PubTransformer

A site to transform Pubmed publications into these bibliographic reference formats: ADS, BibTeX, EndNote, ISI used by the Web of Knowledge, RIS, MEDLINE, Microsoft's Word 2007 XML.

glycosylation - Top 30 Publications

A concise synthesis of rhamnan oligosaccharides with alternating α-(1→2)/(1→3)-linkages and repeating α-(1→3)-linkages by iterative α-glycosylation using disaccharide building blocks.

A concise synthetic route to rhamnan oligosaccharides with alternating α-(1→2)/(1→3)-linkages and repeating α-(1→3)-linkages is reported. This synthesis was achieved by iterative α-glycosylation using disaccharide building blocks and through orthogonal coupling between thioglycosides of L-rhamnose. To investigate the detailed structure-activity relationship of rhamnan sulfate from Monostroma nitidum against herpes simplex virus type 2, the synthesized oligosaccharides, bearing different orthogonal protecting groups (i.e., benzoyl, benzyl, 2-naphthylmethyl, and/or p-methoxybenzyl) are expected to be suitable for conversion into a range of rhamnan structures with diverse sulfation patterns.

Endoplasmic reticulum stress-induced cell death in podocytes.

Endoplasmic reticulum (ER) stress occurs in a variety of pathophysiological mechanisms, and there has been great interest in managing this pathway for the treatment of clinical diseases. Increased ER stress can block integrin-β1 glycosylation, decrease integrin-β1 protein expression and enhance cell death in podocytes. Autophagy is closely interconnected with ER stress to counteract the possible injurious effects related to the impairment of protein folding and is one of the intracellular protein degradation systems. Studies have shown that podocytes exhibit a high rate of autophagy to maintain as terminally differentiated cells. We have attempted to induce autophagy in podocytes with ER stress to increase the longevity of proteins and the degradation of damaged organelles. However, regardless of ER stress or autophagy that protects the cells at early stages, cells cannot adapt to this situation when the stress is already well established, and podocytes will undergo severe injury finally. In summary, ER stress may induce cell death in podocyte, and autophagy mediate to salvage the injuries caused by ER stress in the short term. It seems that adequate, but not excessive, autophagy is crucial to help maintain the cell viability of podocytes.

Structural Evidence for a Role of the Multi-functional Human Glycoprotein Afamin in Wnt Transport.

Afamin, a human plasma glycoprotein and putative transporter of hydrophobic molecules, has been shown to act as extracellular chaperone for poorly soluble, acylated Wnt proteins, forming a stable, soluble complex with functioning Wnt proteins. The 2.1-Å crystal structure of glycosylated human afamin reveals an almost exclusively hydrophobic binding cleft capable of harboring large hydrophobic moieties. Lipid analysis confirms the presence of lipids, and density in the primary binding pocket of afamin was modeled as palmitoleic acid, presenting the native O-acylation on serine 209 in human Wnt3a. The modeled complex between the experimental afamin structure and a Wnt3a homology model based on the XWnt8-Fz8-CRD fragment complex crystal structure is compelling, with favorable interactions comparable with the crystal structure complex. Afamin readily accommodates the conserved palmitoylated serine 209 of Wnt3a, providing a structural basis how afamin solubilizes hydrophobic and poorly soluble Wnt proteins.

Glycomics of human embryonic stem cells and human induced pluripotent stem cells.

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.

The Cyanopivaloyl Ester in the Automated Solid Phase Synthesis of Oligorhamnans.

The development of effective protecting group chemistry is an important driving force behind the progress in the synthesis of complex oligosaccharides. Automated solid phase oligosaccharide synthesis is an attractive technique for the rapid assembly of oligosaccharides build up of repetitive elements. The fact that (harsh) reagents are used in excess in multiple reaction cycles make this technique extra demanding on the protecting groups used. Here, the synthesis of a set of oligorhamnan fragments is reported using the cyanopivaloyl (PivCN) ester, to ensure effective neighboring group participation during the glycosylation events. The PivCN group combines the favorable characteristics of the parent pivaloyl (Piv) ester, stability, minimal migratory aptitude, minimal orthoester formation, while it can be cleaved under mild conditions. We show that the remote CN-group in the PivCN renders the PivCN carbonyl more electropositive and thus susceptible to nucleophilic cleavage. This feature is built upon in the automated solid phase assembly of the oligorhamnan fragments. Where the use of a Piv-protected building block failed because of incomplete cleavage, PivCN-protected synthons performed well and allowed the generation of oligorhamnans, up to 16 monosaccharides in length.

Glycosyl Cross-Coupling of Anomeric Nucleophiles - Scope, Mechanism and Applications in the Synthesis of Aryl C-glycosides.

Stereoselective manipulations at the C1 anomeric position of saccharides are one of the central goals of preparative carbohydrate chemistry. Historically, the majority of reactions forming a bond with anomeric carbon has focused on reactions of nucleophiles with saccharide donors equipped with a leaving group. Here, we describe a novel approach to stereoselective synthesis of C-aryl glycosides capitalizing on the highly stereospecific reaction of anomeric nucleophiles. First, methods for the preparation of anomeric stannanes have been developed and optimized to afford both anomers of common saccharides in high anomeric selectivities. We established that oligosaccharide stannanes could be prepared from monosaccharide stannanes via O-glycosylation with Schmidttype donors, glycal epoxides, or under dehydrative conditions with C1 alcohols. Second, we identified a general set of catalytic conditions with Pd2(dba)3 (2.5 mol%) and a bulky ligand (JackiePhos, 10 mol%) controlling the b-elimination pathway. We demonstrated the glycosyl cross-coupling results in consistently high anomeric selectivities for both anomers with mono- and oligosaccharides, deoxysugars, saccharides with free hydroxyl groups, pyranose and furanose substrates. The versatility of the glycosyl crosscoupling reaction was probed in the total synthesis of salmochelins (siderophores) and commercial anti-diabetic drugs (gliflozins). Combined experimental and computational studies revealed that the b-elimination pathway is suppressed for biphenyl-type ligands due to the shielding of Pd(II) by sterically demanding JackiePhos whereas smaller ligands, which allow for the formation of a Pd-F complex, predominantly result in a glycal product. Similar steric effects account for the diminished rates of cross-couplings of 1,2cis C1-stannanes with aryl halides. DFT calculations also revealed that the transmetalation occurs via a cyclic transition state with retention of configuration at the anomeric position. Taken together, facile access to both anomers of various glycoside nucleophiles, a broad reaction scope, and uniformly high transfer of anomeric configuration make the glycosyl cross-coupling reaction a practical tool for the synthesis of bioactive natural products, drug candidates, allowing for late-stage glycodiversification studies with small molecules and biologics.

NAD+-dependent dehydrogenase PctP and PLP-dependent aminotransferase PctC catalyze the first post-glycosylation modification of sugar intermediate in pactamycin biosynthesis.

The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from D-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Here, we examined the timing of introduction of an amino group into certain sugar-derived intermediates using recombinant enzymes that are encoded in the pactamycin biosynthesis gene cluster. We found that the NAD+-dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate-dependent aminotransferase PctC convert N-acetyl-D-glucosaminyl-3-aminoacetophonone to 3'-amino-3'-deoxy-GlcNAc-3AAP. Further, GlcNAc-3-aminophenyl-β-oxopropanoic acid ethyl ester was converted to the corresponding 3'-amino-derivative. However, PctP did not oxidize most tested D-glucose derivatives including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.

N-Glycan Profile and Kidney Disease in Type 1 Diabetes.

Poorer glycemic control in type 1 diabetes may alter N-glycosylation patterns on circulating glycoproteins, and these alterations may be linked with diabetic kidney disease (DKD). We investigated associations between N-glycans and glycemic control and renal function in type 1 diabetes.

iPTMnet: an integrated resource for protein post-translational modification network discovery.

Protein post-translational modifications (PTMs) play a pivotal role in numerous biological processes by modulating regulation of protein function. We have developed iPTMnet (http://proteininformationresource.org/iPTMnet) for PTM knowledge discovery, employing an integrative bioinformatics approach-combining text mining, data mining, and ontological representation to capture rich PTM information, including PTM enzyme-substrate-site relationships, PTM-specific protein-protein interactions (PPIs) and PTM conservation across species. iPTMnet encompasses data from (i) our PTM-focused text mining tools, RLIMS-P and eFIP, which extract phosphorylation information from full-scale mining of PubMed abstracts and full-length articles; (ii) a set of curated databases with experimentally observed PTMs; and iii) Protein Ontology that organizes proteins and PTM proteoforms, enabling their representation, annotation and comparison within and across species. Presently covering eight major PTM types (phosphorylation, ubiquitination, acetylation, methylation, glycosylation, S-nitrosylation, sumoylation and myristoylation), iPTMnet knowledgebase contains more than 654 500 unique PTM sites in over 62 100 proteins, along with more than 1200 PTM enzymes and over 24 300 PTM enzyme-substrate-site relations. The website supports online search, browsing, retrieval and visual analysis for scientific queries. Several examples, including functional interpretation of phosphoproteomic data, demonstrate iPTMnet as a gateway for visual exploration and systematic analysis of PTM networks and conservation, thereby enabling PTM discovery and hypothesis generation.

A novel, simplified strategy of relative quantification N-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q.

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R(2)=0.9999; d8/d0, R(2)=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC-MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.

Prevalence of Genetic Disorders and GLUT1 Deficiency in a Ketogenic Diet Clinic.

Between July of 2012 and December of 2014, 39 patients were enrolled prospectively to investigate the prevalence of glucose transporter 1 (GLUT1) deficiency in a ketogenic diet clinic. None of them had GLUT1 deficiency. All patients seen in the same clinic within the same period were reviewed retrospectively. A total of 18 of these 85 patients had a genetic diagnosis, including GLUT1 deficiency, pathogenic copy number variants, congenital disorder of glycosylation, neuronal ceroid lipofuscinosis type II, mitochondrial disorders, tuberous sclerosis, lissencephaly, and SCN1A-, SCN8A-, and STXBP1-associated epileptic encephalopathies. The prevalence of genetic diagnoses was 21% and prevalence of GLUT1 deficiency was 2.4% in our retrospective cohort study.

Glycosylation Repurposes Alpha-1 Antitrypsin for Resolution of Community-acquired-pneumonia.

Structural basis of nucleotide sugar transport across the Golgi membrane.

Glycosylation is a fundamental cellular process that, in eukaryotes, occurs in the lumen of both the Golgi apparatus and the endoplasmic reticulum. Nucleotide sugar transporters (NSTs) are an essential component of the glycosylation pathway, providing the diverse range of substrates required for the glycosyltransferases. NSTs are linked to several developmental and immune disorders in humans, and in pathogenic microbes they have an important role in virulence. How NSTs recognize and transport activated monosaccharides, however, is currently unclear. Here we present the crystal structure of an NST, the GDP-mannose transporter Vrg4, in both the substrate-free and the bound states. A hitherto unobserved requirement of short-chain lipids in activating the transporter supports a model for regulation within the highly dynamic membranes of the Golgi apparatus. Our results provide a structural basis for understanding nucleotide sugar recognition, and provide insights into the transport and regulatory mechanism of this family of intracellular transporters.

Gastric Cancer Cell Glycosylation as a Modulator of the ErbB2 Oncogenic Receptor.

Aberrant expression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2) constitute crucial molecular events underpinning gastric neoplastic transformation. Despite ErbB2 extracellular domain being a well-known target for glycosylation, its glycosylation profile and the molecular mechanisms through which it actively tunes tumorigenesis in gastric cancer (GC) cells remain elusive. We aimed at disclosing relevant ErbB2 glycan signatures and their functional impact on receptor's biology in GC cells. The transcriptomic profile of cancer-relevant glycosylation enzymes, and the expression and activation of the ErbB receptors were characterized in four GC cell lines. Cellular- and receptor-specific glycan profiling of ErbB2-overexpressing NCI-N87 cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis a (SLe(a)) antigen. The expression of SLe(a) and key enzymes integrating its biosynthetic pathway were strongly upregulated in this GC cell line. An association between the expression of ERBB2 and FUT3, a central gene in SLe(a) biosynthesis, was disclosed in GC patients, further highlighting the crosstalk between ErbB2 and SLe(a) expression. Moreover, cellular deglycosylation and CA 19.9 antibody-mediated blocking of SLe(a) drastically altered ErbB2 expression and activation in NCI-N87 cells. Altogether, NCI-N87 cell line constitutes an appealing in vitro model to address glycan-mediated regulation of ErbB2 in GC.

Marginal stability drives irreversible unfolding of large multi-domain family 3 glycosylhydrolases from thermo-tolerant yeast.

Protein folding is an extremely complex and fast, yet perfectly defined process, involving interplay of many intra and inter-molecular forces. In vitro, these molecular interactions are reversible for many proteins e.g., smaller and monomeric, organized into single domains. However, refolding of larger multi-domain/multimeric proteins is much more complicated, proceeds in a hierarchal way and is often irreversible. In a comparative study on two large, multi-domain and multimeric isozymes, β -glucosidase I (BGLI) and β-glucosidase II (BGLII) from Pichia etchellsii, we studied spontaneous and assisted refolding under three denaturing conditions viz. GdnHCl, alkaline pH and heat. During refolding, higher refolding yields were obtained for BGLII in case of pH induced unfolding (13.89%±0.25) than BGLI (6%±0.85) while for GdnHCl induced unfolding, refolding was marginal (BGLI=5% ±0.5; BGLII=6%±0.69). Thermal unfolding was irreversible while assisted refolding also showed little structural gain for both proteins. When the apparent free energies of unfolding (ΔGU(app)) were calculated from GdnHCl unfolding data, their values were strikingly found to be lower (BGLI ΔGU(app)=3.02kcal/mol; BGLII ΔGU(app)=2.99kcal/mol) than reported for globular (ΔGU=5-15kcal/mol)/multimeric proteins (ΔGU=23-29kcal/mol) indicating marginal stability results in low refolding.

Prospects from Systems Serology Research.

Antibodies are highly functional glycoproteins capable of providing immune protection through multiple mechanisms, including direct pathogen neutralization and the engagement of their Fc-portions with surrounding effector immune cells and immune components that induce anti-pathogenic responses. Small modifications to multiple antibody biophysical features induced by vaccines and other therapeutic regimens can significantly alter functional immune outcomes, though it is difficult to predict which combinations confer protective immunity. In order to give insight into the highly complex and dynamic processes that drive an effective humoral immune response, here we discuss recent applications of "Systems Serology", a new approach that uses data-driven (also called 'machine learning') computational analysis and high-throughput experimental data to infer networks of important antibody features associated with protective humoral immunity and/or Fc functional activity. This approach offers the ability to understand humoral immunity beyond single correlates of protection, assessing the relative importance of multiple biophysical modifications to antibody features with multivariate computational approaches. Systems Serology has the exciting potential to help identify novel correlates of protection from infection and may generate a more comprehensive understanding of the mechanisms behind protection, including key relationships between specific Fc functions and antibody biophysical features (e.g. antigen recognition, isotype, subclass and/or glycosylation events). Reviewed here are some of the experimental and computational technologies available for Systems Serology research and evidence that the application has broad relevance to multiple different infectious diseases including viruses, bacteria, fungi and parasites. This article is protected by copyright. All rights reserved.

Stable isotope labeling and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora.

The THSG biosynthetic pathway in F. multiflora was characterized, and enzymatic activities responsible for the resveratrol synthesis, hydroxylation, and glycosylation reactions involved in THSG biosynthesis were confirmed in vitro. The biosynthetic origin of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (THSG) and the enzymes involved in THSG biosynthesis in Fallopia multiflora were studied using stable isotope labeling and biocatalytic methods. UPLC-MS-based analyses were used to unravel the isotopologue composition of the biosynthetic intermediates and products, as well as to detect the products of the enzyme assay experiments. In this study, (13)C-labeled L-phenylalanine (L-PHE), sodium pyruvate (SP), and sodium bicarbonate (SB) were used as putative precursors in the feeding experiment. Labeling of polydatin (PD) and THSG using [(13)C9]L-PHE and [(13)C1]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [(13)C] SB and [2, 3-(13)C2] SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation. We developed methods using total crude protein extracts (soluble and microsomal) for comprehensive and simultaneous analysis of resveratrol synthase, glycosyltransferase, and hydroxylase activities in various tissue types of wild F. multiflora and callus cultures. The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro. The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F. multiflora.

The α1,3-fucosyltransferase FUT7 regulates IL-1β-induced monocyte-endothelial adhesion via fucosylation of endomucin.

Monocyte-endothelial adhesion is a hallmark feature of atherosclerosis at early stage and emerging evidence suggests that the glycosylation of vascular adhesive molecules and its ligands is involved in this process. Nevertheless, the mechanism underlying this process remains incompletely elucidated. In this study, we reported that treatment with inflammatory factors interleukin-1β (IL-1β) pronouncedly upregulated α1,3-fucosyltransferase VII gene (FUT7) mRNA and protein expression level in EA.hy926 endothelial cells. Moreover, FUT7 overexpression significantly promoted monocyte-endothelial adhesion, while FUT7 knockdown obviously inhibited IL-1β-induced monocyte-endothelial adhesion. Further analysis demonstrated that fucosylation of selectin ligand endomucin was directly involved in IL-1β-induced monocyte-endothelial adhesion. Finally, we demonstrated that p38 and extracellular signal-regulated kinase (ERK) MAPK signaling pathway was activated by IL-1β, while inhibition of p38/ERK signaling pathway decreased FUT7 expression level and IL-1β-induced monocyte-endothelial adhesion. In summary, these results provide a novel insight that FUT7-mediated fucosylation contribute to the initiation and progression of atherosclerosis.

Structural and functional changes of fibrinogen due to aging.

Different factors affect coagulation process. Since fibrinogen is the main coagulation factor, the influence of aging on fibrinogen structure and function was investigated in this study. Fibrinogen was isolated from plasma obtained from healthy persons in the age range 21-83 and examined. Lectin microarray analysis demonstrated increased glycosylation of fibrinogen due to aging, with predominant increase in high-mannose or hybrid type N-glycans, as well as tri-/tetraantennary complex N-glycans with greater content of galactose and N-acetylglucosamine residues. Spectrofluorimetric analysis indicated that fibrinogen molecules have more densely packed structure, but there are no additional advanced glycation end products with increasing age. According to the results of functional analysis, fibrinogen molecules isolated from older persons exhibited reduced clotting time, with significant positive correlation with age, but there were no differences in clotting speed, maximal optical density of fibrin clot, diameter of fibrin fibres, fibrin porosity or reactivity with the insulin-like growth factor binding protein 1. Glycosylation changes of fibrinogen in healthy aging most likely affect its structure and function, namely clotting time. Structural and functional studies of proteins in relation to healthy aging contribute to deeper understanding of mechanisms responsible for longevity.

The role of the carbohydrates in plasmatic membrane.

In the following paper, authors describe glycans present on cell membranes as they affect the folding, the spatial arrangement, the behavior and the interaction with the substrate of some membrane proteins. Authors describe the synthesis and assembly of a glycan on a protein, the formation of N-glycans, the maturation of an N-glycan in different cellular compartments, the structure of the glycocalyx and how it interacts with any pathogens. The study of the E-cadherin and the potassium channel to demonstrate how glycans affect the spatial arrangement, the stability and activity of the glycoproteins on the membranes. Subsequently, authors analyze the correlation between disorder glycosylation and human health. Authors define glycosylation disorders as a genetic defect that alter the structure or biosynthesis of glycans (sugar chains) in one or more biosynthetic pathways. Human glycosylation disorders reflect the disruption of early steps in the pathways of glycan biosynthesis. More in details, authors analyze the role of glycoprotein in tumor cell adhesion, in particular, in cells MCF-7 and MDA-MB-231 on zeolite scaffold. In the same time, the role of metalloproteinase is described in the mobilization of cancer cells and in metastasis.

Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections.

Flaviviruses are enveloped, single-stranded RNA viruses that widely infect many animal species. The envelope protein, a structural protein of flavivirus, plays an important role in host cell viral infections. It is composed of three separate structural envelope domains I, II, and III (EDI, EDII, and EDIII). EDI is a structurally central domain of the envelope protein which stabilizes the overall orientation of the protein, and the glycosylation sites in EDI are related to virus production, pH sensitivity, and neuroinvasiveness. EDII plays an important role in membrane fusion because of the immunodominance of the fusion loop epitope and the envelope dimer epitope. Additionally, EDIII is the major target of neutralization antibodies. The envelope protein is an important target for research to develop vaccine candidates and antiviral therapeutics. This review summarizes the structures and functions of ED I/II/III, and provides practical applications for the three domains, with the ultimate goal of implementing strategies to utilize the envelope protein against flavivirus infections, thus achieving better diagnostics and developing potential flavivirus therapeutics and vaccines.

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis - Mass spectrometry: Assessment and method validation.

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.

Organelle Specific O-Glycosylation Drives MMP14 Activation, Tumor Growth, and Metastasis.

Cancers grow within tissues through molecular mechanisms still unclear. Invasiveness correlates with perturbed O-glycosylation, a covalent modification of cell-surface proteins. Here, we show that, in human and mouse liver cancers, initiation of O-glycosylation by the GALNT glycosyl-transferases increases and shifts from the Golgi to the endoplasmic reticulum (ER). In a mouse liver cancer model, expressing an ER-targeted GALNT1 (ER-G1) massively increased tumor expansion, with median survival reduced from 23 to 10 weeks. In vitro cell growth was unaffected, but ER-G1 strongly enabled matrix degradation and tissue invasion. Unlike its Golgi-localized counterpart, ER-G1 glycosylates the matrix metalloproteinase MMP14, a process required for tumor expansion. Together, our results indicate that GALNTs strongly promote liver tumor growth after relocating to the ER.

Early GalNAc O-Glycosylation: Pushing the Tumor Boundaries.

Glycosylation alterations are frequently observed in cancer cells and shape tumor progression. In this issue of Cancer Cell, Nguyen et al. show that GALNT1 relocation from Golgi to endoplasmic reticulum drives liver tumor growth and invasion, due to enhanced glycosylation and consequential activation of the extracellular matrix-degrading metalloproteinase MMP14.

Identification of plasma membrane glycoproteins specific to human glioblastoma multiforme cells using lectin arrays and LC-MS/MS.

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low- and high-grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low-grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma-specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC-MS/MS. The identified proteins from the two cell types were quantified using label-free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using western blot and GSI lectin-based immunosorbent assay. This article is protected by copyright. All rights reserved.

A dystroglycan mutation (p.Cys667Phe) associated to Muscle-Eye-Brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α-DG and the transmembrane β-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of α-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the β-DG ectodomain has been identified in a patient affected by Muscle-Eye-Brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering (SAXS) data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the β-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy. This article is protected by copyright. All rights reserved.

Animal Cell Expression Systems.

The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenza A virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenza A virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to control. Over the past decade, however, there have been various commercial influenza vaccines made available from cell technology using animal host cells. Analysis of glycosylation control shows that the type of host cell has the greatest influence on the final analyzed glycan profile. Other factors such as the virus strain, the cultivation system, or various process parameters have been shown to have only a minor effect on the glycosylation pattern. We predict that the analysis of glycan profiles in viral vaccines will become increasingly important in the development and consistent manufacturing of safe and potent vaccines. Graphical Abstract.

Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.

Pseudomonas aeruginosa defends against phages through type IV pilus glycosylation.

Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR-Cas, restriction-modification and abortive infection systems (1-4) . Such strategies are effective, but the phage genome-which encodes potentially inhibitory gene products-is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P) (5) , two of which are glycosylated with O-antigen units (6) or polymers of D-arabinofuranose (7-9) . We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.

Immune response of interferon-γ-inducible lysosomal thiol reductase (GILT) from Chinese sturgeon (Acipenser sinensis) to microbial invasion and its antioxdative activity in lipopolysaccharides-treated mammalian dentritic cells.

Interferon-γ-inducible lysosomal thiol reductase (GILT) plays an important role in the major histocompatibility complex-restricted antigen processing of endocytosed proteins via catalyzing the disulfide bond reduction in the endocytic pathway. Here, the cDNA of Chinese sturgeon (Acipenser sinensis) GILT (CsGILT) was cloned. It contained an open reading frame of 762 nucleotides encoding a protein of 254 amino acids with an estimated molecular weight of 28.1 kDa. The characteristic structural features, including a signature sequence CQHGX2ECX2NX4C, a CXXC motif, two potential N-glycosylation sites, and eight conserved cysteines were detected in the deduced amino acid sequence of CsGILT. CsGILT was widely expressed in Chinese sturgeon with the highest expression in the spleen, and CsGILT mRNA expression was significantly up-regulated when Chinese sturgeons were challenged with polyinosinic polycytidylic acid or Vibrio anguillarum. The recombinant CsGILT displayed obvious thiol reductase activity demonstrated by catalyzing the reduction of mouse IgG(H+L) by dithiothreitol into heavy chain and light chain. CsGILT also displayed significant antioxidant activity in mouse dentritic cells as indicated by its increasing GSH level and GSH/GSSG ratio, decreasing intracellular reactive oxygen species and nitric oxide levels and lipid peroxidation, as well as enhancing the activities of the antioxidative redox enzymes including catalase and superoxide dismutase. Our results suggested an important role for CsGILT in the immune response in Chinese sturgeon to pathogen invasion possibly via a conserved functional mechanism throughout vertebrate evolution, contributing to our understanding the immune biology and protection of Chinese sturgeon.