Complex N-linked Oligosaccharides Research Articles

Improving the cryoprotective effect of antifreeze proteins from Daucus carota on plant-based meat by eliminating N-glycosylation

As a novel animal meat alternative, plant-based meat (PBM) frequently suffers from quality problems as a result of freeze–thaw cycles in commercial transportation and household storage. There is a need to reduce the deterioration of PBM attributes, such as water holding capacity, as a result of these freeze–thaw cycles. In this study, Daucus carota antifreeze protein (DcAFP) and its deglycosylated mutant DcAFP-N294G were heterologously expressed in Komagataella phaffii X33. The effects of pretreatment with recombinant AFPs (rAFPs) on the microstructure, rheological properties, water mobility, and water distribution of PBM were assessed. The rDcAFP-N294G-treated PBM samples had superior viscoelasticity and water distribution features compared to the rDcAFP-treated group because the complex N-linked oligosaccharides did not interfere with the binding of rAFPs to ice molecules. In addition, rAFP pretreatment resulted in a smoother and flatter surface of the high-moisture protein extrudate matrix compared to the commercial cryoprotectant trehalose. Deglycosylated DcAFP has potential applications as a new effective cryoprotectant in meat alternatives.

The Roles of Envelope Glycoprotein M in the Life Cycle of Some Alphaherpesviruses.

The envelope glycoprotein M (gM), a surface virion component conserved among alphaherpesviruses, is a multiple-transmembrane domain-containing glycoprotein with a complex N-linked oligosaccharide. The gM mediates a diverse range of functions during the viral life cycle. In this review, we summarize the biological features of gM, including its characterization and function in some specicial alphaherpesviruses. gM modulates the virus-induced membrane fusion during virus invasion, transports other proteins to the appropriate intracellular membranes for primary and secondary envelopment during virion assembly, and promotes egress of the virus. The gM can interact with various viral and cellular components, and the focus of recent research has also been on interactions related to gM. And we will discuss how gM participates in the life cycle of alphaherpesviruses.

N-acetylglucosaminyltransferase I promotes glioma cell proliferation and migration through increasing the stability of the glucose transporter GLUT1.

Abnormal alteration of N-glycosylation structure contributes to glioma progression. N-acetylglucosaminyltransferase I (MGAT1) plays an essential role in the conversion of processed high-mannose cores into complex or hybrid N-linked oligosaccharide structures. The function of MGAT1 in glioma development remains largely unknown. Here, we found that the expression of MGAT1 is higher in glioblastoma compared to normal brain tissues. Inhibition of EGFR signalling pathway or serum starvation reduces MGAT1 expression. Knockdown of MGAT1 inhibits glioma cell proliferation and migration. Furthermore, MGAT1 promotes complex N-glycosylation of glucose transporter 1 (Glut1) and increases Glut1 protein levels. In summary, our findings indicate that MGAT1 is highly expressed in glioblastoma and promotes glioma cells at least partly through upregulation of Glut1 protein.

4. The Role of Glycosylation in Cardiac Arrhythmias

Long QT syndrome (LQTS) is a debilitating cardiac arrhythmia, and mutations or malfunctions in the human ether-a-go-go–related gene (hERG) are the most common cause of LQTS. hERG is responsible for the cardiac potassium current (IKr), which is important for cardiac repolarization. It has 6 transmembrane domains, ranging from S1 to S6, and a complex N-linked oligosaccharide chain resides on the extracellular S5 pore linker region. The role of this glycosylation chain in hERG function has not yet been well distinguished and in the current study we aim to investigate the relationship between hERG protease susceptibility and the presence of the glycosylation chain. Using molecular techniques, we demonstrate that de-glycosylated hERG channels are degraded by the serine protease Proteinase K at a much faster rate than their glycosylated counter parts. Additionally, through removal of the end-chain sialic acid residues using the enzyme neuraminidase, we conclude that it is likely the physical blockade of susceptible protease sites that grants wild type hERG this glycosylation-dependent protection as opposed to charge repulsion facilitated by specific sugars in the carbohydrate chain. Our data show that the glycosylation chain found on hERG plays a role in protecting the channel from protease degradation, and could provide valuable insight into the development of the cardiac arrhythmia Long QT Syndrome.

Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation

ABSTRACTTo survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.

THE ROLE OF GLYCOSYLATION IN CARDIAC REPOLARIZATION

The human ether-a-go-go-related gene (hERG) encodes the pore-forming alpha subunit of the rapidly activating delayed rectifier potassium current (IKr), which is important for cardiac repolarization. Dysfunction of hERG due to drug block or mutations can result in reduced IKr, which is associated with the development of Long QT Syndrome (LQTS). LQTS is a cardiac arrhythmia seen on an ECG as an increased QT interval, and can result in syncope or sudden death. hERG has 6 transmembrane domains, ranging from S1 to S6, and a complex N-linked oligosaccharide chain which resides on the extracellular S5 pore linker region. Glycosylation chains have been associated with numerous roles in cell and protein function, such as protection from protease-induced degradation. The role of the glycosylation chain found on hERG in the channel’s function and expression has not yet been fully revealed, and could provide insight into the cellular mechanisms behind hERG-related LQTS. In the current study, we aim to investigate the relationship between hERG susceptibility to protease degradation and the presence of the glycosylation chain. Human Embryonic Kidney Cells expressing hERG channels were cultured with the glycosylation-reducing agents tunicamycin (an antibiotic that prevents the addition of N-linked glycosylation) and neuraminidase (an exoglycosidase that removes sialic acid residues). Tunicamycin and neuraminidase-treated cells were then subjected to protease degradation by Proteinase K (PK) and Calpain-1. hERG protease susceptibility was measured either through Western blot fragment intensity quantification or through whole cell voltage clamp analysis. Scorpion toxin BeKm-1 was added to cell culture media with PK, then washed out prior to protease susceptibility analysis. We demonstrate that unglycosylated hERG channels are more susceptible to protease-induced degradation by PK and Calpain-1. Removal of the end-chain sialic acid residues using the enzyme neuraminidase did not alter the glycosylation-mediated protease protection afforded to wild-type hERG channels. Additionally, we showed that the scorpion toxin BeKm-1 requires the glycosylation chain to protect hERG channels from PK cut. Our findings indicate that the glycosylation chain found on mature hERG channels plays a key role in protecting the channel from protease digestion, and suggests a glycosylation-mediated mechanism for the BeKm-1-mediated hERG channel protection from PK cut.

Glycosylation patterns of proteins in trophoblast and decidua of the cat placenta

Glycosylation patterns of proteins in trophoblast and decidua of the cat placenta

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Human herpesvirus 6 glycoprotein M is essential for virus growth and requires glycoprotein N for its maturation

Human herpesvirus 6 (HHV-6) is a T-lymphotropic virus belonging to the betaherpesvirus family. Several HHV-6-encoded glycoproteins are required for cell entry and virion maturation. Glycoprotein M (gM) is conserved among all herpesviruses, and therefore thought to have important functions; however, the HHV-6 g has not been characterized. Here, we examined the expression of HHV-6 g, and examined its function in viral replication, using a mutant and revertant gM. HHV-6 g was expressed on virions as a glycoprotein modified with complex N-linked oligosaccharides. As in other herpesviruses, HHV-6 g formed a complex with glycoprotein N (gN), and was transported from the endoplasmic reticulum to the trans-Golgi network only when part of this complex. Finally, a gM mutant virus in which the gM start codon was destroyed was not reconstituted, although its revertant was, indicating that HHV-6 g is essential for virus production, unlike the gM of alphaherpesviruses.

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions

Members of the peroxidase–cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase–cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase–cyclooxygenase family, including the catalysis of dityrosine formation.

Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases.

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galbeta1-4GlcNAc units terminated by (alpha2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (alpha1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (alpha2-6)-linked sialic acid residues.

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK–tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI–TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI–TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl-agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galbeta1-3GlcNAc) and type 2 (Galbeta1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k(ass) and k(diss) values are 5.14x10(4) M(-1) s(-1) and 2.9x10(-3) s(-1), respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.

Varicella-Zoster Virus Glycoprotein M Homolog Is Glycosylated, Is Expressed on the Viral Envelope, and Functions in Virus Cell-to-Cell Spread

Although envelope glycoprotein M (gM) is highly conserved among herpesviruses, the varicella-zoster virus (VZV) gM homolog has never been investigated. Here we characterized the VZV gM homolog and analyzed its function in VZV-infected cells. The VZV gM homolog was expressed on virions as a glycoprotein modified with a complex N-linked oligosaccharide and localized mainly to the Golgi apparatus and the trans-Golgi network in infected cells. To analyze its function, a gM deletion mutant was generated using the bacterial artificial chromosome system in Escherichia coli, and the virus was reconstituted in MRC-5 cells. VZV is highly cell associated, and infection proceeds mostly by cell-to-cell spread. Compared with wild-type VZV, the gM deletion mutant showed a 90% reduction in plaque size and 50% of the cell-to-cell spread in MRC-5 cells. The analysis of infected cells by electron microscopy revealed numerous aberrant vacuoles containing electron-dense materials in cells infected with the deletion mutant virus but not in those infected with wild-type virus. However, enveloped immature particles termed L particles were found at the same level on the surfaces of cells infected with either type of virus, indicating that envelopment without a capsid might not be impaired. These results showed that VZV gM is important for efficient cell-to-cell virus spread in cell culture, although it is not essential for virus growth.

Enhanced binding affinity for FcγRIIIa of fucose-negative antibody is sufficient to induce maximal antibody-dependent cellular cytotoxicity

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be an important therapeutic function for clinical efficacy of monoclonal antibodies. Recent studies have revealed two methods to increase binding affinity for FcγRIIIa and enhance ADCC more efficiently for antibodies: (i) fucose removal from antibody N-linked complex oligosaccharides and (ii) amino acid mutations in the antibody Fc region. In this study, we compare the biological activities of the methods of generating high ADCC antibodies. We used a fucose-negative antibody and two antibodies with sets of mutations, demonstrated previously to optimally enhance ADCC using the chimeric anti-CD20 antibody, rituximab, as the model. Both amino acid mutant antibodies showed a significantly higher affinity for recombinant FcγRIIIa than fucose-negative antibody when compared using biosensor analysis. The removal of fucose from the antibodies bearing amino acid mutations exhibited a further enhancement of binding to recombinant FcγRIIIa and significantly increased binding to natural killer (NK) cells. Despite the differences manifested in binding for the FcγR, ADCCs were indistinguishable between methods and even when the methods were combined. These results indicate that the affinity of binding to FcγRIIIa does not predict ADCC beyond a certain threshold and that each method alone is sufficient to induce maximal ADCC of the antibody.

Rapid analysis of oligosaccharides derived from glycoproteins by microchip electrophoresis

A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (μ-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G 2), cellobiose ( G ′ 2 ), maltriose (G 3) and panose ( G ′ 3 ) as oligosaccharide isomer models. In μ-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, α 1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by μ-CE, indicating that the present μ-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.

Characterization of caprine herpesvirus 1 glycoprotein D gene and its translation product

Caprine herpesvirus 1 (CpHV-1) is responsible of systemic infection in neonatal kids as well as abortion and fertility disorders in adult goats. This virus is closely related to bovine herpesvirus 1 (BoHV-1) which causes infectious bovine rhinotracheitis. Glycoprotein D (gD) mediates important functions in alphaherpesviruses and is also a main immunogen. The sequence of CpHV-1 gD gene and the biochemical properties of its translation product were analyzed and compared to those of BoHV-1 and other alphaherpesviruses. A relatively high homology was found between CpHV-1 and BoHV-1 glycoproteins D amino acid sequences (similarity of 68.8%). Moreover, six cysteine residues are conserved by CpHV-1 gD and the other studied alphaherpesviruses. CpHV-1 gD has a molecular mass similar to BoHV-1 gD and contains complex N-linked oligosaccharides. In contrast to the BoHV-1 gD, CpHV-1 gD is expressed as a late protein. In spite of the observed differences which could influence its biological functions, CpHV-1 gD shares most characteristics with other alphaherpesviruses and especially BoHV-1.

Improved Effector Functions of a Therapeutic Monoclonal Lewis Y-Specific Antibody by Glycoform Engineering

The aim of the present study was to produce glycosylation variants of the therapeutic Lewis Y-specific humanized IgG1 antibody IGN311 to enhance cell-killing effector function. This was achieved via genetic engineering of the glycosylation machinery of the antibody-producing host. Antibody genes were transiently cotransfected with acetyl-glycosaminyltransferase-III genes into human embryonic kidney-EBV nuclear antigen cells. A control wild-type antibody, IGN311wt, was expressed in the same host using identical expression vectors, but without cotransfection of genes for acetyl-glycosaminyltransferase-III expression. Both expression products were purified to homogeneity and characterized. The glyco-engineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, non-fucosylated and agalactosylated form carrying a bisecting GlcNAc-group. Wild-type expression product (IGN311wt) on the other hand was glycosylated by a multitude of different core-fucosylated complex-type structures of variable degrees of galactosylation. Target affinity of the glyco-engineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a approximately 10-fold increase of antibody-dependent cellular cytotoxicity of the glyco-engineered antibody using different Lewis Y-positive target cancer cell lines (SK-BR-3, SK-BR-5, OVCAR-3, and Kato-III). Complement-mediated cytotoxicity of IGN312-Glyco-I was 0.4-fold reduced using SK-BR-5 as target cell line. The reduction of complement activation could be prevented and even converted into a slight increase of activity by using a different molecular-biological approach directing the glycosylation towards increased levels of complex N-linked oligosaccharides of bisected, non-fucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.

Biosynthesis of the IFN-γ binding protein of ectromelia virus, the causative agent of mousepox

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-γ binding protein (IFN-γBP) with homology to the ligand binding domains of the IFN-γ high affinity receptor (IFN-γR1). Unlike the cellular receptor, the IFN-γBP binds IFN-γ from several species. The IFN-γBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with M r of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-γBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-γBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Regulation of ganglioside biosynthesis in the nervous system

Regulation of ganglioside biosynthesis in the nervous system