Transcript Epitope mapping of gliadin – a trigger of celiac disease

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
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 kDa
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 kDa
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 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)
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:
• 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
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
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
4
3
A405-650
•
2
1
control peptide
background
CPRFSPITIDGMTSLVGMNIP
CSGPVPPSACPPRFSPITIDG
CRRYPGPLHHQAQRFRLDNLL
CLLSQLYQSPNRRYPGPLHHQ
CKGLNGQKPSGATEPITVKFA
CKTMTQKELEQLFSQYGRIIT
CDANLYVSGLPKTMTQKELEQ
CNYLPQNMTQEEFRSLFGSIG
CTDDSKTNLIVNYLPQNMTQE
0
CCPSPMQTGATTDDSKTNLIV
No interaction between mAb
anti-gliadin random selected
peptides
~ interaction is specific
CSNGPSSNNRNCPSPMQTGAT
•
Results
- Interaction between mAb anti-gliadin and PQPELPY
sequence examined by inhibition ELISA
2.5
A405-650
2.0
1.5
1.0
0.5
0.0
PQPELPY
Control
•
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
2.0
1.5
1.0
0.5
0.0
Q
PQ
QPQ
PQPQ
YPQPQ
PYPQPQ
LPYPQPQ
ELPYPQPQ
PELPYPQPQ
QPELPYPQPQ
PQPELPYPQPQ
FPQPELPYPQPQ
PFPQPELPYPQPQ
QPFPQPELPYPQPQ
LQPFPQPELPYPQPQ
Background
Amino Acid sequence
Q
PQ
QPQ
PQPQ
YPQPQ
PYPQPQ
LPYPQPQ
ELPYPQPQ
PELPYPQPQ
QPELPYPQPQ
PQPELPYPQPQ
FPQPELPYPQPQ
PFPQPELPYPQPQ
QPFPQPELPYPQPQ
LQPFPQPELPYPQPQ
A405-650
Peptide no
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
•
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
Results
- Interaction between mAb anti-gliadin and epitope
candidates examined by inhibition ELISA
•
Screening using resin-bound
peptides ~ QPELPYPQPQ
4
Control
KLQPFPQPELPYPQPQ
C-terminal Q (glutamine) and
N-terminal P (proline) is
essential for antibody binding
QPFPQPELPYPQPQ
•
0
QPELPYPQPQ
Minimum epitope ~ PELPYPQPQ
1
PELPYPQPQ
•
74 %
76 %
ELPYPQPQ
Center of the peptide does not
inhibit antibody binding
56 %
ELPYPQP
•
2
ELPYPQ
8
9
10
14
Amino Acid sequence
ELPYP
ELPYPQ
ELPYPQP
ELPYPQPQ
PELPYPQPQ
QPELPYPQPQ
QPFPQPELPYPQPQ
KLQPFPQPELPYPQPQ
ELPYP
Peptide no
A405-650
3
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)
Results
- Interaction between mAb anti-gliadin and gliadin
peptide examined by elution ELISA
AU could reduce the
interaction together
~ionic bonds and hydrogen
bonds are essential for
antibody-peptide interaction
2.0
1.5
1.0
0.5
Eluents (1M)
UT
AT
T
U
A
AU
0.0
Control
•
Tween together with urea or
ammoniumacetate could not
reduce binding
2.5
AUT
•
Neither of the eluents could
reduce the interaction on
their own
A405-650
•
Results
- Interaction between mAb anti-gliadin and gliadin
peptide examined by elution ELISA
A405-650
3
2
1
0,015625
0,03125
0,0625
0,125
0,25
0,5
1
2
0
Ammoniumacetate/Urea concentration in mol/L
•
AU solution used as eluent
•
mAb 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 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?
Thank you for your attention