Insect Glycoproteins Research Articles

Identification and characterization of an α-1,3 mannosidase from Elizabethkingia meningoseptica and its potential attenuation impact on allergy associated with cross-reactive carbohydrate determinant

Core α-1,3 mannose is structurally near the core xylose and core fucose on core pentasaccharide from plant and insect glycoproteins. Mannosidase is a useful tool for characterization the role of core α-1,3 mannose in the composition of glycan related epitope, especially for those epitopes in which core xylose and core fucose are involved. Through functional genomic analysis, we identified a glycoprotein α-1,3 mannosidase and named it MA3. We used MA3 to treat allergen horseradish peroxidase (HRP) and phospholipase A2 (PLA2) separately. The results showed that after MA3 removed α-1,3 mannose on HRP, the reactivity of HRP with anti-core xylose polyclonal antibody almost disappeared. And the reactivity of MA3-treated PLA2 with anti-core fucose polyclonal antibody decreased partially. In addition, when PLA2 was conducted enzyme digestion by MA3, the reactivity between PLA2 and allergic patients’ sera diminished. These results demonstrated that α-1,3 mannose was an critical component of glycan related epitope.

Transmission parameters of pepper whitefly-borne vein yellows virus (PeWBVYV) by Bemisia tabaci and identification of an insect protein with a putative role in polerovirus transmission

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission.

Cross-reactive carbohydrate determinant-specific IgE obscures true atopy and exhibits ⍺-1,3-fucose epitope-specific inverse associations with asthma.

BackgroundIn high-income, temperate countries, IgE to allergen extracts is a risk factor for, and mediator of, allergy-related diseases (ARDs). In the tropics, positive IgE tests are also prevalent, but rarely associated with ARD. Instead, IgE responses to ubiquitous cross-reactive carbohydrate determinants (CCDs) on plant, insect and parasite glycoproteins, rather than to established major allergens, are dominant. Because anti-CCD IgE has limited clinical relevance, it may impact ARD phenotyping and assessment of contribution of atopy to ARD.MethodsUsing an allergen extract-based test, a glycan and an allergen (glyco)protein microarray, we mapped IgE fine specificity among Ugandan rural Schistosoma mansoni (Sm)-endemic communities, proximate urban communities, and importantly in asthmatic and nonasthmatic schoolchildren.ResultsOverall, IgE sensitization to extracts was highly prevalent (43%-73%) but allergen arrays indicated that this was not attributable to established major allergenic components of the extracts (0%-36%); instead, over 40% of all participants recognized CCD-bearing components. Using glycan arrays, we dissected IgE responses to specific glycan moieties and found that reactivity to classical CCD epitopes (core β-1,2-xylose, α-1,3-fucose) was positively associated with sensitization to extracts, rural environment and Sm infection, but not with skin reactivity to extracts or sensitization to their major allergenic components. Interestingly, we discovered that reactivity to only a subset of core α-1,3-fucose-carrying N-glycans was inversely associated with asthma.ConclusionsCCD reactivity is not just an epiphenomenon of parasite exposure hampering specificity of allergy diagnostics; mechanistic studies should investigate whether specific CCD moieties identified here are implicated in the protective effect of certain environmental exposures against asthma.

Cross-reactive carbohydrate determinant interference in cellulose-based IgE allergy tests utilizing recombinant allergen components.

BackgroundCross-reactive carbohydrate determinant (CCD) structures found in plant and insect glycoproteins are commonly recognized by IgE antibodies as epitopes that can lead to extensive cross-reactivity and obscure in vitro diagnostic (IVD) serology results. With the introduction of component resolved diagnosis (CRD), recombinant non-glycosylated components have been utilized to mitigate the risk of CCD-specific IgE (sIgE) detection. However, a recent study has shown that CCD-sIgE may bind directly to the cellulose solid phase matrix used in certain in vitro diagnostic assays, eliminating the advantage of CRD over traditional extract-based testing. The aim of this study is to further investigate the prevalence of CCD-sIgE interference on a commonly-used in vitro sIgE automated platform which employs a cellulose-based matrix to immobilize CCD-free recombinant components.MethodsSera from patients sensitized to peanut, silver birch, and/or timothy grass were analyzed for CCD-sIgE reactivity on ImmunoCAP/Phadia and NOVEOS autoanalyzers against the MUXF3 carbohydrate component. Positive CCD-sIgE sera were further analyzed against non-glycosylated recombinant components bound to the ImmunoCAP solid phase in the absence and presence of a soluble CCD inhibitor. For comparison, sera were then analyzed on NOVEOS, a non-cellulose based automated sIgE assay.ResultsSera from 35% of the sensitized population tested in this study were positive (≥0.35 kU/L) for CCD-sIgE. Of those positives, 17% resulted in CCD-sIgE-positive (false positive) results on ImmunoCAP using non-glycosylated allergosorbents that were negative on NOVEOS. Sera producing false-positive results on ImmunoCAP had varying levels of CCD-sIgE from 0.67 kU/L to 36.52 kU/L. The incidence of CCD interference was predominantly delimited to low-positive IgE results (0.35 kUA/L– 3.00 kUA/L).ConclusionFalsely elevated diagnostic allergen-sIgE results can commonly occur due to the presence of CCD-sIgE using assays that employ a carbohydrate matrix-based allergosorbent. Even the use of non-glycosylated recombinant allergenic components coupled to cellulose matrices do not reduce their risk of detection. The risk of CCD interference that compromises quantitative IgE results can be mitigated by the addition of a soluble CCD inhibitor to positive CCD-sIgE containing sera or by alternatively using a non-cellulose based sIgE assay, such as the NOVEOS assay.

Microarray assessment of N-glycan-specific IgE and IgG profiles associated with Schistosoma mansoni infection in rural and urban Uganda

Core β-1,2-xylose and α-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core β-1,2-xylose and α-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core β-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core α-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core β-1,2-xylose- and α-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core β-1,2-xylose and α-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.

Cross-Reactive Carbohydrate Determinants (CCDs) Interference with Component Resolved Diagnosis on NOVEOS™*, ImmunoCAP® and IMMULITE® 2000.

Allergen specific IgE (sIgE) can bind CCD structures on plant and insect glycoproteins as well as peptide epitopes. These carbohydrate structures can display structural homology across many allergen groups leading to extensive cross-reactivity, obscure in vitro diagnostic results, and debatable clinical relevance. With the introduction of Component Resolved Diagnosis (CRD), non-glycosylated components can be utilized to prevent the detection of anti-CCD IgE and may improve clinical relevance. The aim of this study is to confirm anti-CCD IgE will not be detected on non-glycosylated components on NOVEOS, ImmunoCAP, and IMMULITE 2000. A panel of CCD-positive samples was selected based on their reactivity to several recombinant components. Samples were tested before and after inhibition with a commercially-available CCD inhibitor on NOVEOS, ImmunoCAP, and IMMULITE 2000. ImmunoCAP IgE binding was reduced by samples pre-incubated with a CCD inhibitor while NOVEOS and IMMULITE 2000 resulted in minimal reduction. The reduction of IgE binding to a non-glycosylated recombinant component from samples with a CCD inhibitor on ImmunoCAP suggests trace amount of CCD are still present in the test. This confirms other studies that have indicated the cellulose used as an allergen carrier on ImmunoCAP may elevate specific IgE result. NOVEOS, a microparticle-based chemistry, and IMMULITE 2000, bead-based technology, resulted in minimal IgE reduction with and without CCD inhibitor.

Evolutionarily conserved and species-specific glycoproteins in the N-glycoproteomes of diverse insect species

N-glycosylation is one of the most abundant and conserved protein modifications in eukaryotes. The attachment of N-glycans to proteins can modulate their properties and influences numerous important biological processes, such as protein folding and cellular attachment. Recently, it has been shown that protein N-glycosylation plays a vital role in insect development and survival, which makes the glycans an interesting target for pest control. Despite the importance of protein N-glycosylation in insects, knowledge about insect N-glycoproteomes is scarce. To fill this gap, the N-glycosites were identified in proteins from three major pest insects, spanning different insect orders and diverging in post-embryonic development, feeding mechanism and evolutionary ancestry: Drosophila melanogaster (Diptera), Tribolium castaneum (Coleoptera) and Acyrthosiphon pisum (Hemiptera). The N-glyco-FASP method for isolation of N-glycopeptides was optimized to study the insect N-glycosites and allowed the identification of 889 N-glycosylation sites in T. castaneum, 941 in D. melanogaster and 1338 in A. pisum. Although a large set of the corresponding glycoproteins is shared among the three insects, species- and order-specific glycoproteins were also identified. The functionality of the insect glycoproteins together with the conservation of the N-glycosites throughout evolution is discussed. This information can help in the elaboration of novel pest insect control strategies based on interference in insect glycosylation.

Reduction of cross-reactive carbohydrate determinants in plant foodstuff: elucidation of clinical relevance and implications for allergy diagnosis.

BackgroundA longstanding debate in allergy is whether or not specific immunoglobulin-E antibodies (sIgE), recognizing cross-reactive carbohydrate determinants (CCD), are able to elicit clinical symptoms. In pollen and food allergy, ≥20% of patients display in-vitro CCD reactivity based on presence of α1,3-fucose and/or β1,2-xylose residues on N-glycans of plant (xylose/fucose) and insect (fucose) glycoproteins. Because the allergenicity of tomato glycoallergen Lyc e 2 was ascribed to N-glycan chains alone, this study aimed at evaluating clinical relevance of CCD-reduced foodstuff in patients with carbohydrate-specific IgE (CCD-sIgE).Methodology/Principal FindingsTomato and/or potato plants with stable reduction of Lyc e 2 (tomato) or CCD formation in general were obtained via RNA interference, and gene-silencing was confirmed by immunoblot analyses. Two different CCD-positive patient groups were compared: one with tomato and/or potato food allergy and another with hymenoptera-venom allergy (the latter to distinguish between CCD- and peptide-specific reactions in the food-allergic group). Non-allergic and CCD-negative food-allergic patients served as controls for immunoblot, basophil activation, and ImmunoCAP analyses. Basophil activation tests (BAT) revealed that Lyc e 2 is no key player among other tomato (glyco)allergens. CCD-positive patients showed decreased (re)activity with CCD-reduced foodstuff, most obvious in the hymenoptera venom-allergic but less in the food-allergic group, suggesting that in-vivo reactivity is primarily based on peptide- and not CCD-sIgE. Peptide epitopes remained unaffected in CCD-reduced plants, because CCD-negative patient sera showed reactivity similar to wild-type. In-house-made ImmunoCAPs, applied to investigate feasibility in routine diagnosis, confirmed BAT results at the sIgE level.Conclusions/SignificanceCCD-positive hymenoptera venom-allergic patients (control group) showed basophil activation despite no allergic symptoms towards tomato and potato. Therefore, this proof-of-principle study demonstrates feasibility of CCD-reduced foodstuff to minimize ‘false-positive results’ in routine serum tests. Despite confirming low clinical relevance of CCD antibodies, we identified one patient with ambiguous in-vitro results, indicating need for further component-resolved diagnosis.

Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins

Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance. We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies. IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance. From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG). Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

Differentiation of Isomeric N-Glycan Structures by Normal-Phase Liquid Chromatography−MALDI-TOF/TOF Tandem Mass Spectrometry

The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.

The Drosophila fused lobes Gene Encodes an N-Acetylglucosaminidase Involved in N-Glycan Processing

Most processed, e.g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNA corresponding to fused lobes (fdl), a putative beta-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the alpha1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-beta-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdl-deficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.

Spiroplasma citri Spiralin Acts In Vitro as a Lectin Binding to Glycoproteins from Its Insect Vector Circulifer haematoceps

ABSTRACT In order to understand the molecular mechanisms underlying transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps, we screened leafhopper proteins as putative S. citri-binding molecules using a spiroplasma overlay assay of protein blots (Far-western assay). Insect proteins were separated by one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted, and probed with S. citri proteins. In this in vitro assay, we found that spiroplasma proteins exhibited affinity for seven leafhopper proteins. The interactions between S. citri proteins and insect proteins with molecular masses of 50 and 60 kDa were found to be sugar sensitive. These insect proteins were identified as high mannose N-glycoproteins, which support an interaction of glycoprotein-lectin type with S. citri proteins. Lectin detection in S. citri has revealed only one protein of 24 kDa. Using a leafhopper protein overlay assay on an S. citri protein blot, one spiroplasma protein with a similar molecular mass of 24 kDa was shown to display an insect protein-binding capacity. This protein was identified as the spiralin, which is the most abundant membrane protein of S. citri. Far-western experiments performed with purified spiralin and insect glycoproteins confirmed the binding of spiralin to the insect glycoproteins of 50 and 60 kDa. Thus, the spiralin could play a key role in the transmission of S. citri by mediating spiroplasma adherence to epithelial cells of insect vector gut or salivary gland.

The N‐linked oligosaccharides of aminopeptidase N from Manduca sexta

Mass spectrometric studies on the N-linked glycans of aminopeptidase 1 from Manduca sexta have revealed unusual structures not previously observed on any insect glycoprotein. Structure elucidation of these oligosaccharides was carried out by high-energy collision-induced dissociation (CID) using a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometer. These key experiments revealed that three out of the four N-linked glycosylation sites in this protein (Asn295, Asn623 and Asn752) are occupied with highly fucosylated N-glycans that possess unusual difucosylated cores. Cross-ring fragment ions and 'internal' fragment ions observed in the CID spectra, showed that these fucoses are found at the 3-position of proximal GlcNAc and at the 3-position of distal GlcNAc in the chitobiose unit. The latter substitution has only been previously observed in nematodes. In addition, these core structures can be decorated with novel fucosylated antennae composed of Fucalpha(1-3)GlcNAc. Key fragment ions revealed that these antennae are predominantly found on the upper 6-arm of the core mannose. The paucimannosidic N-glycan (Man(3)GlcNAc(2)), commonly found on other insect glycoproteins, is the predominant oligosaccharide found at the remaining N-glycosylation site (Asn609).

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin.

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.

Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains.

350-kDa royal jelly glycoprotein (apisin), which stimulates proliferation of human monocytes, bears the beta1-3galactosylated N-glycan: analysis of the N-glycosylation site.

While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galbeta1-3GlcNAcbeta1-4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and (1)H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galbeta1-3GlcNAcbeta1-4(GlcNAcbeta1-2)Manalpha1-3 (Manalpha1-3Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both beta1-3 galactosyl and beta1-4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifying an insect glycoprotein bearing the beta1-3 galactosylated N-glycan.

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins.

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins. By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.

Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R.

To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selected from a panel of anti-glycan mAbs generated from Schistosoma-infected or immunized mice because it recognized both a plant glycoprotein horseradish peroxidase and phospholipase A2 from honeybee venom. On further characterization, it was shown that mAb 100-4G11-A recognizes the truncated biantennary N-glycan Man3GlcNAc2-R. Immunocytochemical analysis and immunoblotting with this mAb demonstrated that Man3GlcNAc2-R structures occur on many glycoproteins of schistosomes and other invertebrates. Remarkably, Man3GlcNAc2-R is also expressed on a restricted number of vertebrate glycoproteins. Our data indicate that this truncated N-glycan is immunogenic in mice during the course of infection. Nevertheless, no elevated antibody levels against this glycan epitope could be detected in sera of individuals infected with Schistosoma mansoni.

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.

Purification, cDNA Cloning, and Expression of GDP-l-Fuc:Asn-linked GlcNAc α1,3-Fucosyltransferase from Mung Beans

Substitution of the asparagine-linked GlcNAc by alpha1,3-linked fucose is a widespread feature of plant as well as of insect glycoproteins, which renders the N-glycan immunogenic. We have purified from mung bean seedlings the GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase (core alpha1,3-fucosyltransferase) that is responsible for the synthesis of this linkage. The major isoform had an apparent mass of 54 kDa and isoelectric points ranging from 6. 8 to 8.2. From that protein, four tryptic peptides were isolated and sequenced. Based on an approach involving reverse transcriptase-polymerase chain reaction with degenerate primers and rapid amplification of cDNA ends, core alpha1,3-fucosyltransferase cDNA was cloned from mung bean mRNA. The 2200-base pair cDNA contained an open reading frame of 1530 base pairs that encoded a 510-amino acid protein with a predicted molecular mass of 56.8 kDa. Analysis of cDNA derived from genomic DNA revealed the presence of three introns within the open reading frame. Remarkably, from the four exons, only exon II exhibited significant homology to animal and bacterial alpha1,3/4-fucosyltransferases which, though, are responsible for the biosynthesis of Lewis determinants. The recombinant fucosyltransferase was expressed in Sf21 insect cells using a baculovirus vector. The enzyme acted on glycopeptides having the glycan structures GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4GlcNAcbeta1-Asn, GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1- 6)Manbeta1-4GlcNAcbet a1-4(Fucalpha1-6)GlcNAcbeta1-Asn, and GlcNAcbeta1-2Manalpha1-3[Manalpha1-3(Manalpha1-6 )Manalpha1-6]Manbeta1 -4GlcNAcbeta1-4GlcNAcbeta1-Asn but not on, e.g. N-acetyllactosamine. The structure of the core alpha1,3-fucosylated product was verified by high performance liquid chromatography of the pyridylaminated glycan and by its insensitivity to N-glycosidase F as revealed by matrix-assisted laser desorption/ionization time of flight mass spectrometry.