Complex-type N-linked Oligosaccharides Research Articles

Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of Its Conjugated Peptide.

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.

PrPc: The Normal Prion

PrPc is a normal cell‐surface glycoprotein that is conformationally characterized by two alpha helices and two complex‐type N‐linked oligosaccharide chains. This protein is unique in its propensity to misfold into a neurodegenerative disease‐causing proteinaceous infectious particle, known as a prion. PrPc can undergo conversion into PrPSc through spontaneous misfolding, a genetic mutation of the human PRNP gene, or exposure to a prion from an external source. When this happens, its composition shifts from an alpha‐helical, soluble protein low in beta pleated sheets to an insoluble, protease‐resistant particle with a high percentage of beta sheets. This resulting misfolded form, known formally as PrPSc, is pathogenic and aggregates in neural tissue by recruiting and converting more PrPc to PrPSc, ultimately causing neuronal cell dysfunction followed by cell death. The prion's form of “self‐replication” without utilizing genetic material is particularly fascinating, as it appears to defy the central dogma of biology and differs from all other pathogenic particle infection mechanisms. It is notable that some prions have evolved to act as transmissible infectious agents, similar to viruses, despite the absence of genetic material. This distinguishes some prion diseases, such as chronic wasting disease (CWD) and scrapie, from their inherited disease counterparts. The biosynthesis pathway of PrPc is similar to other cellular proteins, with replication occurring on the ribosomes on the endoplasmic reticulum. However, whereas malfunctioning proteins are normally tagged by ubiquitin and then degraded in a proteasome, the ubiquitin‐proteasome complex is inhibited during prion formation, allowing PrPSc to form and survive degradation, leading to its buildup in cytosol and subsequent neurotoxicity. Human and animal prion diseases have been classified into three broad categories‐‐genetic, sporadic, and acquired‐‐based on properties of the PrP proteins and prion infection morphology. Physical symptoms of prion infection include deviant behavior, dementia, ataxia, insomnia, paraplegia, and paresthesias. Commonly discussed prion‐induced diseases (differentiated via host type and slight conformational discrepancies between misfolded proteins) include scrapie, kuru, CJD, bovine spongiform encephalopathy (mad cow disease), and CWD. Here, we present current research on prions, with a focus on CWD, which was first found in Colorado and affects deer and other cervids.The Olathe North High School Team 1 MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships and histology of PrPc, or the normal cellular prion.Support or Funding InformationThis project is a joint venture between the Milwaukee School of Engineering and the Medical Professions Academy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

Protecting-Group-Free Synthesis of Glycopolymers Bearing Sialyloligosaccharide and Their High Binding with the Influenza Virus.

Glycopolymers having pendant triazole-linked sialyloligosaccharides were successfully synthesized from free saccharides without any protection of the hydroxy and carboxy groups on the saccharides. The glycomonomers were synthesized by the direct azidation of free saccharides using 2-chloro-1,3-dimethylimidazolinium chloride as a condensing agent followed by copper(I)-catalyzed azide-alkyne cycloaddition. The resultant glycomonomers were copolymerized with acrylamide by a reversible addition-fragmentation chain transfer technique. Each of the glycopolymers were obtained and then immobilized on a gold-coated sensor of quartz crystal microbalance to analyze their binding behavior with the lectin. The glycopolymers strongly bound with the corresponding lectin without nonspecific adsorption in aqueous solution. In addition, the glycopolymer bearing a complex-type sialyl N-linked oligosaccharide was found to strongly bind with both human and avian influenza A viruses. The strong binding, observed using the hemagglutination inhibition assay, was attributed to the glycocluster effect of the glycopolymer and the biantennary structure of the N-linked oligosaccharide.

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60kDa accompanied by a broad band of 60–90kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn161 and Asn241 on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.

Cytotoxicity and Glycan-Binding Profile of a d-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (Aplysia kurodai)

A divalent cation-independent 16 kDa D-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized D-galactose and D-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl D-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k(ass) and k(diss) values are 2.4 × 10³ M⁻¹ s⁻¹ and 3.8 × 10⁻³ s⁻¹, respectively. AKL-2 appeared cytotoxicity against both Burkitt's lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.

Synthesis of the glycosylated polypeptide chain of an inducible costimulator on T-cells

The glycoprotein AILIM/ICOS (Activation inducible lymphocyte immunomediately molecule/Inducible co-stimulator) on T-cells was identified in 1998 as a member of the CD28/CTLA4 family. The three-dimensional structure of the AILIM/ICOS extracellular domain has not been solved, and therefore we have examined the preparation of homogeneous glycosylated polypeptide chains of this domain having two homogeneous N-linked complex type oligosaccharides for use in folding experiments. To synthesize the glycosylated whole polypeptide chain of the AILIM/ICOS extracellular domain, the target polypeptide chain was divided into four segments, each containing a cysteine residue. Those peptide segments were synthesized by conventional SPPS, followed by thioesterification of the C-terminus. The oligosaccharide moiety, a biantennary complex type disialyloligosaccharide, was attached to the cysteine thiol in the peptide backbone using the haloacetamide method. These peptides, as well as a glycosylated peptide, were sequentially coupled by use of native chemical ligation. This process successfully afforded the desired polypeptide chain having homogeneous oligosaccharides.

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.

Strategic Glycan Elution Map for the Production of Human-TypeN-Linked Oligosaccharides: The Case of Hen Egg Yolk and White

Glycans play important roles in various biological phenomena, but the lack of a systematic procedure for producing complex structures of glycans severely restricts their application in the medical and industrial fields. In this paper, we propose a basic strategy for the preparation of substantial amounts (>100 mg) of N-linked oligosaccharides, where the structure of each glycan is mapped with its elution position in liquid chromatography as well as the empirical yield. In model experiments using hen egg white and yolk as starting materials, the former provided a series of agalactosylated complex-type and hybrid-type N-linked oligosaccharides containing bisecting N-acetylglucosamine (GlcNAc) in addition to two high-mannose type glycans. In contrast, egg yolk gave predominantly alpha2-6sialylated biantennary glycans together with a high-mannose type one, reflecting the difference in the origins of the tissues. Due to the total identity of the glycans obtained to human ones, the present strategy should provide a practical scheme for the production of human-type N-linked oligosaccharides.

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.

Cell surface N-glycosylation and sialylation regulate galectin-3-induced apoptosis in human diffuse large B cell lymphoma

Galectin-3 is a soluble endogenous lectin in vertebrates and is implicated in a variety of biological functions, including tumor cell adhesion, proliferation, differentiation, apoptosis, cancer progression and metastasis. In the present study, we analyzed the role of galectin-3 in apoptosis in human malignant lymphoma. Galectin-3 induced cell death in the HBL-2 human diffuse large B cell lymphoma cell line. A morphological examination and annexin V assays revealed that galectin-3-induced cell death is consistent with apoptosis and swainsonine, a potent inhibitor of alpha-mannosidase II, which catalyzes the synthesis of complex type N-linked oligosaccharides, inhibited galectin-3-induced apoptosis in HBL-2 cells. These results suggest that galectin-3 induces apoptosis in HBL-2 cells by interacting with cell surface N-linked oligosaccharides. Furthermore, treatment of cells with Vibrio Cholerae neuraminidase enhanced galectin-3-induced apoptosis, suggesting that cell surface sialylation regulates galectin-3-induced apoptosis in human B cell lymphoma. In conclusion, our results indicate that galectin-3-induced apoptosis is regulated by cell surface expression of N-glycans and sialic acid in human diffuse large B cell lymphoma. This mechanism may be involved in the malignant behavior of human lymphoma cells.

Regulatory roles of altered N- and O-glycosylation of CD45 in galectin-1-induced cell death in human diffuse large B cell lymphoma

To clarify the functions of CD45 N- and O-glycans in diffuse large B cell lymphoma (DLBCL), we analyzed the antiproliferative effects of bovine galectin-1 (beta-galactoside-binding lectin-1), which reacts with CD45 N-glycans and O-glycans, on human lymphoma cells. Bovine galectin-1 induced cell death of the DLBCL cell line HBL-2 in vitro. Swainsonine (SW) is a potent inhibitor of alpha-mannosidase II which catalyzes the synthesis of complex type N-linked oligosaccharides, and benzyl-GalNAc (BZGalNAc) is a potent inhibitor of O-glycosylation. Treatment with SW or BZGalNAc prevented cell death of HBL-2 cells by galectin-1. Western blot analysis revealed SW treatment reduced the molecular weight by about 5 kDa of one isoform at 190 kDa among three isoforms of CD45 which have N-linked oligosaccharide ligands for galectin-1. BZGalNAc treatment reduced the molecular weight of another isoform by about 15 kDa. These data suggest that the amount of CD45 N-glycans or O-glycans was reduced by SW and BZGalNAc treatment, respectively, and that reduction of CD45 N-glycans or O-glycans may prevent the interaction between CD45 and galectin-1. Alteration in CD45 N-glycans or O-glycans may regulate cell death of lymphoma cells through the interaction between CD45 N-glycans or O-glycans and galectin-1 in DLBCL.

Altered N-glycosylation in CD45 and regulatory roles of altered N-glycosylation in galectin-1-induced growth inhibition in human diffuse large B cell lymphoma

Beta1-6 branching of L-PHA reactive oligosaccharides, one of the N-glycan structures, plays an important role in the biological behavior of various tumor cell lines. We reported previously that the expression of L-PHA reactive oligosaccharides was closely associated with the prognosis of patients with human diffuse large B cell lymphoma (DLBCL). In the present study, by Western blotting, we analyzed the N-glycosylation patterns in CD45 having L-PHA reactive oligosaccharides. In two cases of DLBCL which do and do not express non-sialylated L-PHA reactive oligosaccharides CD45 was found to be about 180-210 kDa and 180-200 kDa, respectively. Furthermore, after endoglycosidase F3 treatment the CD45 in both cases was found to be 190 or 160 kDa. Therefore, the differences in CD45 molecular weight between the two cases is due to differences in the amount of N-glycosylation. To clarify the biological functions of CD45 N-glycans in DLBCL, we analyzed the antiproliferative effects on human lymphoma cells of bovine galectin-1 (beta-galactoside-binding lectin-1), which reacts with CD45 N-glycans. Bovine galectin-1 stimulation of the DLBCL cell line HBL-2 resulted in inhibition of its growth in vitro. Swainsonine (SW) is a potent inhibitor of alpha-mannosidase II, which catalyzes the synthesis of complex type N-linked oligosaccharides. Reduction in expression of N-linked oligosaccharides, including L-PHA reactive oligosaccharides, on the cell surface by SW treatment prevented the growth inhibition of HBL-2 cells by galectin-1. On Western blots one 190 kDa isoform of the three CD45 isoforms which have N-linked oligosaccharide ligands for galectin-1, was detected with a reduction in molecular weight of about 5 kDa after SW treatment. These data suggested that the amount of CD45 N-glycans is reduced by SW treatment, and that this reduction of N-glycans prevents the interaction between CD45 and galectin-1. Alteration in N-glycosylation of CD45 may regulate lymphoma cell growth in DLBCL through the interaction between the N-glycans of CD45 and galectin-1.

Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans

The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

Regulatory roles of N-glycosylation of immunoglobulin M in CD40-CD40L-mediated cell survival of human diffuse large B cell lymphoma

L-PHA reactive oligosaccharides are found on the surface of HBL-2 cells, a lymphoma cell line, established from a human diffuse large B cell lymphoma (DLBCL). Swainsonine (SW) is a potent inhibitor of alpha-mannosidase II which catalyzes the biosynthesis of complex type N-linked oligosaccharides in human cells. CD40L stimulation of HBL-2 cells leads to their prolonged survival. Reduction in the expression of N-linked oligosaccharides, including L-PHA reactive oligosaccharides, on the cell surface by SW treatment resulted in enhancement of HBL-2 cell survival by CD40L stimulation. From an Annexin V assay the enhancement of CD40L-mediated HBL-2 cell survival by SW treatment may have resulted from anti-necrotic effects after 48 h of incubation. Bcl-2 enzyme linked immuno sorbent assay (ELISA) data showed that the expression of bcl-2 protein was enhanced by CD40L stimulation alone and also by CD40L stimulation along with SW treatment. However, there were no significant differences in the amount of bcl-2 protein with these treatments. Therefore, the enhancement of CD40L-mediated cell survival by SW treatment did not depend on the enhancement of bcl-2 protein expression. Furthermore, SW treatment of HBL-2 cells led to degradation of the heavy chain of IgM and rescued HBL-2 cells from anti-IgM-induced growth inhibition. Anti-IgM induced growth inhibition of HBL-2 cells prevented the inhibition of cell death by CD40L. From the present results it is possible that reduction of N-glycosylation of the heavy chain of IgM by SW treatment may reduce anti-IgM-induced growth inhibition, and reduction in anti-IgM-induced growth inhibition due to altered N-glycosylation may enhance CD40-CD40L-mediated cell survival through TRAF2 which interacts with both IgM and CD40 in HBL-2 cells.

Biosynthesis in vitro of Caenorhabditis elegans phosphorylcholine oligosaccharides.

The biosynthesis in vitro of phosphorylcholine oligosaccharides in Caenorhabditis elegans has been investigated. Here we show that extracts of C. elegans' microsomes transfer phosphorylcholine from L-alpha-dipalmitoyl phosphatidylcholine to hybrid and complex type N-linked oligosaccharides containing mannose residues disubstituted with N-acetylglucosamine. The reaction products are consistent with structures reported for C. elegans as well those found in the filarial nematodes Acanthocheilonema viteae, Onchocerca volvulus, and Brugia malayi, strongly supporting the concept that the phosphorylcholine oligosaccharide biosynthetic enzymes are conserved in this group of organisms. Because it is thought that phosphorylcholine substitution of oligosaccharides modulates host immune response in filarial infections, this in vitro system may help in gaining an understanding of the basis for this response.

The carbohydrate moiety of serum IgM from Atlantic cod (Gadus morhua L.)

The carbohydrate moiety of cod serum IgM was analysed using oligosaccharide sequencing techniques. The carbohydrate moiety constituted about 10% of the molecular weight of cod IgM, was associated with the constant region of the heavy chains (Fc), and was composed of N-linked complex type oligosaccharides. Considerable heterogeneity was observed. Sixteen different glycan structures were identified, over 60% were sialylated and 40% contained core fucose. The carbohydrate moiety of cod IgM was shown to provide protection against protease digestion, and partial deglycosylation abolished the antigen binding property of natural cod anti-TNP-BSA antibody.

Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with α1–6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and α- and β-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactoseβ1–3/4/6 N-acetylglucosamine-, sialic acidα2–3/6galactose-, fucoseα1–2galactose- and galactoseα1–3galactose- were not detected. Terminal α-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies

Effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells.

We have investigated the effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells to elucidate the role of carbohydrate in the triggering of T cell function. The inhibitors of glycoprotein processing, especially mannnosidase inhibitors, enhanced the anti CD3-induced production of interleukin-2 (IL-2), which is a cytokine without the linkage sequence of N-linked oligosaccharides. On the other hand, N-methyl-1-deoxynojirimycin (NMdNM, an inhibitor of processing glucosidase 1), 1-deoxynojirimycin (dNM, an inhibitor of processing glucosidase I and II) and bromoconduritol (BCD, an inhibitor of processing glucosidase II) inhibited the secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma), or interleukin-5 (IL-5) into culture supernatants of anti CD3-stimulated T cells, which had N-linked oligosaccharides. Mannosidase inhibitors, 1-deoxymannojirimycin (dMAN, an inhibitor of processing mannosidase I) and swainsonine (SWN, an inhibitor of processing mannosidase II) did not inhibit the secretion or production of IL-4, IFN-gamma and IL-5. To confirm the inhibition of N-linked oligosaccharide processing in the cytokines by the above inhibitors, the binding of IFN-gamma to lectins with various sugar-binding specificities was investigated. All inhibitors reduced the binding of IFN-gamma to PHA E4, which had a high affinity to bi- or tri-antennary complex type N-linked oligosaccharides with bisecting N-acetylglucosamine. Similarly, all inhibitors reduced the binding of IFN-gamma to PHA L4, which had high affinity to tri- or tetra-antennary complex type N-linked oligosaccharides with beta1-6-linked branching. SWN and dMAN increased the binding of IFN-gamma to concanavalin A (ConA), which had a high affinity to bi-antennary complex type, hybrid type and high-mannose type N-linked oligosaccharides. These results suggest that the processing inhibitors used here inhibit the N-linked oligosaccharide processing of cytokines, and the inhibition of processing enzyme glucosidases I and II induces a decreased secretion of cytokines with N4-linked oligosaccharides.

Molecular cloning and characterization of cDNA coding for beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum.

In plants as well as in animals beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides. Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs. The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system. Using pyridylaminated Man(5)- or Man(3)GlcNAc(2)as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain. Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR. Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N. tabacum and N.benthamiana.