Biotoxicity Assays for Fruiting Body Lectins and Other Cytoplasmic Proteins

Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O.; Aebi, Markus

doi:10.1016/s0076-6879(10)80007-2

Cited by 21 publications

(36 citation statements)

References 30 publications

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“…We next tested recombinant Lb-Tec2 for toxicity against Drosophila melanogaster, Aedes aegypti, and C. elegans using previously described assays (19)(20)(21). Recombinant Lb-Tec2 was not toxic for the tested insects (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

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Methylated glycans as conserved targets of animal and fungal innate defense

Wohlschlager

Butschi

Grassi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultsmentioning

confidence: 99%

“…Expression was induced with 0.5 mM IPTG at an OD 600 of 0.6 for 24 h at 10°C. Expression and solubility of recombinant proteins were checked as previously described (19).…”

Section: Methodsmentioning

confidence: 99%

Methylated glycans as conserved targets of animal and fungal innate defense

Wohlschlager

Butschi

Grassi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…These biotoxicity assays involved feeding the organisms MOA-expressing E. coli as described previously (10) and revealed a clear toxicity of MOA toward C. elegans and A. castellanii ( Fig. 1 and supplemental Fig.…”

Section: Moa Inhibits C Elegans Development and Amoebal Growthmentioning

confidence: 98%

“…Our results suggest that MOA is a fungal toxin that protects the fruiting body from predators such as fungivorous nematodes. C. elegans Toxicity Assays and Statistical Analysis-Biotoxicity assays with C. elegans were performed as described (10). Results were analyzed using a t test for pairwise comparisons.…”

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confidence: 99%

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

Wohlschlager¹,

Butschi

Zurfluh³

et al. 2011

Journal of Biological Chemistry

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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.

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“…Biotoxicity of the fungal BBPs was assessed by feeding the recombinant E. coli cells to C. elegans and Acanthamoeba sp. as previously described (15,20) and by adding purified recombinant tamavidin 2 to the rearing medium of D. melanogaster as described in Method S3 in the supplemental material and in reference 21.…”

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confidence: 99%