Epitope mapping of Gliadin A trigger of Celiac

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

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

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

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?

Introduction - Antibody response and autoimmune diseases Antigen • 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

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

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

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

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

Introduction - Antibody response and autoimmune diseases Antibodies • 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

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

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

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

Introduction - Antibody response and autoimmune diseases Self protein (auto-antigen) Antigen presenting cell T-cell 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

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

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

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

• Using this knowledge Skovbjerg and colleagues produced a monoclonal antibody directed against the deamidated gliadin peptide 58 -73, LQPFPQPELPYPQPQ Glutamine Glutamic acid • The glutamine in position 65 was replaced by glutamic acid • Thus, a m. Ab 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)

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

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

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 Synthetic peptides • truncated resin-bound peptides • modified amino acids • secondary structures

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

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

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)

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

Results • Experimental data is based on: • m. Ab 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

Results - m. Ab anti-gliadin and gliadin peptide interaction examined by Luminex • Interaction between m. Ab anti-gliadin and the gliadin peptide is concentration dependent ~ interaction is specific

Results - Interaction between m. Ab 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 m. Ab anti-gliadin random selected peptides ~ interaction is specific

Results - Interaction between m. Ab 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

Results - Screening of epitope using N-terminally truncated resin-bound peptides examined by modified ELISA Peptide no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Amino Acid sequence Q PQ QPQ PQPQ YPQPQ PYPQPQ LPYPQPQ ELPYPQPQ PELPYPQPQ QPELPYPQPQ PQPELPYPQPQ FPQPELPYPQPQ PFPQPELPYPQPQ QPFPQPELPYPQPQ LQPFPQPELPYPQPQ • The peptide ELPYPQPQ was the first peptide to interact with m. Ab anti-gliadin • The epitope is located in the first 8 -10 amino acids of the gliadin peptide

Results - Interaction between m. Ab anti-gliadin and epitope candidates examined by inhibition ELISA • Screening using resin-bound peptides ~ QPELPYPQPQ Peptide no 8 9 10 14 Amino Acid sequence ELPYPQPQ PELPYPQPQ QPFPQPELPYPQPQ KLQPFPQPELPYPQPQ • 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 %

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

Results - Interaction between m. Ab 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 (1 M)

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

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

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 m. Ab 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?

Thank you for your attention