Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum.

BACKGROUND: The porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible. METHODS: Different enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC. RESULTS: Whereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered 'male', while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc2-4PC2-4 motifs not observed in our previous studies on other nematodes. CONCLUSION: The interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation. GENERAL SIGNIFICANCE: Unusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.

Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.

Glycan arrays have become a technique of choice to screen glycan-protein interactions in a high-throughput manner with high sensitivity and low sample consumption. Here, the synthesis of a new multifunctional fluorescent linker for glycan labeling via aminoxy ligation and immobilization is described; the linker features a fluorescent naphthalene group suitable for highly sensitive high-performance liquid chromatography-based purification and an azido- or amino-modified pentanoyl moiety for the immobilization onto solid supports. Several glycoconjugates displaying small sugar epitopes via chemical or chemoenzymatic synthesis were covalently attached onto a microarray support and tested with lectins of known carbohydrate binding specificity. The glycan library was extended using glycosyltransferases (e.g. galactosyl-, sialyl- and fucosyltransferases); the resulting neoglycoconjugates, which are easily detected by mass spectrometry, mimic antennal elements of N- and O-glycans, including ABH blood group epitopes and sialylated structures. Furthermore, an example natural plant N-glycan containing core α1,3-fucose and ß1,2-xylose was also successfully conjugated to the fluorescent linker, immobilized and probed with lectins as well as antihorseradish peroxidase. These experiments validate our linker as being a potentially valuable tool to study glycozyme and lectin specificities, sensitive enough to allow purification of natural glycans.

Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin.

Interest in powerful, nanosized tools to analyze in detail glycan-protein interactions has increased significantly over recent years. Here, we report two complementary approaches to characterize such interactions with high sensitivity, low sample consumption, and without the need for sample labeling, namely, surface plasmon resonance (SPR) and an approach that combines limited proteolysis and mass spectrometry. Combination of these two approaches to investigate glycan-protein interactions allows (1) to characterize interactions through kinetic and thermodynamic parameters, (2) to capture efficiently the carbohydrate-binding protein, and (3) to identify the interacted protein and its carbohydrate binding site by mass spectrometry. As a proof of principle, the interaction of the galactose-specific legume lectin Erythrina cristagalli agglutinin with several sugars has been characterized in-depth by means of these two approaches.

Highly modified and immunoactive N-glycans of the canine heartworm.

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF-MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.

Neo-glycopeptides: the importance of sugar core conformation in oxime-linked glycoprobes for interaction studies.

Carbohydrate binding proteins, such as lectins, are crucial in numerous biological recognition processes. While binding may be mediated by a single monosaccharide, several lectins have shown exquisite epimer and linkage recognition indicating that a larger structure is essential for optimal interaction. Several approaches have been described for their detailed study, including lectinosorbent assays, microarrays and surface plasmon resonance (SPR). Most of these approaches ignore that the aglycon-bound monosaccharide is often in a non-natural conformation that affects the occurring binding event. In this paper we demonstrate that oxime-bound glycans, employed in such approaches, occur predominantly in the open form (approximately 70%). Through the use of a secondary amine, the aglycon-bound monosaccharide in the resulting neo-glycopeptide probe is forced into the ring-form. Resulting structures were analyzed by means of nuclear magnetic resonance and differential derivatization experiments. The impact of ring closure was further demonstrated through interaction studies using SPR and various lectins with distinct binding specificities.

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues.

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.

Recent progress in the field of neoglycoconjugate chemistry.

Glycosylation is probably the most complex secondary gene event that affects the vast majority of proteins in nature resulting in the occurrence of a heterogeneous mixture of glycoforms for a single protein. Many functions are exerted by single monosaccharides, well-defined oligosaccharides, or larger glycans present in these glycoproteins. To unravel these functions it is of the utmost importance to prepare well-defined single glycans conjugated to the underlying aglycon. In this review, the most recent developments are described to address the preparation of carbohydrate-amino acid (glyco-conjugates). Naturally occurring N- and O-linked glycosylation are described and the preparation of non-natural sugar-amino acid linkages are also included.

Optimized synthesis of aminooxy-peptides as glycoprobe precursors for surface-based sugar-protein interaction studies.

An improved procedure for solid phase coupling of Boc-aminooxyacetic acid to peptides is described. By avoiding base-containing activation mixtures which cause over-acylation, it practically suppresses this unwanted side reaction and leads to near quantitative yields of Aoa-peptides, useful as glycoprobe precursors in glycomic studies.