Expression of glutenases in wheat (Triticum aestivum L.) endosperm and increase in the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as an alternative therapy for celiac patients.

摘要

Detoxification of wheat, barley and rye prolamins constitute a challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of glutenases as food supplement is an attractive alternative to deliver the therapeutic agents directly to the small intestine, where the induction of immune system and downstream reactions take place. The aim of the present research is to evaluate the capacity of wheat grain to express and store enzymes capable of detoxifying immunogenic gluten proteins. In this direction to identify the most suitable combination of glutenases capable of degrading gluten proteins and in silico digestion approach was attempted where wheat, barley and rye gluten sequences available in the public domain were cleaved using various enzyme combinations. Digestion of prolamins with barley or wheat germinating grain endoprotease combined with Flavobacterium meningosepticum prolyl endopeptidase (Fm-PEP) significantly decreased the number of proteolytically resistant peptides from all prolamin families. The next step was to produce wheat plants expressing the selected glutenases in wheat endosperm and characterize transformants expressing these genes in their grains under in vitro conditions. To achieve this objective wheat embryonic calli were transformed with constructs encoding for Fm-PEP and barley cysteine endoprotease B2 using the biolistic approach. Four lines showed correct inheritance of transgene to the T 2 generation. After analysis of gluten extracts under simulated gastro-intestinal conditions via PAGE, RP-HPLC one of the transformants designated FME108-10 showed near-complete degradation of immunogenic peptides in the gliadin fraction.;Since we proposed to use wheat grains expressing 'glutenases' as an ingredient of the daily bakery products it will be convenient to have thermostable enzymes that retain activity at and over 90°C. In order to achieve this objective a site saturation mutagenesis approach was followed. Introduction of substitutions at amino acid residues 412, 413, 414 and 415 increased thermostability of Fm-PEP, while maintaining its digestive properties under simulated gastro-intestinal conditions. In summary the results of present research suggest that it is possible to produce celiac safe wheat cultivars expressing high quantities of thermostable glutenases, which will allow detoxification of gluten proteins in human gut after consumption.