1. Epitope mapping of Gliadin - A trigger of Celiac Disease Ph.D. Student Nicole H. Petersen Bioorganic Chemistry KU LIFE

2. Presentation of speaker Who am I ? Nicole H. Petersen Ph.D. student at KU LIFE, Bioorganic Chemistry group I started my Ph.D. project in December 2009 Project title: ”Characterization of antibody response using epitope libraries” Experimental work is performed at KU LIFE and at Statens Serum Institut, in the department of Clinical Biochemistry and Immunology Supervisers: Paul R. Hansen and Gunnar Houen

3. Outline Brief introduction to antibody response and autoimmune diseases Celiac Disease Epitope mapping and different strategies for epitope mapping Solid-phase peptide synthesis Results with epitope mapping of a gliadin peptide Why is this relevant and how can it be applied Conclusion and future perspectives

4. Introduction - Antibody response and autoimmune diseases Antibody response ~ antibodies produced upon exposure to an antigen The interaction between an antibody and its antigen is the heart of antibody response What is an antigen? What is an antibody? How is an antibody response generated? How are these things related to autoimmune diseases?

5. Introduction - Antibody response and autoimmune diseases Antigen is recognized by the immune system and stimulates an antibody response Can be a bacteria, virus, a protein… Originally the term antigen came from antibody generator Antigen

6. Introduction - Antibody response and autoimmune diseases An Antigen is recognized as foreign by the immune system and is engulfed by antigen presenting cells, such as macrophages, monocytes and dendritic cells Antigen presenting cell Antigen

7. The antigen is degrated into smaller fragments and displayed on the cell surface together with specialized glycoproteins Antigen presenting cell Introduction - Antibody response and autoimmune diseases Antigen

8. T-cells recognize the antigen-glycoprotein complex Antigen presenting cell Introduction - Antibody response and autoimmune diseases T-cell Antigen

9. T-cells stimulate B-cells to produce antibodies towards the specific antigen Antigen presenting cell Introduction - Antibody response and autoimmune diseases T-cell B-cell Antibodies Antigen

10. Introduction - Antibody response and autoimmune diseases Antibodies are antigen-binding immunoglobulin proteins They are composed of 4 peptide chains, which are connected through disulfide bonds. These interactions give the antibody molecule a characteristic Y-shaped structure The antigen-binding site is located in the N-terminal region of the antibody Antibodies

11. T-cells stimulates B-cells to produce antibodies towards the specific antigen Antigen presenting cell Introduction - Antibody response and autoimmune diseases T-cell B-cell Antibodies Antigen

12. Antigen presenting cell Introduction - Antibody response and autoimmune diseases Epitope T-cell B-cell Antibodies The antibodies interact with the antigen The region of an antigen that interact with the antibody is defined as an epitope Antigen

13. Antigen presenting cell Introduction - Antibody response and autoimmune diseases T-cell B-cell Antigen Antibodies Finally the antigen is neutralized Antigen

14. Introduction - Antibody response and autoimmune diseases Antigen presenting cell T-cell Self protein (auto-antigen) B-cell Auto-antibodies Abnormal functioning of the immune system, it fails to recognize protein/tissue ~ antibody response is produced against these A disease that results from such an immune response is termed an autoimmune disease

15. Celiac Disease Celiac disease: is triggered by the ingestion of wheat gluten – especially the wheat protein gliadin leads to inflammation in the small intestine is characterized by the presence of antibodies directed against gliadin and the enzyme transglutaminase(auto-antigen), which is involved in the digestion of gliadin upon ingestion of gliadin, the gliadin protein is fragmented and central glutamines are deamidated to glutamic acid, some of these deamidated peptide fragments have shown to induce celiac disease in sensitive patiens occurs in 1 % of the population throughout Europe and America, more predominant among females than males by 3:1 ratio Gluten-free diet is currently the only effective mode of treatment

16. Celiac Disease - Gliadin Gliadin is a wheat protein with a molecular weight of 30 kDa Short N-terminal domain Central repetitive domain, rich in Pro and Gln Long C-terminal domain, containing several charged amino acid residues

17. Celiac Disease - Gliadin Gliadin is a wheat protein with a molecular weight of 30 kDa Several peptide fragments which trigger celiac disease are located in the central domain Especially a deamidated peptide, which corresponds to amino acids 58-73 from the central domain induce celiac disease. This peptide contains the motif PQPELPY, which has been suggested to be an immunodominant epitope of gliadin Short N-terminal domain Central repetitive domain, rich in Pro and Gln Long C-terminal domain, containing several charged amino acid residues

18. Using this knowledge Skovbjerg and colleagues produced a monoclonal antibody directed against the deamidated gliadin peptide 58-73, LQPFPQPELPYPQPQ The glutamine in position 65 was replaced by glutamic acid Thus, a mAb anti-gliadin antibody has been generated, but how do we identify the epitope on the peptide that interact with the antibody? (Skovbjerg et al., 2004) Glutamine Glutamic acid

19. Epitope mapping Epitope mapping is the process of identifying the binding sites of antibodies on their target antigens Several strategies for epitope mapping exist: recombinant proteins X-ray co-crystallography phage-display peptide scanning truncated resin-bound peptides

20. Epitope mapping Epitope mapping is the process of identifying the binding sites of antibodies on their target antigens Several strategies for epitope mapping exist: recombinant proteins X-ray co-crystallography phage-display peptide scanning truncated resin-bound peptides

21. Epitope mapping Epitope mapping is the process of identifying the binding sites of antibodies on their target antigens Several strategies for epitope mapping exist: recombinant proteins X-ray co-crystallography phage-display peptide scanning truncated resin-bound peptides Synthetic peptides modified amino acids secondary structures

22. Different strategies for epitope mapping - Peptide scanning Peptide scanning is used for linear epitope mapping of entire proteins/large sequences This method involves a series of overlapping linear peptides that cover the sequence in question Protein sequence Overlapping peptides

23. Different strategies for epitope mapping - Peptide scanning Protein sequence Overlapping peptides Peptide scanning is used for linear epitope mapping of entire proteins/large sequences This method involves a series of overlapping linear peptides that cover the sequence in question

24. Different strategies for epitope mapping - Resin-bound peptides Mainly used for epitope mapping of small sequences and to distinguish closely related epitopes Truncated versions of peptides, usually N- or C-terminal, are synthetised on a solid support and examined for reactivity on this support First described in 1985, where the approach was used for epitope mapping of rat cytochrome C Example of N-terminal truncated peptide library. A number of peptides is synthesized in one batch, by removing resin after each coupling cycle (Patersen, Y. 1985)

25. Different strategies for epitope mapping - Solid-phase peptide synthesis SPPS is based on: addition of α-amino and side-chain protected amino acids to an insoluble support Removal of N-terminal protection Activation and coupling of the next amino acid Resultant peptide is cleaved from the resin to yield a free peptide Synthesized from the C- to the N-terminal

26. Results Experimental data is based on: mAb anti-gliadin produced by Skovbjerg and colleagues deamidated gliadin peptide, LQPFPQPELPYPQPQ corresponding to amino acids 58-73 in the gliadin protein, except that the glutamine in position 65 was replaced by glutamic acid

27. Results - mAb anti-gliadin and gliadin peptide interaction examined by Luminex Interaction between mAb anti-gliadin and the gliadin peptide is concentration dependent ~ interaction is specific

28. Results - Interaction between mAb anti-gliadin and random selected peptides examined by ELISA Peptides were selected based on their content of amino acids found in the gliadin peptide, such as Pro, Gln, Glu No interaction between mAb anti-gliadin random selected peptides ~ interaction is specific

29. Results - Interaction between mAb anti-gliadin and PQPELPY sequence examined by inhibition ELISA Assumption ~ PQPELPY may be an epitope of the gliadin peptide A vague inhibition in antibody binding was observed (15 %) ~ the PQPELPY peptide does not constitute the actual epitope

30. Results - Screening of epitope using N-terminally truncated resin-bound peptides examined by modified ELISA The peptide ELPYPQPQ was the first peptide to interact with mAb anti-gliadin The epitope is located in the first 8-10 amino acids of the gliadin peptide Peptide noAmino Acid sequence 1Q 2PQ 3QPQ 4PQPQ 5YPQPQ 6PYPQPQ 7LPYPQPQ 8ELPYPQPQ 9PELPYPQPQ 10QPELPYPQPQ 11PQPELPYPQPQ 12FPQPELPYPQPQ 13PFPQPELPYPQPQ 14QPFPQPELPYPQPQ 15LQPFPQPELPYPQPQ

31. Results - Interaction between mAb anti-gliadin and epitope candidates examined by inhibition ELISA Screening using resin-bound peptides ~ QPELPYPQPQ Center of the peptide does not inhibit antibody binding Minimum epitope ~ PELPYPQPQ C-terminal Q (glutamine) and N-terminal P (proline) is essential for antibody binding 56 % 74 % 76 % Peptide noAmino Acid sequence 8 ELPYP ELPYPQ ELPYPQP ELPYPQPQ 9PELPYPQPQ 10QPELPYPQPQ 14QPFPQPELPYPQPQ KLQPFPQPELPYPQPQ

32. Results - Interaction between mAb anti-gliadin and gliadin peptide examined by elution ELISA Current theory ~ ionic and hydrogen bonds are essential for antibody-antigen interaction Three ELISA Eluents (1M) Urea (U) ~ hydrogen bonds Tween (T) ~ hydrophilic and hydrophobic interactions Ammoniumacetate (A) ~ ionic bonds (Rubinstein et al., 2008)

33. Results - Interaction between mAb anti-gliadin and gliadin peptide examined by elution ELISA Neither of the eluents could reduce the interaction on their own Tween together with urea or ammoniumacetate could not reduce binding AU could reduce the interaction together ~ionic bonds and hydrogen bonds are essential for antibody-peptide interaction (1M)

34. Results - Interaction between mAb anti-gliadin and gliadin peptide examined by elution ELISA AU solution used as eluent mAb anti-gliadin – gliadin peptide interaction, relative high AU concentration to see elution effect ~ strong interaction

35. Why is this relevant and how can it be applied The precise localization of epitopes is essential in the development of new and improved biological applications such as: designed vaccines diagnostic immuno-therapeutics Characterization of epitopes is fundamental to the understanding of immunological discrimination between self and non-self and in mechanisms of bio-recognition in general Hopefully these results in the long term will contribute to the determine the etiology of celiac disease Improve existing treatment

36. Conclusion and future perspective Minimum immunodominant epitope was identified as PELPYPQPQ C-terminal glutamine and N-terminal proline is essential for antibody binding ionic bonds and hydrogen bonds are essential in regard to mAb anti-gliadin and gliadin peptide interaction Examine the secondary structure of some of the reactive peptides How do these data relate to patient sera, is the identified epitope of the gliadin peptide a natural epitope as well?

37. Thank you for your attention