Nematotoxicity of Marasmius oreades Agglutinin (MOA) Depends on Glycolipid Binding and Cysteine Protease Activity

Abstract

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Gal␣1,3Gal/ GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca 2؉ concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Entomotoxic b (Cordara et al 2011;Grahn et al 2007;Grahn et al 2009;Rempel et al 2002;Winter et al 2002;Wohlschlager et al 2011) na Entomotoxic (Brzin et al 2000;Renko et al 2010;Sabotič et al 2007a;Sabotič et al 2006;Sabotič et al 2011;Šmid et al 2015 (91 % sequence similarity) and are approximately 32 % identical (43 % similar) to CGL2. Galectins CGL1 and CGL2 are highly abundant in young fruiting bodies but hardly produced in the vegetative mycelium of C. cinerea (Boulianne et al 2000;Plaza et al 2014).…”

“…These proteins are analogous to bacterial and plant AB toxins including the previously mentioned plant toxin ricin. The best characterized examples of this lectin family are MOA and LSL from the saprophytic mushrooms Marasmius oreades (Cordara et al 2011;Grahn et al 2007;Grahn et al 2009;Wohlschlager et al 2011) and Laetiporus sulphureus (Mancheno et al 2005;Mancheno et al 2010;Tateno and Goldstein 2003). These proteins consist of a single N-terminal β-trefoil-type lectin domain fused to a cysteine protease (MOA) and to an aerolysin-type pore-forming domain (LSL).…”

“…These proteins consist of a single N-terminal β-trefoil-type lectin domain fused to a cysteine protease (MOA) and to an aerolysin-type pore-forming domain (LSL). The numbers and specificities of the carbohydrate-binding sites of the β-trefoil domains differ in that all three canonical binding sites of MOA are functional and bind to glycans carrying terminal Gal-α1,3-Gal/ GalNAc-β epitopes (Grahn et al 2007;Grahn et al 2009;Wohlschlager et al 2011) while only two of the three canonical binding sites of LSL appear to be functional and specific for β-galactosides including lactose and LacNAc (Angulo et al 2011;Mancheno et al 2005). Homologs of both proteins from other dikaryotic fungi have been partially characterized (Chumkhunthod et al 2006;Kadirvelraj et al 2011;Plaza et al 2014;Wohlschlager et al 2011).…”

“…The numbers and specificities of the carbohydrate-binding sites of the β-trefoil domains differ in that all three canonical binding sites of MOA are functional and bind to glycans carrying terminal Gal-α1,3-Gal/ GalNAc-β epitopes (Grahn et al 2007;Grahn et al 2009;Wohlschlager et al 2011) while only two of the three canonical binding sites of LSL appear to be functional and specific for β-galactosides including lactose and LacNAc (Angulo et al 2011;Mancheno et al 2005). Homologs of both proteins from other dikaryotic fungi have been partially characterized (Chumkhunthod et al 2006;Kadirvelraj et al 2011;Plaza et al 2014;Wohlschlager et al 2011). The MOA homologs PSL and SCA (Schizophyllum commune agglutinin), from the saprophytic mushrooms Polyporus squamosus and S. commune, differ from MOA in that PSL harbors a lower number of functional carbohydrate-binding sites with a different specificity toward terminal Neu5Ac-α2,6-Gal-β epitopes but with the catalytic triad of the cysteine protease domain conserved (Kadirvelraj et al 2011;Wohlschlager et al 2011).…”

“…Homologs of both proteins from other dikaryotic fungi have been partially characterized (Chumkhunthod et al 2006;Kadirvelraj et al 2011;Plaza et al 2014;Wohlschlager et al 2011). The MOA homologs PSL and SCA (Schizophyllum commune agglutinin), from the saprophytic mushrooms Polyporus squamosus and S. commune, differ from MOA in that PSL harbors a lower number of functional carbohydrate-binding sites with a different specificity toward terminal Neu5Ac-α2,6-Gal-β epitopes but with the catalytic triad of the cysteine protease domain conserved (Kadirvelraj et al 2011;Wohlschlager et al 2011). SCA exhibits the carbohydrate-binding specificity of MOA but not its catalytic activity ).…”