The Structural Difference of Isobaric N-Glycans of Two Microalgae Samples Reveals Taxonomic Distance.

Microalgae of the Chlorella clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed Chlorella serves as wholesome food additive. A recent study on 80 commercial Chlorella preparations revealed an unexpected variety of protein-linked N-glycan patterns with unprecedented structural features, such as the occurrence of arabinose. Two groups of products exhibited a characteristic major N-glycan isobaric to the Man2GlcNAc2XylFuc N-glycan known from pineapple stem bromelain, but tandem mass spectrometry (MS/MS) analysis pointed at two types of N-glycan different from the bromelain structure, as well as from each other. Here we report the exact structures of these two novel N-glycan structures, elucidated by nuclear magnetic resonance spectroscopy and MS/MS, as well as on their phylogenetic context. Despite their humble size, these two N-glycans exhibited a very different design with structural features unrelated to those recently described for other Chlorella-clade strains. The major glycans of this study presented several novel structural features such as substitution by arabinose or xylose of the internal N-acetylglucosamine, as well as methylated sugars. ITS1-5.8S-ITS2 rDNA barcode analyses revealed that the xylose-containing structure derived from a product primarily comprising Scenedesmus species, and the arabinose-containing glycan type related to Chlorella species (SAG211-34 and FACHB-31) and to Auxenochlorella. This is another example where characteristic N-glycan structures distinguish phylogenetically different groups of microalgae.

Thorsmoerkia curvula gen. et spec. nov. (Trebouxiophyceae, Chlorophyta), a semi-terrestrial microalga from Iceland exhibits high levels of unsaturated fatty acids.

A terrestrial green alga was isolated at Iceland, and the strain (SAG 2627) was described for its morphology and phylogenetic position and tested for biotechnological capabilities. Cells had a distinctly curved, crescent shape with conical poles and a single parietal chloroplast. Phylogenetic analyses of 18S rDNA and rbcL markers placed the strain into the Trebouxiophyceae (Chlorophyta). The alga turned out to belong to an independent lineage without an obvious sister group within the Trebouxiophyceae. Based on morphological and phylogenetic data, the strain was described as a new genus and species, Thorsmoerkia curvula gen. et sp. nov. Biomass was generated in column reactors and subsequently screened for promising metabolites. Growth was optimized by pH-regulated, episodic CO2 supplement during the logarithmic growth-phase, and half of the biomass was thereafter exposed to nitrogen and phosphate depletion. The biomass yield reached up to 53.5 mg L-1 day-1. Fatty acid (FA) production peaked at 24 mg L-1 day-1 and up to 83% of all FAs were unsaturated. At the end of the log phase, approximately 45% of dry mass were lipids, including eicosapentaenoic acid. Carotenoid production reached up to 2.94 mg L-1 day-1 but it was halted during the stress phase. The N-linked glycans of glycoproteins were assessed to reveal chemotaxonomic patterns. The study demonstrated that new microalgae can be found at Iceland, potentially suitable for applied purposes. The advantage of T. curvula is its robustness and that significant amounts of lipids are already accumulated during log phase, making a subsequent stress exposure dispensable.

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during in vitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. OBJECTIVE: We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. METHODS: IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). RESULTS: Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r = 0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n = 15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r = 0.61) and to all blank ImmunoCAPs (r > 0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. CONCLUSIONS: Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

Monoclonal antibody therapy for Junin virus infection.

Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Characterization of plants expressing the human ß1,4-galactosyltrasferase gene.

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human ß1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.

Rhamnogalacturonan II structure shows variation in the side chains monosaccharide composition and methylation status within and across different plant species.

A paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild-type plants, up to 45% of the l-fucose is substituted by l-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than l-fucose to l-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The ß-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.

A new allergen from ragweed (Ambrosia artemisiifolia) with homology to art v 1 from mugwort.

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Reassessing the role of hyaluronidase in yellow jacket venom allergy.

BACKGROUND: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in yellow jacket allergy. It shows 50% homology with the hyaluronidase from honeybee venom, Api m 2. Recently, IgE binding to YJ-HYA and cross-reactivity with Api m 2 has been shown to be due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: We sought to quantify the importance of YJ-HYA in yellow jacket allergy and the cross-reactivity with Api m 2 by discriminating between carbohydrate and peptide epitopes. METHODS: IgE binding to Vespula species venom was studied by means of Western blotting in 136 patients with yellow jacket allergy (31 in vitro single positive to yellow jacket venom and 105 in vitro double-positive to yellow jacket-honeybee). Inhibition studies were carried out with MUXF-BSA (isolated bromelain glycopeptides linked to bovine serum albumin) and purified Api m 2. RESULTS: Among yellow jacket single-positive sera, only 1 of 31 bound with YJ-HYA, whereas this was the case in 87% of 105 double-positive sera. Of 83 patients in whom inhibitions were performed, 65% reacted with hyaluronidase through CCDs alone, 27% reacted with both CCDs and peptide epitopes, and 8% reacted only with the hyaluronidase peptide. The protein-specific reactivity with YJ-HYA was cross-inhibited by Api m 2 in 48% (14/29). Antigen 5 and phospholipase A(1) were each recognized by around 90% of sera from both groups, together identifying 97% of patients. CONCLUSION: Hyaluronidase is a minor yellow jacket venom allergen, and only 10% to 15% of patients with yellow jacket allergy are estimated to have IgE against the hyaluronidase protein. Peptide-specific cross-reactivity with Api m 2 occurs in half of these sera. Component-resolved diagnosis with antigen 5 and phospholipase would detect virtually all patients with yellow jacket venom allergy.

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Sialic acid concentrations in plants are in the range of inadvertent contamination.

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-D: -manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.

Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding.

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz (1)H and (13)C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core, which is substituted by a variable number (5-28) of alpha-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) beta-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp(4) motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with alpha-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked beta-arabinose residues appeared to react with the antibodies. Mono-beta-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.

Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens.

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain.

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.