Structure–function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity

Abstract

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of two antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle scattering of X-rays (SAXS). One carbohydrate binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94 and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. While no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

A lectin gene is involved in the defense of Pleurotus ostreatus against the mite predator Tyrophagus putrescentiae

“…Six groups of these proteins were reported to be involved in the fungal defense of the fungal fruit body, including galectins, Coprinus-like, ricin-like, Clitocybe-like, and fungal fruit body lectins ( Varrot et al, 2013 ; Tayyrov et al, 2018 ; Bleuler-Martinez et al, 2022 ; Figure 1 ). For example, lectins with entomotoxic activity, CNL from Clitocybe nebularis is Clitocybe-like lectin family, MPL from Macrolepiota procera are ricin-like lectin family, CCL2 from Coprinopsis cinerea is galectins lectin family, and XCL from Xerocomellus chrysenteron is fungal fruit body lectin family ( Wohlschlager et al, 2014 ; Bleuler-Martinez et al, 2022 ).…”

“…Six groups of these proteins were reported to be involved in the fungal defense of the fungal fruit body, including galectins, Coprinus-like, ricin-like, Clitocybe-like, and fungal fruit body lectins ( Varrot et al, 2013 ; Tayyrov et al, 2018 ; Bleuler-Martinez et al, 2022 ; Figure 1 ). For example, lectins with entomotoxic activity, CNL from Clitocybe nebularis is Clitocybe-like lectin family, MPL from Macrolepiota procera are ricin-like lectin family, CCL2 from Coprinopsis cinerea is galectins lectin family, and XCL from Xerocomellus chrysenteron is fungal fruit body lectin family ( Wohlschlager et al, 2014 ; Bleuler-Martinez et al, 2022 ). Furthermore, the ricin-like lectin family is the major group (46.7%) of 137 proteins, those mostly contain β-trefoil fold structures from Schizophyllum commune , Coprinellus micaceus , Flammula alnicola , etc., in more closely related branches, which are likely to be of value in providing potential insecticidal activity.…”

“…Massive structural lectin families from fungal fruit bodies have been characterized, mainly galectin, β-trefoil-type, β-propeller-type, actinoporin-type, cyanovirin-N-type, and immunoglobulin-type. Some of these have been reported to have insecticidal properties ( Bleuler-Martínez et al, 2011 ; Plaza et al, 2015 ; Bleuler-Martinez et al, 2017 ; Lebreton et al, 2021 ; Bleuler-Martinez et al, 2022 ). Lectins abundant in P. ostreatus have been widely reported for their anti-tumor activity ( Vajravijayan et al, 2020 ).…”

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors

“…Many of the lectins that were most potent in decreasing adhesion to mucin, e.g. MOA, AAL, ABL, CML1, and Tec2, have a glycan-binding target that is present on mucins and the surface of C. jejuni ( Kruger et al., 2002 ; Wimmerova et al., 2003 ; Carrizo et al., 2005 ; Corcoran and Moran, 2007 ; Grahn et al., 2007 ; Olausson et al., 2008 ; Wohlschlager et al., 2014 ; Turonova et al., 2016 ; Bleuler-Martinez et al., 2022 ). The main glycans of mucin type II are GalNAc, GlcNAc, Fuc, Gal, and Neu5Ac ( Schömig et al., 2016 ).…”

“…The following lectins were produced as His-tagged recombinant proteins in E. coli BL21(DE3) using ZYM-5052 autoinduction medium for 4 h at 37°C and 20 h at 18°C ( Studier, 2005 ): Coprinopsis galectin 2 (CGL2, UniProt ID: Q9P4R8 [ Walser et al., 2004 )], Coprinopsis galectin 3 (CGL3, UniProt ID: Q206Z5 [ Wälti, 2008 )], Marasmius oreades agglutinin [MOA, UniProt ID: Q8X123 ( Kruger et al., 2002 ; Grahn et al., 2007 )], Agaricus bisporus lectin [ABL, UniProt ID: Q00022 ( Carrizo et al., 2005 )], the Sordaria macrospora transcript associated with perithecial development [TAP1, UniProt ID: F7VWP8 ( Nowrousian and Cebula, 2005 )], Aleuria aurantia lectin [AAL, UniProt ID: P18891 ( Wimmerova et al., 2003 ; Olausson et al., 2011 )], Coprinopsis cinerea lectin 2 [CCL2, UniProt ID: B3GA02 ( Schubert et al., 2012 )], C. cinerea mucin-binding lectin 1 [CML1, UniProt ID: B3VS76 ( Bleuler-Martinez et al., 2022 )], and Laccaria bicolor tectonin 2 [Tec2, UniProt ID: B0CZL6 ( Wohlschlager et al., 2014 )].…”