The Drosophila neurally altered carbohydrate mutant has a defective Golgi GDP-fucose transporter.

Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac(1) flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mistargeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac(1) mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac(1) Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac(1) mutant. These results validate the Drosophila nac(1) mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.

Discovery and structural characterization of fucosylated oligomannosidic N-glycans in mushrooms.

L-fucose is a common constituent of Asn-linked glycans in vertebrates, invertebrates, and plants, but in fungal glycoproteins, fucose has not been found so far. However, by mass spectrometry we detected N-glycans and O-glycans containing one to six deoxyhexose residues in fruit bodies of several basidiomycetes. The N-glycans of chanterelles (Cantharellus cibarius) contained a deoxyhexose chromatographically identical to fucose and sensitive to α-L-fucosidase. Analysis of individual glycan species by tandem MS, glycosidase digestion, and finally (1)H NMR revealed the presence of L-fucose in α1,6-linkage to an α1,6-mannose of oligomannosidic N-glycans. The substitution by α1,6-mannose of α1,2-mannosyl residues of the canonical precursor structure was yet another hitherto unknown modification. No indication for the occurrence of yet other modifications, e.g. bisecting N-acetylglucosamine, was seen. Besides fucosylated N-glycans, short O-linked mannan chains substituted with fucose were present on chanterelle proteins. Although undiscovered so far, L-fucose appears to represent a prominent feature of protein-linked glycans in the fungal kingdom.

Development of Dictyostelium discoideum is associated with alteration of fucosylated N-glycan structures.

The social amoeba Dictyostelium discoideum has become established as a simple model for the examination of cell-cell interactions, and early studies suggested that shifts in glycosylation profiles take place during its life cycle. In the present study, we have applied HPLC and mass spectrometric methods to show that the major N-glycans in axenic cultures of the AX3 strain are oligomannosidic forms, most of which carry core fucose and/or intersecting and bisecting N-acetylglucosamine residues, including the major structure with the composition Man8GlcNAc4Fuc1. The postulated alpha1,3-linkage of the core fucose correlates with the cross-reactivity of Dictyostelium glycoproteins with a horseradish peroxidase antiserum; a corresponding core alpha1,3-fucosyltransferase activity capable of modifying oligomannosidic N-glycans was detected in axenic Dictyostelium extracts. The presence of fucose on the N-glycans and the reactivity to the antiserum, but not the fucosyltransferase activity, are abolished in the fucose-deficient HL250 strain. In later stages of development, N-glycans at the mound and culmination stages show a reduction in both the size and the degree of modification by intersecting/bisecting residues compared with mid-exponential phase cultures, consistent with the hypothesis that glycosidase and glycosyltransferase expression levels are altered during the slime mould life cycle.

Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method.

Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.

Neutral N-glycan patterns of the gastropods Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica.

The N-glycosylation potentials of Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica were analysed by investigation of the N-glycan structures of the skin and viscera glycoproteins by a combination of HPLC and mass-spectrometry methods. It is one of the first steps to enlarge the knowledge on the glycosylation abilities of gastropods, which may help to establish new cell culture systems, to uncover new means for pest control for some species, and to identify carbohydrate-epitopes which may be relevant for immune response. All snails analysed contained mainly oligomannosidic and small paucimannosidic structures, often terminated with 3-O-methylated mannoses. The truncated structures carried modifications by beta1-2-linked xylose to the beta-mannose residue, and/or an alpha-fucosylation, mainly alpha1,6-linked to the innermost N-acetylglucosaminyl residue of the core. Many of these structures were missing the terminal N-acetylglucosamine, which has been shown to be a prerequisite for processing to complex N-glycans in the Golgi. In some species (Planorbarius corneus and Achatina fulica) traces of large structures, terminated by 3-O-methylated galactoses and carrying xylose and/or fucose residues, were also detected. In Planorbarius viscera low amounts of terminal alpha1-2-fucosylation were determined. Combining these results, gastropods seem to be capable to produce all kinds of structures ranging from those typical in mammals through to structures similar to those found in plants, insects or nematodes. The detailed knowledge of this very complex glycosylation system of the gastropods will be a valuable tool to understand the principle rules of glycosylation in all organisms.

Molecular and immunological characterization of the glycosylated orange allergen Cit s 1.

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) binds a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analyzed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognize a range of glycoproteins and neoglycoconjugates containing beta1,2-xylose and core alpha1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients' sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients' IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1, using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein compared with the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Owing to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically relevant allergen.

N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues.

In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focused on its glycosphingolipids, which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and western blotting, we have now analyzed the peptide N-glycosidase A-released N-glycans of adults of this species. The presence of hybrid bi- and triantennary N-glycans, some modified by core alpha1,6-fucose and peripheral phosphorylcholine, was demonstrated by LC/electrospray ionization (ESI)-Q-TOF-MS/MS, as was the presence of paucimannosidic N-glycans, some of which carry core alpha1,3-fucose, and oligomannosidic oligosaccharides. Western blotting verified the presence of protein-bound phosphorylcholine and core alpha1,3-fucose, whereas glycosyltransferase assays showed the presence of core alpha1,6-fucosyltransferase and Lewis-type alpha1,3-fucosyltransferase activities. Although, the unusual tri- and tetrafucosylated glycans found in the model nematode Caenorhabditis elegans were not found, the vast majority of the N-glycans found in A. suum represent a subset of those found in C. elegans; thus, our data demonstrate that the latter is an interesting glycobiological model for parasitic nematodes.

Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants.

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering). The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439). To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122). In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.