Plasticity of the β-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Abstract

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.

Author Summary

All multicellular organisms have developed mechanisms to defend themselves against predators, parasites and pathogens. As a common mechanism, animals, plants and fungi use a large arsenal of carbohydrate-binding proteins (lectins) to protect themselves from predation and parasitism. The success of this type of innate defence mechanism critically depends on the diversity of specific recognition of foreign carbohydrate structures by the host lectins. In this study, we use NMR structure determination to show that part of this diversity is created by the plasticity of common protein folds. The identified fungal lectin that is toxic to nematodes and insects, adopts a common lectin fold but is remarkable in terms of its specificity and affinity for the recognized foreign carbohydrate structure, the number and location of the carbohydrate binding sites on the protein and the degree of oligomerization. Since the identified in vivo target of the fungal lectin is characteristic for invertebrates, our results may be exploited to develop novel approaches for the control of animal and human parasites.