Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.

The human anticoagulant factor, Protein C, is a plasma glycoprotein that has reported anti-ischaemic and anti-inflammatory properties. To explore potential mechanisms for these reported activities, we examined the effect of Protein C on the process of cell adhesion to vascular endothelial cells, which plays a critical role during inflammatory responses. We show that both human plasma-derived and human cell-produced recombinant Protein C inhibit E-selectin-mediated cell adhesion. This effect was not mediated through the serine protease activity of Protein C, but through its carbohydrates. Using oligosaccharides isolated from human cell-produced Protein C, we have defined a polylactosamine structural determinant that inhibits adhesion. This uncharged determinant appears to be a more potent ligand for E-selectin than the sialylated Lewis X antigen. Our data suggest a potential mechanism for the reported anti-inflammatory effects of Protein C and describe a new ligand for selectin-mediated adhesion.

Pristionchus pacificus is a free-living nematode increasingly used as an organism for comparison to the more familiar model Caenorhabditis elegans. In this study, we examined the N-glycans of this organism isolated after serial release with peptide:N-glycosidases F and A; after fluorescent labelling with 2-aminopyridine, chromatographic fractionation by three types of reversed-phase HPLC (with either classical C18, fused core C18 or alkylamide bonded phases) followed by mass spectrometric analyses revealed key features of its N-glycome. In addition to paucimannosidic and oligomannosidic glycans typical of invertebrates, N-glycans with two core fucose residues were detected. Furthermore, a range of glycans carrying up to three phosphorylcholine residues was observed whereas, unlike C. elegans, no tetrafucosylated N-glycans were detected. Structures with three fucose residues, unusual methylation of core α1,3-fucose or with galactosylated fucose motifs were found in low amounts; these features may correlate with a different ensemble or expression of glycosyltransferase genes as compared to C. elegans. From an analytical perspective, both the alkylamide RP-amide and fused core C18 columns, as compared to a classical C18 material, offer advantages in terms of resolution and of elution properties, as some minor pyridylamino-labelled glycans (e.g., those carrying phosphorylcholine) appear in earlier fractions and so potential losses of such structures due to insufficient gradient length can be avoided.

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.

The root-knot nematode (RKN) is one of the most dangerous and widespread types of nematodes affecting tomatoes. There are few methods for controlling nematodes in tomatoes. Nature resistance genes (R-genes) are important in conferring resistance against nematodes. These genes that confer resistance to the RKN have already been identified as Mi-1, Mi-2, Mi-3, Mi-4, Mi-5, Mi-6, Mi-7, Mi-8, Mi-9, and Mi-HT. Only five of these genes have been mapped. The major problem is that their resistance breaks down at high temperatures. Some of these genes still work at high temperatures. In this paper, the mechanism and characteristics of these natural resistance genes are summarized. Other difficulties in using these genes in the resistance and how to improve them are also mentioned.

Although countless genomes have now been sequenced, the glycomes of the vast majority of eukaryotes still present a series of unmapped frontiers. However, strides are being made in a few groups of invertebrate and unicellular organisms as regards their N-glycans and N-glycosylation pathways. Thereby, the traditional classifi cation of glycan structures inevitably approaches its boundaries. Indeed, the glycomes of these organisms are rich in surprises, including a multitude of modifi cations of the core regions of N-glycans and unusual antennae. From the actually rather limited glycomic information we have, it is nevertheless obvious that the biotechnological, developmental and immunological relevance of these modifi cations, especially in insect cell lines, model organisms and parasites means that deciphering unusual glycomes is of more than just academic interest.

Background: Galactosylation of core fucose residues is a peculiar feature of invertebrate N-glycans. Results: Two variants of galactosylated fucose were observed in a Caenorhabditis elegans mutant and in two nematode parasites. Conclusion: Galactosylation of fucose in nematodes is more widespread than previously thought and is affected by processing hexosaminidases. Significance: Galactosylated fucose motifs may present new targets for anthelminthic agents.

The N-glycosylation of the model nematode Caenorhabiditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that ‘simple’ organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic sub-pool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a ‘bisecting’ β-galactose which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Not only do these residues constitute novel modifications of N-glycans, but these features may well be targets of nematoxic proteins produced as defence molecules by various fungal species.