Methylated glycans as conserved targets of animal and fungal innate defense

Abstract

Effector proteins of innate immune systems recognize specific nonself 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.

Development of a competitive binding assay for the Burkholderia cenocepacia lectin BC2L-A and structure activity relationship of natural and synthetic inhibitors

“…For the selective methylation of calcium-coordinating hydroxy groups, we used the previously reported 34 3-O-methyl mannoside 36 (Table 3) homolog LecB from P. aeruginosa. Surprisingly, no competitive binding was observed for any of these derivatives up to concentrations of more than 650 µM, a concentration more than 10-100-fold above the affinities of the corresponding derivatives with unmodified hydroxy groups.…”

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Cospin, for example, appears to be toxic to D. melanogaster but not to other diptera, nematodes, amoebozoa, fungi, or bacteria Sabotič, unpublished observations), whereas other toxic proteins provide a general protection against different antagonists . LbTec2, for example, binds a glycan modification that has been observed in different phyla ranging from bacteria to plants and animals (Wohlschlager et al 2014). Another level of diversity is found in the regulation of the biosynthesis of these fungal toxins.…”

“…The three homologous proteins: AAL, AOL 1 and AFL, differ slightly in their specificity for the connectivity of the bound fucose residues (Matsumura et al 2008) and are structurally similar to fucose-binding hololectins from bacteria, including RSL from Ralstonia solanacearum (Sudakevitz et al 2002). The primary sequence of hololectin LbTec2 is unrelated to that of AAL but is similar to those of proteins from filamentous bacteria (actinobacteria), slime moulds, and animals and is predicted to also adopt a six-bladed β-propeller fold with six carbohydrate-binding sites (Wohlschlager et al 2014). LbTec2 binds to Sepharose and is specific for 2-O-Mefucose and 3-O-Me-mannose residues.…”

“…Members of the β-propeller-type family of mushroom lectins include hololectin AAL from the orange peel mushroom Aleuria aurantia (Fujihashi et al 2003;Wimmerova et al 2003) and its homologs from various ascomycetous molds such as AOL 1 from Aspergillus oryzae, and AFL from Aspergillus fumigatus (Houser et al 2015;Matsumura et al 2008) as well as PVL and LbTec2 from the basidiomycetes Psathyrella velutina and Laccaria bicolor (Cioci et al 2006;Wohlschlager et al 2014). Basidiomycetous PVL and LbTec2 show very low sequence homology to each other and to ascomycetous AAL (below 15 % sequence identity and 30 to 45 sequence similarity), while AFL and AOL 1 are highly homologous to each other (82 % sequence identity and 90 % similarity) and 30 % identical (60 % similar) to AAL.…”

“…Based on the lack of toxicity resistance of various C. elegans strains with mutations in N-glycan and glycosphingolipid biosynthesis, fucosylated O-glycans have been hypothesized as most likely target glycan of AAL in this organism. LbTec2 was recently shown to be toxic to C. elegans and genetic evidence, in combination with glycome analysis, showed that the nematotoxicity is dependent on 2-O-Me-fucose and 3-O-Me-mannose in C. elegans Nglycans (Wohlschlager et al 2014). According to a recent report on C. elegans N-glycan structure, LbTec2 most likely binds to 2-O-Me-Fuc-α1,2-Gal-β1,4 and 3-O-Me-Man-α1,3 on the core β-mannose of C. elegans N-glycans (Yan et al 2015).…”