• Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.

      Butschi, Alex; Titz, Alexander; Wälti, Martin A; Olieric, Vincent; Paschinger, Katharina; Nöbauer, Katharina; Guo, Xiaoqiang; Seeberger, Peter H; Wilson, Iain B H; Aebi, Markus; et al. (2010-01)
      The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.
    • Crystal Structures of Fungal Tectonin in Complex with O-Methylated Glycans Suggest Key Role in Innate Immune Defense.

      Sommer, Roman; Makshakova, Olga N; Wohlschlager, Therese; Hutin, Stephanie; Marsh, May; Titz, Alexander; Künzler, Markus; Varrot, Annabelle; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-03-06)
      Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction.
    • Expression, Purification, and Functional Characterization of Tectonin 2 from Laccaria bicolor: A Six-Bladed Beta-Propeller Lectin Specific for O-Methylated Glycans.

      Wohlschlager, Therese; Titz, Alexander; Künzler, Markus; Varrot, Annabelle; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer, 2020-04-19)
      Tectonins are conserved defense proteins of innate immune systems featuring a β-propeller fold. Tectonin 2 from Laccaria bicolor, Lb-Tec2, is the first fungal representative of the tectonin superfamily that has been described. In-depth characterization revealed a specificity for O-methylated glycans and identified a unique sequence motif and binding site architecture underlying this unusual specificity. This chapter provides information on how to produce and purify recombinant Lb-Tec2, characterize its interaction with O-methylated glycans and demonstrate its biological function.
    • Methylated glycans as conserved targets of animal and fungal innate defense.

      Wohlschlager, Therese; Butschi, Alex; Grassi, Paola; Sutov, Grigorij; Gauss, Robert; Hauck, Dirk; Schmieder, Stefanie S; Knobel, Martin; Titz, Alexander; Dell, Anne; et al. (2014-07-08)
      Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans, suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1, coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus, showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.
    • O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

      Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),Saarland Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2016)
      Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.