Identification of gliadin-binding peptides by phage display

Chen et al. BMC Biotechnology 2011, 11:16 http://www.biomedcentral.com/1472-6750/11/16 RESEARCH ARTICLE Open Access Identification of gliadin-binding peptides by phage display Tingsu Chen1,4,5, Karolina Hoffmann2, Sofia Östman3, Ann-Sofie Sandberg2, Olof Olsson4* Abstract Background: Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products. Results: Phage from a random oligomer display library were enriched by repeated pannings against immobilised gliadin proteins. Phage from the final panning round were plated, individual plaques picked, incubated with host bacteria, amplified to a population size of 1011 to 1012 and purified. DNA was isolated from 1000 purified phage populations and the region covering the 36 bp oligonucleotide insert from which the displayed peptides were translated, was sequenced. Altogether more than 150 different peptide-encoding sequences were identified, many of which were repeatedly isolated under various experimental conditions. Amplified phage populations, each expressing a single peptide, were tested first in pools and then one by one for their ability to inhibit binding of human anti-gliadin antibodies in ELISA assays. These experiments showed that several of the different peptideexpressing phage tested inhibited the interaction between gliadin and anti-gliadin antibodies. Finally, four different peptide-encoding sequences were selected for further analysis, and the corresponding 12-mer peptides were synthesised in vitro. By ELISA assays it was demonstrated that several of the peptides inhibited the interaction between gliadin molecules and serum anti-gliadin antibodies. Moreover, ELISA competition experiments as well as dot-blot and western blot revealed that the different peptides interacted with different molecular sites of gliadin. Conclusions: We believe that several of the isolated and characterised gliadin-binding peptides described here could provide valuable tools for researchers in the field of CD by facilitating studies on localisation and uptake of various gliadin peptides in the small intestine. In future work, the potential of these peptides to detoxify gluten will be investigated. Background Coeliac disease (CD) is a common and complex inflammatory disorder of the small intestine that affects genetically susceptible individuals carrying HLA-DQ2 or -DQ8 haplotypes. Symptoms develop after ingestion of gluten storage proteins (prolamins) from wheat (gliadins), barley (hordeins), rye (secalins), and their crossbred varieties [1,2]. CD can be diagnosed at any age. It can either be asymptomatic or present with a broad spectrum of clinical manifestations. The classical * Correspondence: 4 Department of Plant and Environmental Sciences, University of Gothenburg, SE-40530, Gothenburg, Sweden Full list of author information is available at the end of the article (typical) form of CD is usually characterized by gastrointestinal symptoms like flatulence, vomiting, constipation or persistent diarrhoea, general failure to thrive, mineral and vitamin deficiencies, and weight loss due to malabsorption. Atypical forms, on the other hand, present predominantly with extra-intestinal manifestations that include a blistering skin disease (Dermatitis herpetiformis), iron-deficiency anaemia, osteoporosis, fatigue and neurological complaints [3-6]. The prevalence of CD is estimated to be about 1% in the Western populations [7,8]. Moreover, in recent years the total disease prevalence has increased. The reason for the observed raise is currently unknown and cannot be explained by the © 2011 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chen et al. BMC Biotechnology 2011, 11:16 http://www.biomedcentral.com/1472-6750/11/16 increase of CD diagnosis that occurred after introduction of antibody screening [9,10]. In CD patients, peptides that originate from incomplete digestion of gluten prolamins, either in their native form or deamidated by tissue transglutaminase (tTG), bind to HLA-DQ2 or -DQ8 receptors of antigen presenting cells that activate the lamina propria infiltrating CD4+ T cells. As a response the CD4+ T cells release pro-inflammatory cytokines, in particular g-interferon. Ultimately, this leads to profound tissue remodelling characterised by the atrophy of the small intestinal villi and hyperplasia of crypts [2,11-14]. Active CD is also characterised by high levels of antibodies against tTG and gliadin in the patients’ sera. The role of anti-tTG IgA class antibodies is still unclear. However, it has been proposed that they may be involved in the development of mucosal damage [15]. Also IgG class antigliadin antibodies have been shown to contribute to the pathogenesis by activating the complement system or inducing antibody-mediated cytotoxicity [16]. T cell epitopes in wheat gluten proteins have been characterised within both gliadins and glutenins. A hierarchy exists within these epitopes. The majority of CD patient-derived intestinal T cell clones recognise a-gliadins, and less frequently g-gliadins and glutenins [17-20]. The most prominent peptide is a 33-mer of agliadins (residues 57-89) that contains six T-cell epitopes. Another fragments, also found in a-gliadins (residues 31-43 and 44-55), seem to be important for the activation of the innate immunity system [18,21-23]. In a recent study gluten-specific T cells from peripheral blood of CD patients challenged either with wheat, barley, rye or a combination of the three cereals were used to identify the immunostimulatory sequences in these grains [24]. The a-gliadin 33-mer was found immunogenic only after the wheat challenge while sequences from ω-gliadin (wheat) and C-hordein (barley) were found to be immunodominant despite the grain consumed. Currently there is no cure for CD. The only existing therapy is a life-long adherence to a gluten-free (GF) diet [3]. However, several strategies that may in the future serve as alternatives to the GF diet have been proposed. T cell activation may be inhibited by molecules that block peptide binding to HLA-DQ2. Alternatively, inhibition of tissue transglutaminase may prevent gluten deamidation [25]. Supplementation with prolyl endopeptidases (PEPs), enzymes derived from moulds and bacterial strains, or with a mixture of PEP and cysteine endoprotease from germinating bar (...truncated)