Diter von Wettstein - US grants

[unreadable] DESCRIPTION (provided by applicant): Celiac disease is the most common food-sensitive enteropathological condition in humans and caused by an autoimmune reaction against wheat, barley and rye prolamins. In human leukocyte antigen (HLA) DQ2 (or DQ8)-positive individuals exposure to gluten can lead to a painful chronic erasure of the microvilli of the epithelium in the intestine and to a permanent intolerance of dietary gluten. It is commonly detected in early childhood in cases with severe symptoms. In many patients symptoms arise only later in life and, if untreated, it is associated with increased morbidity and mortality. Despite its prevalence in most populations (up to 1 in 200), the only effective therapy is strict dietary abstinence from these food grains. Recent research has established that the autoimmune response results from the resistance to digestion of certain prolamine/glutamine rich peptides of the prolamins by gastric, pancreatic and brush-border membrane proteases. These peptides are taken up through the intestinal mucosa and initiate the autoimmune response. In this proposal we wish to address the following testable hypotheses for removal of the prolamin T-cell stimulating epitopes in barley and wheat. 1) The fully viable barley mutation lys3a prevents transcription of all prolamin genes requiring de-methylation of their promoters in the developing endosperm except of the single gene with a CpG island encoding the High Molecular (HMW) D-hordein. This prolamin is highly homologous to the wheat HMW prolamins that are responsible for baking quality. We ask the question if we can exploit this mutation to obtain barley grain that provides celiac safe food products. 2) The close evolutionary relationship of barley and wheat makes it likely that the two cereals employ the same transcriptional control system for the synthesis of the storage proteins in the grain. We want to test this by selecting ortholog mutations of lys3a in wheat and ask the question if it prevents the transcription of the gliadin genes but not the 6 HMW genes present in hexaploid wheat. 3) Proline/glutamine rich peptides containing T-cell stimulating epitopes can be detoxified by prolyl endopeptidases and the barley cysteine endoprotease EP-B in vitro and in vivo with rat intestinal perfusion experiments. We want to test if it is possible to synthesize these enzymes in transgenic barley and wheat during endosperm development, target the enzymes into the storage vacuoles and detoxify the prolamins during grain development.Celiac disease or sprue is the most common food-sensitive enteropathy in humans resulting from chronic inflammatory injury to the mucosa of the small intestine after ingestion of proline- and glutamine-rich prolamins present in wheat gluten as well as in barley and rye grain storage proteins. To date the only effective treatment of this debilitating disease is life-long strict abstinence from the staple food grains. The goal of this research is to eliminate the celiac causing epitopes of prolamins by exploiting the cell biological features of prolamin synthesis in the endosperm of barley and wheat. [unreadable] [unreadable] [unreadable]