| 1. |
- Chen, Tingsu, et al.
(författare)
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Identification of gliadin-binding peptides by phage display
- 2011
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 11
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Tidskriftsartikel (refereegranskat)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 peptide-expressing 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.
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| 2. |
- Hoffmann, Karolina, 1980, et al.
(författare)
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Blocking peptides decrease tissue transglutaminase processing of gliadin in vitro
- 2009
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 57:21, s. 10150-10155
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Tidskriftsartikel (refereegranskat)abstract
- Tissue transglutaminase (tTG) plays an important role in celiac disease pathology as it catalyzes deamidation and cross-linking of specific gluten peptides and converts them into potent epitopes recognized by intestinal T-cells. We investigated whether synthetic peptides with high affinity to gliadin could alter tTG activity on gliadin and whole gluten digest. The immobilized substrates were incubated with synthetic peptides identified by the phage display technique and a control peptide with no affinity to gliadin. Transglutaminase activity was measured with time resolved fluorescence. The mean tTG activity, compared to that of the control without the peptides, was reduced by 31, 33, and 36% for three selected gliadin-binding peptides, and 30% for the peptide pool (P <= 0.001-0.004) when gliadin was the substrate. Finally, substrate specificity experiments suggested that avenin was processed in a manner similar that used for gliadin during in vitro assays with tTG. The results showed that the blocking peptides efficiently reduced tTG processing of gliadin in vitro, and this strategy will be further investigated as an alternative therapy for celiac disease.
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| 3. |
- Hoffmann, Karolina, 1980
(författare)
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Gliadin-Blocking Peptides In vitro assessment of their potential to alleviate celiac disease development
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis work aimed to investigate the potential of synthetic peptides as agents to block wheat prolamins (gliadins) that trigger the development of symptoms in celiac disease. The first step of this investigation included selection and synthesis of peptides with high affinity to gliadins and assessment of the potential of these so-called blocking peptides to limit gliadin reactivity in vitro. Wheat proteins targeted by blocking peptides were characterized, and peptides ability to reduce gliadin recognition was evaluated in in vitro assays. It was tested whether blocking peptides could reduce gliadin processing by tissue transglutaminase, and its recognition by anti-gliadin antibodies. The digestive stability of complexes formed by gliadin and blocking peptides was also studied. Finally, their potential to reduce a negative non-immunological effect of gliadin on intestinal mucosal cells was assessed in vitro in a Caco-2 cell line model. A large pool of 12-mer peptides with a high affinity to gliadins was selected with the phage display technology, and two peptides denoted P61, and P64, were chosen for experiments. Blocking peptides expressed an affinity to a broad spectrum of gliadin proteins and to α-amylase/trypsin inhibitors, wheat allergenic proteins that are involved in eliciting an immune response in baker’s asthma. Both blocking peptides significantly reduced the tissue transglutaminase processing of intact gliadin and partially reduced its recognition by anti-gliadin antibodies. The blocking peptides also partially reduced the negative non-immunological effect that gliadin had on a Caco-2 cell line. However, complexes of blocking peptides with gliadin were only partially stable after the pancreatic phase of in vitro digestion, with P64 complex with gliadin being more stable than that of P61. The two chosen blocking peptides, P61 and P64, have proven the potential to bind to gliadin and to partially prevent its toxicity and recognition in in vitro assays. In order to be used in celiac disease therapy, however, more efficient gliadin blocking peptide complexes need to be explored. The large pool of 12-mer peptides obtained during selection with the phage display will further be screened in a search for the most effective peptides.
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| 4. |
- Hoffmann, Karolina, 1980, et al.
(författare)
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In vitro digestive stability of complexes between gliadin and synthetic blocking peptides
- 2011
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Ingår i: Biotechnology and Applied Biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 58:3, s. 190-197
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Tidskriftsartikel (refereegranskat)abstract
- Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions.
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| 5. |
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