrometry. We have assigned over 300 individual N‐glycans for each age group. Moreover, we observed a notable abundance of antennary structures, galactosylation, fucosylation, and sialylation in both age groups. In addition, as indicated from our results, increasing outer arm fucosylation and decreasing α(2,3)‐linked sialylation with aging suggest that these traits are age‐dependent. Lastly, we have focused on an extensive characterization of the N‐glycome of the notochordal cell‐rich NP in aging without inferred degeneration, describing glycosylation changes specific for aging only. Our findings in combination with those of other studies, suggest that the degeneration of the NP does not involve identical processes as aging. [ABSTRACT FROM AUTHOR]

Glycosylation, a prevalent post-translational modification, plays a significant role in diverse biological processes and exhibits associations with various diseases, including cancer. Among the different types of glycosylation, N-glycosylation is particularly prominent. Glycosylation analysis also plays a crucial role in understanding the efficacy of protein biopharmaceuticals, as the majority of these drugs are glycoproteins. The precise detection and characterization of N-glycosylation require a series of enrichment steps. On the other hand, peptide fragmentation methods constitute a crucial step in reducing sample complexity. Moreover, enrichment strategies coupled with mass spectrometry are essential for the analysis and identification of N-glycopeptides. This research aims to enhance the detection methods for N-glycopeptides and N-glycoproteins by employing strategies that involve peptide fractionation and glycopeptide enrichment. This study targeted to assess and compare the efficacy of an integration method and a direct enrichment approach in terms of identifying glycopeptides from the glycoproteome of human plasma. The results demonstrate that the integration method detects 212 N-glycopeptides and 88 N-glycoproteins, while Cotton-HILIC direct enrichment detects 88 N-glycopeptides and 41 N-glycoproteins. In conclusion, the employment of Cotton-HILIC enrichment with high pH fractionation exhibits greater qualitative abilities in characterizing the structures and functions of glycopeptides.

ABSTRACT In order to produce therapeutic glycoproteins modified with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. In this study, a Pichia pastoris mutant Glyco4, which has a humanized N-glycosylation pathway and could successfully generate the human-like N-glycans, was carefully characterized. Glyco4 displays a significant growth delay and cell wall defects. Comparative transcriptomics reveals that manipulating the N-glycosylation pathway could notably affect the expression pattern of numerous biological pathways in Glyco4. Among the differentially expressed genes, the down-regulation of PpSPI1 was proven to be the main cause of the cell wall defects in Glyco4. Deletion of PpSPI1 in P. pastoris GS115 strain presented growth delay and weaker resistance to cell wall/membrane perturbing agents. PpSpi1 was shown to be a glycosylphosphatidylinositol-anchored cell wall glycoprotein and involved in the formation of the mannoprotein layer on the outer surface of cell wall. Overexpressing PpSPI1 in Glyco4 could partially recover the cell wall defects and also improve its resistance to cell wall perturbing agents and osmotic stress. Thus, overexpression of PpSpi1 is a useful strategy to facilitate P. pastoris industrial applications in the manufacture of human glycoproteins. IMPORTANCE Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Simple Summary: All human cells possess a complex glycan coating, and alterations in cell surface glycans play a role in cancer progression and immune suppression. Colorectal cancer is a heterogenous disease that can be classified into several molecular subtypes, with major variances in prognosis and therapy responses. Two important molecular markers in this regard are microsatellite instability and BRAF gene mutation, which have been largely ignored in previous glycomics studies. We analyzed the N-glycan profiles of local and advanced colorectal cancers consisting of different molecular subtypes to identify possible explanations for their differing behaviors. Our results show that the studied molecular subgroups of colorectal cancer exhibit characteristic glycan profiles, which may explain their tumorigenic properties. Aberrant glycosylation affects cancer progression and immune evasion. Approximately 15% of colorectal cancers (CRCs) demonstrate microsatellite instability (MSI) and display major differences in outcomes and therapeutic responses, as compared to corresponding microsatellite stable (MSS) tumors. We compared the N-glycan profiles of stage II and IV MSI CRC tumors, further subdivided into BRAFV600E wild-type and mutated subgroups (n = 10 in each subgroup), with each other and with those of paired non-neoplastic mucosal samples using mass spectrometry. Further, the N-glycans of BRAFV600E wild-type stage II MSI tumors were compared to corresponding MSS tumors (n = 9). Multiple differences in N-glycan profiles were identified between the MSI CRCs and control tissues, as well as between the stage II MSI and MSS samples. The MSI CRC tumors showed a lower relative abundance of high-mannose N-glycans than did the control tissues or the MSS CRCs. Among MSI CRC subgroups, acidic N-glycans showed tumor stage and BRAF mutation status-dependent variation. Specifically, the large, sulfated/phosphorylated, and putative terminal N-acetylhexosamine-containing acidic N-glycans differed between the MSI CRC subgroups, showing opposite changes in stages II and IV, when comparing BRAF mutated and wild-type tumors. Our results show that molecular subgroups of CRC exhibit characteristic glycan profiles that may explain certain carcinogenic properties of MSI tumors. [ABSTRACT FROM AUTHOR]

Background: Aberrant N-glycosylation and its involvement in pathogenesis have been reported in endometrial cancer (EC). Nevertheless, the serum N-glycomic signature of EC remains unknown. Here, we investigated serum N-glycome patterns of EC to identify candidate biomarkers. Methods: This study enrolled 34 untreated EC patients and 34 matched healthy controls (HC) from Peking Union Medical College Hospital. State-of-the-art MSbased methods were employed for N-glycans profiling. Multivariate and univariate statistical analyses were used to identify discriminative N-glycans driving classification. Receiver operating characteristic analyses were performed to evaluate classification accuracy. Results: EC patients displayed distinct differences in serum N-glycome and had abnormal high-mannose and hybrid-type N-glycans, fucosylation, galactosylation, and linkage-specific sialylation compared with HC. The glycan panel built with the four most discriminative and biologically important derived N-glycan traits could accurately identify EC (random forest model, the area under the curve [AUC]=0.993 [95%CI 0.955-1]). The performancewas validated by two othermodels. Total hybridtype N-glycans significantly associated with the differentiation types of EC could effectively stratify EC into well- or poorly-differentiated subgroups (AUC>0.8). Conclusion: This study provides the initial evidence supporting the utility of serum N-glycomic signature as potential markers for the diagnosis and phenotyping of EC. [ABSTRACT FROM AUTHOR]

Objectives: Exploring the role of OPN N‐glycosylation in osteoblasts and osteoclasts. Methods: Immunohistochemistry was used to detect the expression of OPN in mice with apical periodontitis. The asparagine at position 79 of the OPN protein was mutated to glutamine, and the above plasmids were transfected into osteoblasts and osteoclasts. The effect of OPN N‐glycosylation on proliferation of osteoblasts and osteoclasts was detected by CCK8 assays. Western blotting was used to detect the expression of OPN N‐glycosylation on osteoclasts and osteoblasts. Detection of N‐glycosylation of OPN activated the NF‐κB signaling pathway to regulate osteoblasts and osteoclasts. Results: OPN increased the expression in a mice model of apical periodontitis. The expression curve of OPN resembled a reverse V shape. The OPN N‐glycosylation site was identified as 79 by MS. N‐glycosylation of OPN promoted the proliferation of osteoclasts. But the N79 glycosylation site of mutant OPN could not increase the proliferation of osteoblasts. OPN N‐glycosylation modulated the expression of osteoclast‐ and osteoblast‐associated factors through the NF‐κB signaling pathway. N‐glycosylation of OPN promoted nuclear translocation of NF‐κB in osteoclasts and osteoblasts. Conclusions: The N‐glycosylation site of OPN is 79. N‐glycosylation of OPN played an important role in the biological function of OPN protein. [ABSTRACT FROM AUTHOR]

INTRODUCTION: There is an urgent need for novel blood biomarkers for the detection of Alzheimer's disease (AD). We previously showed that levels of the bisecting N‐acetylglucosamine glycan epitope was elevated in cerebrospinal fluid in AD. However, its diagnostic value in blood is unknown. METHODS: We analyzed blood levels of bisecting N‐acetylglucosamine and total tau in a retrospective cohort of 233 individuals. Progression to AD was compared between the groups using Cox regression. The predictive value of the biomarkers was determined by logistic regression. RESULTS: Bisecting N‐acetylglucosamine correlated with tau levels (p < 0.0001). Individuals with an intermediate tau/bisecting N‐acetylglucosamine ratio had elevated AD risk (hazard ratio = 2.06, 95% confidence interval [CI]: 1.18–3.6). Moreover, a combined model including tau/bisecting N‐acetylglucosamine ratio, apolipoprotein E (APOE) ε4 status, and Mini‐Mental State Examination score predicted future AD (area under the curve = 0.81, 95% CI: 0.68–0.93). DISCUSSION: Bisecting N‐acetylglucosamine in combination with tau is a valuable blood biomarker for predicting AD. [ABSTRACT FROM AUTHOR]

Background: The pathological mechanisms of SARS-CoV-2 in humans remain unclear and the unpredictability of COVID-19 progression may be attributed to the absence of biomarkers that contribute to the prognosis of this disease. Therefore, the discovery of biomarkers is needed for reliable risk stratification and to identify patients who are more likely to progress to a critical stage. Methods: Aiming to identify new biomarkers we analysed N-glycan traits in plasma from 196 patients with COVID-19. Samples were classified into three groups according to their severity (mild, severe and critical) and obtained at diagnosis (baseline) and at 4 weeks of follow-up (postdiagnosis), to evaluate their behaviour through disease progression. N-glycans were released with PNGase F and labelled with Rapifluor-MS, followed by their analysis by LC-MS/MS. The Simglycan structural identification tool and Glycostore database were employed to predict the structure of glycans. Results: We determined that plasma from SARS-CoV-2-infected patients display different N-glycosylation profiles depending on the disease severity. Specifically, levels of fucosylation and galactosylation decreased with increasing severity and Fuc1Hex5HexNAc5 was identified as the most suitable biomarker to stratify patients at diagnosis and distinguish mild from critical outcomes. Conclusion: In this study we explored the global plasma glycosignature, reflecting the inflammatory state of the organs during the infectious disease. Our findings show the promising potential of glycans as biomarkers of COVID-19 severity. [ABSTRACT FROM AUTHOR]

Aim: Changes in N-glycosylation have been described in numerous diseases and are being considered as biomarkers of ongoing pathological condition. Previous studies demonstrated the interrelation of N-glycosylation and type 1 diabetes (T1D), particularly linking serum N-glycan changes with complications accompanying the disease. Moreover, the role of complement component C3 in diabetic nephropathy and retinopathy has been implicated, and C3 N-glycome was found to be altered in young T1D patients. Therefore, we investigated associations between C3 N-glycan profiles and albuminuria and retinopathy accompanying T1D, as well as glycosylation connection with other known T1D complication risk factors. Research design and methods: Complement component C3 N-glycosylation profiles have been analyzed from 189 serum samples of T1D patients (median age 46) recruited at a Croatian hospital centre. Using our recently developed high-throughput method, relative abundances of all six of the C3 glycopeptides have been determined. Assessment of C3 N-glycome interconnection with T1D complications, hypertension, smoking status, estimated glomerular filtration rate (eGFR), glycaemic control and duration of the disease was done using linear modelling. Results: Significant changes of C3 N-glycome in severe albuminuria accompanying type 1 diabetes were observed, as well as in T1D subjects with hypertension. All except one of the C3 glycopeptides proved to be associated with measured HbA1c levels. One of the glycoforms was shown to be changed in non-proliferative T1D retinopathy. Smoking and eGFR showed no effect on C3 N-glycome. Furthermore, C3 N-glycosylation profile was shown to be independent of disease duration. Conclusion: This study empowered the role of C3 N-glycosylation in T1D, showing value in distinguishing subjects with different diabetic complications. Being independent of the disease duration, these changes may be associated with the disease onset, making C3 N-glycome a potential novel marker of the disease progression and severity. [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) is an avian pathogen from the Coronavirus family causing major health issues in poultry flocks worldwide. Because of its negative impact on health, performance, and bird welfare, commercial poultry are routinely vaccinated by administering live attenuated virus. However, field strains are capable of rapid adaptation and may evade vaccine-induced immunity. We set out to describe dynamics within and between lineages and assess potential escape from vaccine-induced immunity. We investigated a large nucleotide sequence database of over 1700 partial sequences of the S1 spike protein gene collected from clinical samples of Dutch chickens submitted to the laboratory of Royal GD between 2011 and 2020. Relative frequencies of the two major lineages GI-13 (793B) and GI-19 (QX) did not change in the investigated period, but we found a succession of distinct GI-19 sublineages. Analysis of dN/dS ratio over all sequences demonstrated episodic diversifying selection acting on multiple sites, some of which overlap predicted N-glycosylation motifs. We assessed several measures that would indicate divergence from vaccine strains, both in the overall database and in the two major lineages. However, the frequency of vaccine-homologous lineages did not decrease, no increase in genetic variation with time was detected, and the sequences did not grow more divergent from vaccine sequences in the examined time window. Concluding, our results show sublineage turnover within the GI-19 lineage and we demonstrate episodic diversifying selection acting on the partial sequence, but we cannot confirm nor rule out escape from vaccine-induced immunity. Succession of GI-19 IBV variants in broiler populations. IBV lineages overrepresented in either broiler, or layer production chickens. Ongoing episodic selection at the IBV S1 spike protein gene sequence. Several positively selected codons coincident with N-glycosylation motifs. [ABSTRACT FROM AUTHOR]

Cell wall is a strong and complex net whose function is to provide turgor, pathogens attack protection and to give structural support to the cell. In growing and expanding cells, the cell wall of fruits is changing in space and time, because they are changing according to stage of ripening. Understand which mechanisms to produce significant could help to develop tools to prolong the fruit shelf life. Cell wall proteins (CWPs) with enzymatic activity on cell wall polysaccharides, have been studied widely. Another investigations take place in the study of N-glycosylations of CWPs and enzymes with activity on glycosidic linkages. α-mannosidase (α-Man; EC 3.2.1.24) and β-d-N-acetylhexosaminidase (β-Hex; EC 3.2.1.52), are enzymes with activity on mannose and N-acetylglucosamine sugar presents in proteins as part of N-glycosylations. Experimental evidence indicate that both are closely related to loss of fruit firmness, but in the literature, there is still no review of both enzymes involved fruit ripening. This review provides a complete state-of-the-art of α-Man and β-Hex enzymes related in fruit ripening. Also, we propose a vesicular α-Man (EC 3.2.1.24) name to α-Man involved in N-deglycosylations of CWPs of plants. Key message: Cell wall proteins (CWPs) with enzymatic activity on cell wall polysaccharides, have been studied widely. Another investigations take place in the study of N-glycosylations of CWPs and enzymes with activity on glycosidic linkages. Lysosomal α-mannosidase (α-Man; EC 3.2.1.24) and β-d-N-acetylhexosaminidase (β-Hex; EC 3.2.1.52), are enzymes with activity on mannose and N-acetylglucosamine sugar presents in proteins as part of N-glycosylations. Experimental evidence indicate that both are closely related to loss of fruit firmness, but in the literature, there is still no review of both enzymes involved fruit ripening. This review provides a complete view of lysosomal α-Man and β-Hex enzymes related in fruit ripening. [ABSTRACT FROM AUTHOR]

Next to prostate-specific antigen, no biochemical biomarkers have been implemented to guide patient follow-up after primary therapy for localized prostate cancer (PCa). We evaluated the prognostic potential of urine N-glycome in terms of event-free survival (EFS) in patients undergoing primary therapy for PCa. The prognostic features of the urine N-glycosylation profile at diagnosis, assessed in 77 PCa patients, were determined in terms of EFS next to standard clinical parameters. The majority of patients were diagnosed with International Society of Urological Pathology grade ≤ 3 (82%) T1–2 tumors (79%) and without pelvic lymph node invasion (96%). The patients underwent active surveillance (14%), robot-assisted laparoscopic prostatectomy (48%), or external beam radiotherapy (37%). Decreased ratios of biantennary core-fucosylation were noted in patients who developed an event, which was linked to a shorter EFS in both the intention-to-treat cohort and all subcohort analyses. Combining the urine N-glycan biomarker with the D’Amico Risk Classification for PCa resulted in an improved nomogram for patient classification after primary therapy. The rate of urine N-glycan biantennary core-fucosylation, typically linked to more aggressive disease status, is lower in patients who eventually developed an event following primary therapy and subsequently in patients with a worse EFS. The combination of urine N-glycan biomarkers together with clinical parameters could, therefore, improve the post-therapy follow-up of patients with PCa.

A certified reference material of ricin (CRM-LS-1) was produced by the EuroBioTox consortium to standardise the analysis of this biotoxin. This study established the N-glycan structures and proportions including their loci and occupancy of ricin CRM-LS-1. The glycan profile was compared with ricin from different preparations and other cultivars and isoforms. A total of 15 different oligomannosidic or paucimannosidic structures were identified in CRM-LS-1. Paucimannose was mainly found within the A-chain and oligomannose constituted the major glycan type of the B-chain. Furthermore, the novel primary structure variants E138 and D138 and four different C-termini of the A-chain as well as two B-chain variants V250 and F250 were elucidated. While the glycan proportions and loci were similar among all variants in CRM-LS-1 and ricin isoforms D and E of all cultivars analysed, a different stoichiometry for isoforms D and E and the amino acid variants were found. This detailed physicochemical characterization of ricin regarding the glycan profile and amino acid sequence variations yields unprecedented insight into the molecular features of this protein toxin. The variable attributes discovered within different cultivars present signature motifs and may allow discrimination of the biotoxin’s origin that are important in molecular forensic profiling. In conclusion, our data of in-depth CRM-LS-1 characterization combined with the analysis of other cultivars is representative for known ricin variants.

IgG Fc N-glycosylation is necessary for effector functions and is an important component of quality control. The choice of antibody manufacturing platform has the potential to significantly influence the Fc glycans of an antibody and consequently alter their activity and clinical profile. The Human Contraception Antibody (HCA) is an IgG1 antisperm monoclonal antibody (mAb) currently in clinical development as a novel, non-hormonal contraceptive. Part of its development is selecting a suitable expression platform to manufacture HCA for use in the female reproductive tract. Here, we compared the Fc glycosylation of HCA produced in two novel mAb manufacturing platforms, namely transgenic tobacco plants (Nicotiana benthamiana; HCA-N) and mRNA-mediated expression in human vaginal cells (HCAmRNA). The Fc N-glycan profiles of the two HCA products were determined using mass spectrometry. Major differences in site occupancy, glycan types, and glycoform distributions were revealed. To address how these differences affect Fc function, antibody-dependent cellular phagocytosis (ADCP) assays were performed. The level of sperm phagocytosis was significantly lower in the presence of HCA-N than HCAmRNA. This study provides evidence that the two HCA manufacturing platforms produce functionally distinct HCAs; this information could be useful for the selection of an optimal platform for HCA clinical development and for mAbs in general.

Secretory Immunoglobulin A (SIgA) is central to mucosal immunity: represents one of the main immunological mechanisms of defense against the potential attack of pathogens. During lactation SIgA is produced by plasmablasts in the mammary gland and is present in breast milk, playing a vital role in the passive immunity of the newborn. Interestingly, the different components of SIgA are highly N-glycosylated, and these N-Glycans have an essential role in health maintenance. In this work, we performed a glycomic study to compare N-glycosylation of SIgA purified from mature breast milk and saliva, and plasma IgA from the same lactating participants. Our results revealed a greater diversity than previously reported, with 89 glycan compositions that may correspond to over 250 structures. Among these glycans, 54 glycan compositions were characterized as body-fluid specific. Most of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, whereas in salivary SIgA the unique structures were mainly undecorated complex N-Glycans. In addition, we evaluated the effect of delivery mode on (S)IgA glycosylation. Lactating participants who had given birth by vaginal delivery presented an increased proportion of high mannose and fucosylated glycans in salivary SIgA, and selected high mannose, fucosylated, sialylated, and both fucosylated and sialylated glycans in plasma IgA, indicating that the hormonal changes during vaginal delivery could affect plasma and saliva IgA. These results reveal the structural details that provide a new dimension to the roles of (S)IgA N-Glycans in different tissues, and especially in maternal and new-born protection and infant development. The design of optimal recombinant IgA molecules specifically targeted to protect mucosal surfaces will need to include this dimension of structural detail.

ABSTRACT: Chicken egg yolk granules (EYG) were the precipitated component of egg yolk after water dilution and centrifugation. Compared with egg yolk, EYG are rich in proteins, phospholipids, and minerals. In this study, an integrated proteomic analysis was carried out to in-depth mapping of the proteome, phosphoproteome, and N-glycoproteome of EYGs. After hydrolysis of the EYG total protein, the hydrolyzed peptides or the enriched phosphopeptides/glycopeptides were identified by liquid chromatography-tandem mass spectrometry. A total of 125 phosphorylation sites from 36 phosphoproteins and 244 N-glycosylation sites from 100 N-glycoproteins were identified in EYG. All 3 vitellogenins (precursors of egg yolk high-density lipoprotein) were heavily phosphorylated and N-glycosylated, of which 37 phosphorylation sites and 32 N-glycosylation sites were identified on vitellogenins-2. A Total of 30 N-glycosylation sites were identified on apolipoprotein-B (precursor of egg yolk low-density lipoprotein), but no phosphorylation site was identified. These phosphorylation and N-glycosylation of EYG proteins provide new insights for understanding the assembly structure and functional characteristics of EYG, thus contributing to its development and utilization.

The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of immune cell receptors. Although knowledge in the field of glycoimmunology is still limited, numerous studies point to the key role of glycosylation in maintaining homeostasis, but also in reflecting its disruption. Changes in oligosaccharide patterns can lead to impairment of both innate and acquired immune responses, with important implications in the pathogenesis of diseases, including autoimmunity. B cells appear to be unique within the immune system, since they exhibit both innate and adaptive immune activity. B cell surface is rich in glycosylated proteins and lectins which recognise glycosylated ligands on other cells. Glycans are important in the development, selection, and maturation of B cells. Changes in sialylation and fucosylation of cell surface proteins affect B cell signal transduction through BCRs, CD22 inhibitory coreceptor and Siglec-G. Plasmocytes, as the final stage of B cell differentiation, produce and secrete immunoglobulins (Igs), of which IgGs are the most abundant N-glycosylated proteins in human serum with the conserved N-glycosylation site at Asn297. N-oligosaccharide composition of the IgG Fc region affects its secretion, structure, half-life and effector functions (ADCC, CDC). IgG N-glycosylation undergoes little change during homeostasis, and may gradually be modified with age and during ongoing inflammatory processes. Hyperactivated B lymphocytes secrete autoreactive antibodies responsible for the development of autoimmunity. The altered profile of IgG N-glycans contributes to disease progression and remission and is sensitive to the application of therapeutic substances and immunosuppressive agents. In this review, we focus on the role of N-glycans in B-cell biology and IgG activity, the rearrangement of IgG oligosaccharides in aging, autoimmunity and immunosuppressive therapy.

BackgroundThe pathological mechanisms of SARS-CoV-2 in humans remain unclear and the unpredictability of COVID-19 progression may be attributed to the absence of biomarkers that contribute to the prognosis of this disease. Therefore, the discovery of biomarkers is needed for reliable risk stratification and to identify patients who are more likely to progress to a critical stage.MethodsAiming to identify new biomarkers we analysed N-glycan traits in plasma from 196 patients with COVID-19. Samples were classified into three groups according to their severity (mild, severe and critical) and obtained at diagnosis (baseline) and at 4 weeks of follow-up (postdiagnosis), to evaluate their behaviour through disease progression. N-glycans were released with PNGase F and labelled with Rapifluor-MS, followed by their analysis by LC-MS/MS. The Simglycan structural identification tool and Glycostore database were employed to predict the structure of glycans.ResultsWe determined that plasma from SARS-CoV-2-infected patients display different N-glycosylation profiles depending on the disease severity. Specifically, levels of fucosylation and galactosylation decreased with increasing severity and Fuc1Hex5HexNAc5 was identified as the most suitable biomarker to stratify patients at diagnosis and distinguish mild from critical outcomes.ConclusionIn this study we explored the global plasma glycosignature, reflecting the inflammatory state of the organs during the infectious disease. Our findings show the promising potential of glycans as biomarkers of COVID-19 severity.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) constitute complex signaling hubs that are crucial for the development and homeostasis of the human body. Most of FGFs are released by cells using the conventional secretory pathway and are N-glycosylated, yet the role of FGFs glycosylation is largely unknown. Here, we identify N-glycans of FGFs as binding sites for a specific set of extracellular lectins, galectins − 1, -3, -7 and − 8. We demonstrate that galectins attract N-glycosylated FGF4 to the cell surface, forming a reservoir of the growth factor in the extracellular matrix. Furthermore, we show that distinct galectins differentially modulate FGF4 signaling and FGF4-dependent cellular processes. Using engineered variants of galectins with altered valency we demonstrate that multivalency of galectins is critical for the adjustment of FGF4 activity. Summarizing, our data reveal a novel regulatory module within FGF signaling, in which the glyco-code in FGFs provides previously unanticipated information differentially deciphered by multivalent galectins, affecting signal transduction and cell physiology. 8PPE1YWgWFxc_zmTSXi8tA Video Abstract [ABSTRACT FROM AUTHOR]

SUMMARY: As an essential enzyme in the uridine diphosphate (UDP)‐GlcNAc biosynthesis pathway, the significant role of N‐acetylglucosamine phosphate mutase (AGM) remains unknown in plants. In the present study, a functional plant AGM (AtAGM) was identified from Arabidopsis thaliana. AtAGM catalyzes the isomerization of GlcNAc‐1‐P and GlcNAc‐6‐P, and has broad catalytic activity on different phosphohexoses. UDP‐GlcNAc contents were significantly decreased in AtAGM T‐DNA insertional mutants, which caused temperature‐dependent growth defects in seedlings and vigorous growth in adult plants. Further analysis revealed that protein O‐GlcNAcylation but not N‐glycosylation was dramatically impaired in Atagm mutants due to UDP‐GlcNAc shortage. Combined with the results from O‐GlcNAcylation or N‐glycosylation deficient mutants, and O‐GlcNAcase inhibitor all suggested that protein O‐GlcNAcylation impairment mainly leads to the phenotypic variations of Atagm plants. In conclusion, based on the essential role in UDP‐GlcNAc biosynthesis, AtAGM is important for plant growth mainly via protein O‐GlcNAcylation‐level regulation. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) non-structural protein 1 (NS1) is involved in multiple aspects of the DENV lifecycle. Importantly, it is secreted from infected cells as a hexameric lipoparticle that mediates vascular damage that is a hallmark of severe dengue. Although the secretion of NS1 is known to be important in DENV pathogenesis, the exact molecular features of NS1 that are required for its secretion from cells are not fully understood. In this study, we employed random point mutagenesis in the context of an NS1 expression vector encoding a C-terminal HiBiT luminescent peptide tag to identify residues within NS1 that are essential for its secretion. Using this approach, we identified 10 point mutations that corresponded with impaired NS1 secretion, with in silico analyses indicating that the majority of these mutations are located within the β-ladder domain. Additional studies on two of these mutants, V220D and A248V, revealed that they prevented viral RNA replication, while studies using a DENV NS1-NS5 viral polyprotein expression system demonstrated that these mutations resulted in a more reticular NS1 localisation pattern and failure to detect mature NS1 at its predicted molecular weight by Western blotting using a conformation-specific monoclonal antibody. Together, these studies demonstrate that the combination of a luminescent peptide tagged NS1 expression system with random point mutagenesis enables rapid identification of mutations that alter NS1 secretion. Two such mutations identified via this approach revealed residues that are essential for correct NS1 processing or maturation and viral RNA replication. [ABSTRACT FROM AUTHOR]

Bladder cancer (BC) is the 10th most common malignancy worldwide, with an estimated 573,000 new cases and 213,000 deaths in 2020. Available therapeutic approaches are still unable to reduce the incidence of BC metastasis and the high mortality rates of BC patients. Therefore, there is a need to deepen our understanding of the molecular mechanisms underlying BC progression to develop new diagnostic and therapeutic tools. One such mechanism is protein glycosylation. Numerous studies reported changes in glycan biosynthesis during neoplastic transformation, resulting in the appearance of the so-called tumor-associated carbohydrate antigens (TACAs) on the cell surface. TACAs affect a wide range of key biological processes, including tumor cell survival and proliferation, invasion and metastasis, induction of chronic inflammation, angiogenesis, immune evasion, and insensitivity to apoptosis. The purpose of this review is to summarize the current information on how altered glycosylation of bladder cancer cells promotes disease progression and to present the potential use of glycans for diagnostic and therapeutic purposes. [ABSTRACT FROM AUTHOR]

Glycosylation is a critical quality attribute in biotherapeutics, impacting properties such as protein stability, solubility, clearance rate, efficacy, immunogenicity, and safety. Due to the heterogenic and complex nature of protein glycosylation, comprehensive characterization is demanding. Moreover, the lack of standardized metrics for evaluating and comparing glycosylation profiles hinders comparability studies and the establishment of manufacturing control strategies. To address both challenges, we propose a standardized approach based on novel metrics for a comprehensive glycosylation fingerprint which greatly facilitates the reporting and objective comparison of glycosylation profiles. The analytical workflow is based on a liquid chromatography–mass spectrometry-based multi-attribute method. Based on the analytical data, a matrix of glycosylation-related quality attributes, both at site-specific and whole molecule level, are computed, which provide metrics for a comprehensive product glycosylation fingerprint. Two case studies illustrate the applicability of the proposed indices as a standardized and versatile approach for reporting all dimensions of the glycosylation profile. The proposed approach further facilitates the assessments of risks associated with changes in the glycosylation profile that may affect efficacy, clearance, and immunogenicity. [ABSTRACT FROM AUTHOR]

FGF/FGFR signaling is critical for the development and homeostasis of the human body and imbalanced FGF/FGFR contributes to the progression of severe diseases, including cancers. FGFRs are N-glycosylated, but the role of these modifications is largely unknown. Galectins are extracellular carbohydrate-binding proteins implicated in a plethora of processes in heathy and malignant cells. Here, we identified a precise set of galectins (galectin-1, -3, -7, and -8) that directly interact with N-glycans of FGFRs. We demonstrated that galectins bind N-glycan chains of the membrane-proximal D3 domain of FGFR1 and trigger differential clustering of FGFR1, resulting in activation of the receptor and initiation of downstream signaling cascades. Using engineered galectins with controlled valency, we provide evidence that N-glycosylation-dependent clustering of FGFR1 constitutes a mechanism for FGFR1 stimulation by galectins. We revealed that the consequences of galectin/FGFR signaling for cell physiology are markedly different from the effects induced by canonical FGF/FGFR units, with galectin/FGFR signaling affecting cell viability and metabolic activity. Furthermore, we showed that galectins are capable of activating an FGFR pool inaccessible for FGF1, enhancing the amplitude of transduced signals. Summarizing, our data identify a novel mechanism of FGFR activation, in which the information stored in the N-glycans of FGFRs provides previously unanticipated information about FGFRs’ spatial distribution, which is differentially deciphered by distinct multivalent galectins, affecting signal transmission and cell fate. [ABSTRACT FROM AUTHOR]

Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are rarely documented for oomycetes. In this study, an N-glycosylation inhibitor tunicamycin (TM) hampered the mycelial growth, sporangial release, and zoospore production of Phytophthora capsici, indicating that N-glycosylation was crucial for oomycete growth development. Among the key catalytic enzymes involved in N-glycosylation, the PcSTT3B gene was characterized by its functions in P. capsici. As a core subunit of the oligosaccharyltransferase (OST) complex, the staurosporine and temperature sensive 3B (STT3B) subunit were critical for the catalytic activity of OST. The PcSTT3B gene has catalytic activity and is highly conservative in P. capsici. By using a CRISPR/Cas9-mediated gene replacement system to delete the PcSTT3B gene, the transformants impaired mycelial growth, sporangial release, zoospore production, and virulence. The PcSTT3B-deleted transformants were more sensitive to an ER stress inducer TM and display low glycoprotein content in the mycelia, suggesting that PcSTT3B was associated with ER stress responses and N-glycosylation. Therefore, PcSTT3B was involved in the development, pathogenicity, and N-glycosylation of P. capsici. [ABSTRACT FROM AUTHOR]

Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future. [ABSTRACT FROM AUTHOR]

The strained/frustrated electrostatic interactions between the ion pair of TTBPy+ X– increases the reactivity in both the ions, resulting in the activation of a third molecule like sulfonamides (aromatic/-aliphatic) via hydrogen bonding. This intriguing weak-interactions-based reactivity has been utilized to develop an organocatalytic synthesis of 2-deoxy-sulfonamido-glycosides from glycals. The sulfonamidoglycosylation of glycals using a catalytic amount of 2,4,6-tri- tert -butylpyridinium salts proceeded stereoselectively to provide N -glycosides in good to high yields. This process was demonstrated with l -rhamnal and d -galactal. Besides, IR spectroscopic studies explain that the hindered protonated pyridine cannot behave as a cationic Brønsted acid as is generally perceived. [ABSTRACT FROM AUTHOR]

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30 years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication. [ABSTRACT FROM AUTHOR]

There is a demand for increasing the current manufacturing capacities for recombinant protein-based drugs. Novel expression systems such as plants are being explored as faster, more flexible, and possibly cheaper platforms. Many of these therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. In planta protein sialylation has been achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana , comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. Here we address technical issues that can compromise the efficacy of protein sialylation and how they can be overcome. We used the same reporter protein to compared three strategies to transiently deliver the sialylation pathway-genes evaluating efficacy, heterogeneity and batch-to-batch consistency. In addition, we assess the ability of the single-step method to sialylated additional recombinant proteins with different complexity and number of glycosylation sites. Finally, we show that efficient protein sialylation can be up-scaled for large-scale production of sialylated proteins in plants. [Display omitted] • Glycan-sialylation is important for the biological activity of therapeutic proteins. • The mammalian sialylation pathway can be introduced in plants. • We compared three approaches to transiently deliver the sialylation pathway-genes. • The single multigene vector can simplify the transient expression system. • We show up-scale production of glycoproteins with consistent human-like sialylation. [ABSTRACT FROM AUTHOR]

Wild‐type herpes simplex virus (HSV) strains infrequently mediate cell–cell fusion in cell cultures and barely induce large multinucleated cells. In this study, we established a system to quantify infrequent cell–cell fusion induced by wild‐type HSV strains. The established system clarified that the HSV‐1 envelope glycoprotein B and its N‐glycosylation at asparagine at position 141 were required for efficient cell–cell fusion. This study provides a link between cell–cell fusion induced by wild‐type HSV‐1 and viral pathogenesis in vivo. [ABSTRACT FROM AUTHOR]

Abstract Background N-linked glycans on viral glycoproteins have been shown to be important for protein expression, processing and intracellular transport. The fusion glycoprotein F of Cedar virus (CedV) contains six potential N-glycosylation sites. Findings To investigate their impact on cell surface transport, proteolytic cleavage and biological activity, we disrupted the consensus sequences by conservative mutations (Asn to Gln) and found that five of the six potential N-glycosylation sites are actually utilized. The individual removal of N-glycan g1 (N66), g2 (N79) and g3 (N98) in the CedV F2 subunit had no or only little effect on cell surface transport, proteolytic cleavage and fusion activity of CedV F. Interestingly, removal of N-linked glycan g6 (N463) in the F1 subunit resulted in reduced cell surface expression but slightly increased fusogenicity upon co-expression with the CedV receptor-binding protein G. Most prominent effects however were observed for the disruption of N-glycosylation motif g4 (N413), which significantly impaired the transport of CedV F to the cell surface, thereby also affecting proteolytic cleavage and fusion activity. Conclusions Our findings indicate that the individual N-linked modifications, with the exception of glycan g4, are dispensable for processing of CedV F protein in transfection experiments. However, removal of g4 led to a phenotype that was strongly impaired concerning cell surface expression and proteolytic activation.

Abstract Background N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion. Results Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin. Conclusions In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris.

Abstract Background Rheumatoid arthritis (RA) is a chronic autoimmune disease for which prediction of long-term prognosis from disease’s outset is not clinically feasible. The importance of immunoglobulin G (IgG) and its Fc N-glycosylation in inflammation is well-known and studies described its relevance for several autoimmune diseases, including RA. Herein we assessed the association between IgG N-glycoforms and disease prognosis at 2 years in an early inflammatory arthritis cohort. Methods Sera from 118 patients with early inflammatory arthritis naïve to treatment sampled at baseline were used to obtain IgG Fc glycopeptides, which were then analyzed in a subclass-specific manner by liquid chromatography coupled to mass spectrometry (LC-MS). Patients were prospectively followed and a favorable prognosis at 2 years was assessed by a combined index as remission or low disease activity (DAS28 < 3.2) and normal functionality (HAQ ≤ 0.25) while on treatment with conventional synthetic DMARDs and never used biologic DMARDs. Results We observed a significant association between high levels of IgG2/3 Fc galactosylation (effect 0.627 and adjusted p value 0.036 for the fully galactosylated glycoform H5N4F1; effect −0.551 and adjusted p value 0.04963 for the agalactosylated H3N4F1) and favorable outcome after 2 years of treatment. The inclusion of IgG glycoprofiling in a multivariate analysis to predict the outcome (with HAQ, DAS28, RF, and ACPA included in the model) did not improve the prognostic performance of the model. Conclusion Pending confirmation of these findings in larger cohorts, IgG glycosylation levels could be used as a prognostic marker in early arthritis, to overcome the limitations of the current prognostic tools.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

β-Klotho (KLB), a type I transmembrane protein, serves as an obligate co-receptor determining the tissue-specific actions of endocrine fibroblast growth factors (FGFs). Despite accumulative evidence suggesting the occurrence of N-glycosylation in the KLB protein, the precise N-glycosites, glycoforms, and the impacts of N-glycosylation on the expression and function of the KLB protein remain unexplored. Employing a mass spectrometry-based approach, a total of 12 N-glycosites displaying heterogeneous site occupancy and glycoforms were identified within the extracellular region of the recombinant human KLB protein. Molecular simulation revealed negligible impact of these N-glycans on the overall structure of the KLB protein. However, both pharmacological inhibition of N-glycosylation and mutagenesis targeting N-glycosites reduced mature KLB protein content without impacting KLB mRNA synthesis in cells, underscoring the critical role of N-glycosylation in maintaining the stability of the KLB protein. Further studies revealed that the underglycosylated KLB mutant underwent rapid degradation via both lysosomal and proteasomal pathways and was unable to be efficiently trafficked to the plasma membrane, leading to impaired FGF21 signaling transduction. Collectively, multisite N-glycosylation is essential for the stability and cell surface localization of the KLB protein, representing a novel modulatory mechanism of endocrine FGF signaling. • Decoding N-glycosites and site-specific N-glycans on β-Klotho protein • N-glycans may not significantly alter the structure of β-Klotho protein. • Impaired N-glycosylation causes proteasomal and lysosomal degradation of β-Klotho. • N-glycosylation is required for plasma membrane localization of β-Klotho. [ABSTRACT FROM AUTHOR]