Pathogenesis of Celiac Disease: Identification of Covalent Conjugates Between Tissue Transglutaminase and Gluten Peptides from Different Cereals

• Applicant: Professor Dr. Peter Köhler
• Subject Area: Biological and Biomimetic Chemistry ; Food Chemistry
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 250645717

Celiac disease (CD) is one of the most frequent food intolerances affecting approximately 1 % of the population. It is characterized by a serious damage of the small intestinal mucosa and is accompanied by a drastic decrease of the nutrient uptake. CD is triggered by the ingestion of storage proteins of wheat, rye, barley, and possibly oats, which have been termed gluten in the field of CD. Due to its high proline content gluten is only incompletely digested by gastro-intestinal peptidases leading to the accumulation of medium to long-chain peptides that pass the enterocyte layer and trigger the immunological response leading to the typical symptoms of CD. In the complex pathogenesis of CD the intestinal tissue transglutaminase (TG2) plays a key role. Firstly, TG2 catalyzes deamidation of specific glutamine residues of gluten peptides thus causing a pro-inflammatory immune response. Secondly, TG2 forms covalently linked conjugates with gluten peptides, which induce the generation of antibodies against these conjugates. While the specificity of TG2 in the deamidation of glutamine residues has been elucidated only very limited information is available on the structure and occurrence of TG2-gluten peptide conjugates.Therefore, the aim of this research proposal is to identify binding sites between TG2 and gluten peptides derived from all CD-active protein types of wheat (omega5-, omega1,2-, alpha-, gamma-gliadins, HMW- and LMW-subunits of glutenin), rye (omega-, gamma40k-, HMW- and gamma75k-secalins), barley (C-, gamma-, D- and B-hordeins), and oats (avenins) to shed more light on the pathway of CD pathogenesis.For this purpose CD-active protein types will be isolated in the gram-scale from wheat, rye barley, and oats flours. Isolated proteins will be converted into CD-active peptides by partially hydrolyzing them with gastro-intestinal peptidases (pepsin, trypsin, chymotrypsin). Peptides will then be incubated in the presence of TG2, and conjugates containing isopeptide crosslinks between TG2 and gluten peptides will be enriched by means of gel permeation chromatography. To break down TG2 into peptides of suitable sizes the conjugates will be partially hydrolyzed with trypsin. Isopeptide cross-linked peptides will be identified by liquid chromatography mass-spectrometry (LC-MS) with alternating collision-induced (CID) and electron-transfer dissociation (ETD). Further LC-MS experiments will provide the amino acid sequences and the location of isopeptide bonds in these peptides. The prerequisites to achieve the aim of the study are fulfilled as the working group of the applicant has long-term expertise in the isolation, characterization and partial enzymatic hydrolysis of cereal proteins on the one hand, and in the elucidation of intermolecular crosslinks between proteins or peptides on the other.

• DFG Programme: Research Grants