Abstract. Many West Nile (WN) virus isolates associated with significant outbreaks possess a glycosylation site on the envelope (E) protein. E-protein glycosylated variants of New York (NY) strains of WN virus are more neuroinvasive in mice than the non-glycosylated variants. To determine how E protein glycosylation affects the interactions between WN virus and avian hosts, we inoculated young chicks with NY strains of WN virus containing either glycosylated or non-glycosylated variants of the E protein. The glycosylated variants were more virulent and had higher viremic levels than the non-glycosylated variants. The glycosylation status of the variant did not affect viral multiplication and dissemination in mosquitoes in vivo. Glycosylated variants showed more heat-stable propagation than non-glycosylated variants in mammalian (BHK) and avian (QT6) cells but not in mosquito (C6/36) cells. Thus, E-protein glycosylation may be a requirement for efficient transmission of WN virus from avian hosts to
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Centralized Modularity of N-Linked Glycosylation Pathways in Mammalian Cells. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.
N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.. ...
A majority of all biologically active proteins are glycosylated and various diseases have proven to correlate with alterations in protein glycosylation. Sensitive identification of different glycoprotein glycoforms is therefore of great diagnostic value. Here we describe a method with potential for glycoprotein profiling, based on lectins as capture probes immobilized on particulate substrates in the nm-range. The nanoparticles present high concentrations of attachment sites for specific ligands and cause minimal steric hindrance to binding. In the present model study the mannose-binding lectin ConA has been coupled to polystyrene nanoparticles via a poly(ethyleneoxide) linker which protects the protein conformation and activity and prevents unspecific protein adsorption. The ConA-coated particles are accommodated at different spots on the analytical surface via oligonucleotide linkage. This attachment, which relies on the hybridization of complementary oligonucleotides, allows firm fixation of ...
HIV-1 envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs) and the focus for design of an antibody-based HIV vaccine. The Env trimer is covered by ∼90N-linked glycans, which shield the underlying protein from immune surveillance. bNAbs to HIV develop during infection, with many showing dependence on glycans for binding to Env. The ability to routinely assess the glycan type at each glycosylation site may facilitate design of improved vaccine candidates. Here we present a general mass spectrometry-based proteomics strategy that uses specific endoglycosidases to introduce mass signatures that distinguish peptide glycosites that are unoccupied or occupied by high-mannose/hybrid or complex-type glycans. The method yields ,95% sequence coverage for Env, provides semi-quantitative analysis of the glycosylation status at each glycosite. We find that most glycosites in recombinant Env trimers are fully occupied by glycans, varying in the proportion of ...
Glycosylation, or the attachment of glycans (sugars) to proteins, is the most abundant post-translational modification in nature and plays a pivotal role in protein folding and activity. Glycans are involved in almost every human disease and biological process. Glycosylation is also important in biotechnology; about 70% of protein therapeutics approved or in development are glycosylated. By merging bottom-up engineering design principles with innovative molecular biology methodologies in a cell-free environment, we seek to create a simplified framework for studying and engineering glycosylation. Our envisioned platform will broaden the glycoengineering toolkit, facilitate discovery of the structural and functional consequences of glycan attachment, and enable a new era of applications in glycoprotein therapeutics and conjugate vaccines.. ...
Flavocytochrome b558 of the NADPH oxidase which generates superoxide in phagocytic cells, is a α1β1 heterodimer of gp91phox and p22phox, which together form a membrane-spanning electron-transport chain that transfers electrons from NADPH in the cytosol to oxygen. The C-terminal portion of gp91phox is a member of the ferredoxin-NADP+ reductase family of reductases. Little is known of the organization of the N-terminal section of this molecule, which is associated with the two haem structures. It is N-glycosylated, and site-directed mutagenesis has been used to eliminate the five potential N-linked glycosylation consensus sites. Mutated cDNAs were expressed in vitro. This approach provided evidence for glycosylation of residues Asn131, Asn148 and Asn239, but not of Asn96 and Asn429.. ...
Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand ...
Kathrin Stavenhagen: Glycopeptide analysis remains challenging because of its sample heterogeneity resulting from the degree of glycosylation site occupancy (macroheterogeneity) and the different glycoforms attached to individual glycosylation sites (microheterogeneity).. With respect to the latter one, qualitative site-specific glycosylation information of glycoproteins can be obtained by unspecific protease treatment resulting in small amino acid stretches carrying the glycan. This improves determination of the glycosylation sites. However, detecting these glycopeptides by 1D-LC-ESI-MS/MS is challenging due to insufficient or irreversible retention on the stationary phase and thus multiple analyses with different LC-setups are required. Since biological sample amounts are usually limited, methods for acquiring comprehensive information in a single run are necessary.. To obtain qualitative information of the glycosylation site we set up an integrated C18-porous graphitized carbon ...
Abnormal glycosylation is a hallmark of many cancers that contributes to tumor growth and invasion. There are many protein receptors that are regulated abnormally in cancer due to mutations and/or alterations in glycosylation. Studies to link specific glycosylation changes to signaling outcomes have primarily focused on studies of individual receptors or specific pathways.
The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation ...
Following the footsteps of genomics and proteomics, recent years have witnessed the growth of large-scale experimental methods in the field of glycomics. In parallel, there has also been growing interest in developing Systems Biology based methods to study the glycome. The combined goals of these endeavors is to identify glycosylation-dependent mechanisms regulating human physiology, check points that can control the progression of pathophysiology, and modifications to reaction pathways that can result in more uniform biopharmaceutical processes. In these efforts, mathematical models of N- and O-linked glycosylation have emerged as paradigms for the field. While these are relatively few in number, nevertheless, the existing models provide a basic framework that can be used to develop more sophisticated analysis strategies for glycosylation in the future. The current review surveys these computational models with focus on the underlying mathematics and assumptions, and with respect to their ...
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of ...
patterns, provoking an immune response when treating humans with GPs produced in plants. State of the art approaches are based either on glycosylation mutants or overproduction of the desired protein product (intending to override the cellular glycosylation machinery). Though the mutants either show impaired yield or loss of vitality in comparison to wild type, or the proteins do not terminate in mannose residues, which is essential for the biological uptake via mannose receptors in patients with lysosomal storage diseases. This invention provides a new device to generate GPs with hypo-allergenic properties in a cost-effective way: In contrast to current methods, the new technique enables effective production of heterologous GPs by vital plants without impairments. The technology is based on a genetic modification of the N-glycosylation pathway. It is applicable to Solanaceous and other plants of agronomical interest as transgenic producers of GPs. The suppression of a specific enzyme leads to a ...
Increased understanding of the role of protein- and lipid-linked carbohydrates in a wide range of biological processes has led to interest in drugs that target the enzymes involved in glycosylation. But given the importance of carbohydrates in fundamental cellular processes such as protein folding, therapeutic strategies that modulate, rather than ablate, the activity of enzymes involved in glycosylation are likely to be a necessity. Two such approaches that use imino sugars to affect glycosylation enzymes now show considerable promise in the treatment of viral infections, such as hepatitis B, and glucosphingolipid storage disorders, such as Gaucher disease.
A variety of sulfo-protected monosaccharide donors and acceptors were investigated in glycosylation reactions. Trifluoroethylsulfonate (SO3TFE) group was compatible with a wide range of activation conditions commonly used with fluoride, imidate, and sulfide donors. In addition, the influence of a SO3TFE group, at the critical 2-position in glycosyl donor, on the stereoselectivity of the glycosylation reaction was studied ...
A chemical gycosylation reaction involves the coupling of a glycosyl donor, to a glycosyl acceptor forming a glycoside. If both the donor and acceptor are sugars, then the product is an oligosaccharide. The reaction requires activation with a suitable activating reagent. The reactions often result in a mixture of products due to the creation of a new stereogenic centre at the anomeric position of the glycosyl donor. The formation of a glycosidic linkage allows for the synthesis of complex polysaccharides which may play important roles in biological processes and pathogenesis and therefore having synthetic analogs of these molecules allows for further studies with respect to their biological importance. The glycosylation reaction involves the coupling of a glycosyl donor and a glycosyl acceptor via initiation using an activator under suitable reaction conditions. A glycosyl donor is a sugar with a suitable leaving group at the anomeric position. This group, under the reaction conditions, is ...
There are multiple factors that drive glycosylation and several currently available approaches to influence glycosylation patterns. Notable factors that influence glycosylation include the following: cell culture process conditions, ingredients in cell culture, genetic selection or engineering, and directly impacting the physiological organelles responsible for glycosylation (endoplasmic reticulum - the ER - and Golgi apparatus). In cell line development there are opportunities to use genetic engineering tools to manipulate the glycosylation pathway. In upstream development, cell culture process conditions (ie. temperature and pH) can be manipulated. Lastly, commercially available constituents in the glycan process, such as precursors and enzymes, can be added to cell culture media to further hone glycoprofiles. However, there have been limited available approaches to improve the functionality of the ER and Golgi.. One relatively unexplored lever to improve ER and Golgi functionality, and thus ...
Dr Fahey (Saldova)s main area of expertise is Glycobiology. She focuses on development and utilisation of technologies for glycan analyses, including automated high-throughput glycan analysis as well as detailed glycan characterization.. She also focuses on the role of system biology in regulation of glycosylation in health and disease (disorders of glycosylation, cancer and chronic inflammatory diseases). She has been looking at "Epigenetic regulation of glycosylation and the impact on chemoresistance in cancer" (SFI-SIRG) which follows on from novel published findings demonstrating that changes in DNA methylation in ovarian cancer cells are concomitant with significant alterations in the expression of key enzymes that form part of glycosylation. Epigenetic alterations, tumour hypoxia and glycosylation are integral aspects of carcinogenesis. Determining the mechanism of epigenetic regulation of glycosylation and the link with hypoxia and drug resistance will help to improve the treatment of ...
Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and ...
Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS3 in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylaton that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular ...
The most abundant modification of proteins in eukaryotes is N-linked glycosylation:more than 10000 different acceptor sites are N-glycosylated in the mouse proteome. The process is essential because it underpins the folding and quality control of non-cytoplasmic (organelle-targeted), membrane-embedded, or secreted proteins. N-glycosylation is involved in organism development, the immune response, and host-pathogen interactions. Finally, a multitude of diseases are linked to the dysfunction of this process, including the various congenital disorders of glycosylation (CDGs).This proposal focuses on the central enzyme in the N-glycosylation pathway, oligosaccharyltransferase (OST). OST is a complex molecular machine that is embedded in the membrane of the Endoplasmic Reticulum (ER), where it catalyzes the transfer of a glycan moiety from a lipid-linked oligosaccharide (LLO) onto acceptor proteins that contain a recognition sequence N-X-S/T (the glycosylation sequon). After its covalent attachment ...
To-date, no claim regarding finding a consensus sequon for O-glycosylation has been made. Thus, predicting the likelihood of O-glycosylation with sequence and structural information using classical regression analysis is quite difficult. In particular, if a binary response is used to distinguish between O-glycosylated and non-O-glycosylated sequences, an appropriate set of non-O-glycosylatable sequences is hard to find. Three sequences from similar post-translational modifications (PTMs) of proteins occurring at, or very near, the S/T-site are analyzed: N-glycosylation, O-mucin type (O-GalNAc) glycosylation, and phosphorylation. Results found include: 1) The consensus composite sequon for O-glycosylation is: ~(W-S/T-W), where
AGA of pentasaccharide 5 utilized three different glycosylation modules (A1, B1 and B1*, see Scheme 1 and ESI†). Each module uses three times three equivalents of glycosylating agent followed by an activator wash step with TMSOTf solution added at −30 °C, and the resin is agitated for 1 min before removal of the activator. Glycosylation module A1 activates three equivalents of glycosyl phosphate building block 1 with stoichiometric amounts of TMSOTf at −30 °C. After maintaining −30 °C for 10 min, the temperature is raised to −15 °C and maintained for 25 min. Glycosylation module B1 activates three equivalents of glycosyl phosphate building block 2 at −30 °C and allows 10 min for glycosylation, but raises the temperature to −10 °C and maintains it for 25 min to account for the lower reactivity of 2 caused by the presence of the Lev group. Glycosylation module B1* uses B1 reaction conditions for building block 1 since the C3-hydroxyl of 2 is expected to be a weaker nucleophile ...
Experts say glycosylation analysis is the most important and least precise area of chemical analysis. Moreover, there is no magic bullet for analyzing glycosylation in monoclonal antibodies. The methods for identifying the chemical structures are not well developed at this time. Even two results from the same lab using the same method on the same sample can vary.
Mannose is a succar monomer o the aldohexose series o carbohydrates. Mannose is a C-2 epimer o glucose. Mannose is important in human metabolism, especially in the glycosylation o certain proteins. Several congenital disorders o glycosylation are associated wi mutations in enzymes involved in mannose metabolism.[1]. ...
Profiling of glycosylation gene expression in CHO fed-batch cultures in response to glycosylation-enhancing medium components. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.
DUGi: Viewing Item from repository Recercat: Glycoconjugates constitute a major class of biomolecules which include glycoproteins, glycosphingolipidsand proteoglycans. The enzymatic process in which glycans (sugar chains) are linked toproteins or lipids is called glycosylation. Glycosylation is involved in many biological processes, bothphysiological and pathological, inlcuding host-pathogen interactions, tumour invasion, cell traffickingand signalling. Changes in glycan structure are thought be be at least partly responsible for the developmentof inflammation, infection, arteriosclerosis, immune defects and autoimmunity. Such changeshave been observed in human diseases such as diabetes mellitus, rheumatoid arthritis and AlzheimersDisease. Aberrant patterns of glycosylation are also a universal feature of cancer cells. The field ofglycobiology thus shows great potential for the discovery of glycan biomarkers for disease diagnosisand prognosis.Here we focus specifically on N-glycans, that is, glycans
Glycosylation is a post-translational modification (PTM) that exerts profound structural and functional effects on the modified protein. Glycan synthesis and conjugation to proteins are regulated by a myriad of factors, both genetic and environmental, and are also influenced by external stressors. Glycosylation patterns are known to vary in correlation to a large number of diseases; therefore, it is possible to study such alterations to identify reliable biomarkers and help elucidate mechanisms underlying the disease. For these reasons, the development of analytical methods able to investigate the glycosylation of proteins in complex samples and to measure and characterize disease-related alterations is of great importance.. In this thesis, the development and application of rapid and small-scale methods for the analysis of the glycosylation pattern on specific proteins in biological fluids, with a high degree of automation and potential for parallel sample treatment, is presented.. Paper I ...
In past years, several strategies have been evaluated in our laboratory to express rFel d 1, starting with separate expression of both chains of the molecule in P. pastoris (unpublished data), and subsequently, expression of both chains coupled by a linker sequence (this study). In our hands, this approach resulted in a rFel d 1 preparation with good immune reactivity but an undesirable degree of molecular heterogeneity caused by hyperglycosylation of a fraction of the molecules at the N-linked glycosylation site in the β-chain of Fel d 1. Additionally, some instability of the linker sequence resulted in partial cleavage of the two-chain heterodimer (confirmed by N-terminal sequencing of the resulting fragments). Both post-translational modifications did not significantly affect immune reactivity but the observed heterogeneity is less favorable from a production standpoint. A mutant lacking the N-linked glycosylation site showed decreased heterogeneity, but was still partially cleaved (not ...
Acetylation and glycosylation are widespread posttranslational modifications (PTMs) involved in diverse cellular processes and can affect the structure of proteins. We have demonstrated that proteins can misfold and form amyloid structures upon inhibition of histone deacetylases (HDACs) in clinically relevant concentrations. Acetylation plays a role in the aetiology of ageing-related proteinopathies such as in Alzheimers disease, Parkinsons disease or Huntingtons disease. Furthermore, aggregate formation is fostered by mis-glycosylation of proteins; for instance, mutations in one key enzyme of terminal glycosylation (sialylation) are responsible for the age-dependent GNE (bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase) myopathy. We aim to investigate how acetylation and glycosylation influence protein folding and misfolding, in particular proteins involved in ageing-related diseases. We also analyse, how protein shuttling factors can ameliorate protein misfolding ...
Characterization of post-translational modifications (PTMs) of therapeutic proteins is very important during the bioprocess development to maintain desired product quality and during the submission process to regulatory authorities for product approval. Monitoring glycosylation in pharmacokinetic studies can be useful to evaluate the dependence of clearance rates on different glycoforms. The cost and efficiency of characterization affect the speed to market of biopharmaceutical proteins. A reduction in the number of manual processing steps, cost of reagents and consumption of sample, as well as the time required for chemical analysis, is therefore necessary.. The research presented in this thesis is focused on the potential of using microfluidic discs for automated, miniaturized, parallel and rapid sample preparation for PTM characterization of therapeutic monoclonal antibodies. Paper I describes the method development for N-linked glycosylation profiling. Several sample preparation steps have ...
Malignant transformation of cells is associated with aberrant glycosylation presented on the cell-surface. Commonly observed changes in glycan structures during malignancy encompasses aberrant expression and glycosylation of mucins; abnormal branching of N-glycans; and increased presence of sialic acid on proteins and glycolipids. Accumulating evidence supports the notion that the presence of certain glycan structures correlates with cancer progression by affecting tumor cell invasiveness, ability to disseminate through the blood circulation and to metastasize in distant organs. During metastasis tumor cell-derived glycans enable binding to cells in their microenvironment including endothelium and blood constituents through glycan-binding receptors - lectins. In this review we will discuss current concepts how tumor cell-derived glycans contribute to metastasis with the focus on three types of lectins: siglecs, galectins and selectins. Siglecs are present on virtually all hematopoetic cells and usually
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan (S.O., Y.K., T.F., T.Y., M.N.); CLEA Japan, Fujinomiya, Japan (M.H.); DMPK Research Laboratories, Sohyaku Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Toda, Japan (A.I.); and Discovery Technology Laboratories, Sohyaku Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Toda, Japan (T.K.) ...
The present invention provides methods and compositions for improved expression and production of recombinant antibodies in prokaryotic expression systems. Particularly contemplated are prokaryotic expression and production of full length aglycosylated antibodies. The antibody products of the invention can be used in various aspects of biological research, diagnosis and medical treatment.
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How is low-molecular-mass highly glycosylated protein abbreviated? L-HGP stands for low-molecular-mass highly glycosylated protein. L-HGP is defined as low-molecular-mass highly glycosylated protein rarely.
Asn-linked glycosylation of proteins is a universal cotranslational and posttranslational modification of proteins entering the secretory pathway. In all eukaryotes, a hallmark of N-glycosylation is the en bloc transfer of a common preassembled oligosaccharide (Glc3Man9GlcNAc2) from the lipid carrier dolichol pyrophosphate to selected Asn residues in the sequence Asn-X-Ser/Thr (where X ≠ P) within nascent polypeptides. This transfer takes place in the lumen of the endoplasmic reticulum (ER) and is catalyzed by the oligosaccharyltransferase (OST), a heteromeric membrane protein complex (Kelleher and Gilmore, 2006). In yeast (Saccharomyces cerevisiae) and mammals, OST consists of a catalytically active subunit (STAUROSPORIN AND TEMPERATURE SENSITIVE3 [STT3]) and several different noncatalytic subunits that contribute to N-glycosylation by regulation of the substrate specificity, stability, or assembly of the complex (Yan and Lennarz, 2002; Mohorko et al., 2011). In yeast, the OST complex is ...
The stereoselectivity of glycosylation … … reactions can depend critically on the reactivity of the acceptor glycoside (the nucleophile in the reaction). In their Communication on page 8240 ff., J. D. CodEe et al. report a facile system that maps the relationship between glycosyl acce...
Sevigny M.B., Li C.F., Alas M., Hughes-Fulford M.. Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H(2). COX-2 exists as 72 and 74kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn(580). In this study, Asn(580) was mutated to determine the biological significance of this variable glycosylation. COS-1 cells transfected with the mutant gene were unable to express the 74kDa glycoform and were found to accumulate more COX-2 protein and have five times greater COX-2 activity than cells expressing both glycoforms. Thus, COX-2 turnover appears to depend upon glycosylation of the 72kDa glycoform.. FEBS Lett. 580:6533-6536(2006) [PubMed] [Europe PMC] ...
A purified mammalian proteoglycan, and genetic information encoding such proteoglycans, having a core polypetide molecular weight of about 30 kD to about 35 kD, and comprising a hydrophilic amino terminal extracellular region, a hydrophilic carboxy terminal cytoplasmic region, a transmembrane hydrophobic region between said cytoplasmic and extracellular regions, a protease susceptible cleavage sequence extracellularly adjacent the transmembrane region of the peptide, and at least one glycosylation site for attachment of a heparan sulfate chain to said extracellular region, said glycosylation site comprising a heparan sulfate attachment sequence represented by a formula Xac-Z-Ser-Gly-Ser-Gly, where Xac represents an amino acid residue having an acidic sidechain, and Z represents from 1 to 10 amino acid residues. Additional peptides having this glycosylation site and genetic information useful for preparing a number of variations based on this glycosylation site are also provided.
Toda la información sobre las últimas publicaciones científicas de la Clínica Universidad de Navarra. Acquired potential N-glycosylation sites within the tumor-specific immunoglobulin heavy chains of B-cell malignancies
The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific HA binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44H-Rg and ...
N-linked glycosylation is not required for hIL-6 receptor binding, STAT signaling or cytokine-dependent B9.11 cell proliferation. (A) Calibration of quantities
One of the most common posttranslational modifications of eukaryotic proteins is glycosylation. Glycosylation of proteins can affect many biological activities. For therapeutic glycoproteins, it can modify biological activity, targeting, trafficking, serum half life, clearance, and recognition by receptors (1, 2). For such reasons, biomanufacturers must monitor and characterize the glycosylation patterns of their recombinant therapeutic proteins (3, 4). Therapeutic proteins have two main types of glycosylation: N-linked glycans and O-linked glycans (5). Attachment of an N-glycan starts in the endoplasmic…. ...
The LIP-6 MAb was produced against the undifferentiated cell line bh2-1 and recognizes an antigen expressed on all pre-B and B cell lines tested and some myeloid lineage lines. FACS analysis of normal tissues showed that LIP-6 is expressed on B lineage cells at all stages of differentiation, from bone marrow pre-B to plasma cells. T cells and thymocytes are LIP-6-, and splenic CD11b+ cells are heterogeneous for LIP-6 expression. The LIP-6 MAb was shown to precipitate a major 75-kDa and a minor 85-kDa protein under reducing conditions and a large protein of | 240 kDa under nonreducing conditions. Removal of N-linked sugars from the reduced lysates resulted in a single 65-kDa protein, suggesting that there is differential glycosylation of a single 65-kDa protein that forms disulfide-linked multimers. Finally, the LIP-6 antigen was shown not to be linked to the cell surface via a GPI linkage.
Highly conserved threonine residues were noted near the C-terminus of the external surface glycoproteins of HIV-1, SIV, and influenza A virus; this threonine residue was shown to be the efficient target of O-glycosylation on all three viruses. In all three cases, this O-glycosylated threonine was essential for the infectivity of the virus. We will define the functional role of C-terminal threonine glycosylation for HIV-1 and we will develop assays amenable to high throughput screening for the development of antiviral drugs. We will delineate protein-peptide and peptide-peptide interactions that are dependent on the O-glycosylated threonine of gp120. We will also examine whether there are rare examples of naturally-occurring HIV-1 sequences that are functional without an O-glycosylated threonine at this location ...
Highlights: • Glycosylatable GFP (gGFP) is developed for the use in mammalian cells. • gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. • Differential fluorescence/glycosylation pattern probes membrane protein topology. • Membrane topology of URG7, MRP6{sub 102}, and SP-C was determined by gGFP tagging in vivo. - Abstract: Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6{sub 102}, SP-C(Val) and ...
Structural Analysis of Glycosylated Peptides in Complex Mixtures with Ion Trap MS n Shiaw-Lin Wu Pavel Bondarenko Tom Shaler Paul Shieh and William S. Hancock Protein Glycosylation The data presented here can be acquired using a,Structural,Analysis,of,Glycosylated,Peptides,in,Complex,Mixtures,with,,,,,,,,,,,,Ion,Trap,MSn,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology