Intracellular expression of a single-domain antibody reduces cytoxicity of 15-acetyl-deoxynivalenol in yeast

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DOIResolve DOI: http://doi.org/10.1074/jbc.M109.045047
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TypeArticle
Journal titleJournal Of Biological Chemistry
Volume284
Issue50
Pages3502935039; # of pages: 11
Abstract15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (VHH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and VHH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin- specific VHH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 µg·ml⁻¹. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 µg·ml⁻¹. The proof of concept established within this eukaryotic system suggests that in planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Institute for Biological Sciences
Peer reviewedYes
NPARC number15329257
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Record identifier2a4b02eb-6557-4c13-bc38-57a6530c7cd4
Record created2010-05-21
Record modified2016-05-09
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