HLA-DQA1 and Celiac
Disease
Presented by Cassie Bac
Image: http://www.scientificamerican.com/article.cfm?id=celiac-diseaseinsights
Background on Celiac Disease (CD)
•
•
•
•
Autoimmune disease that
creates a toxic immune
response to gluten-- damaging
the lining of the small intestine
Affects 1 in 133 healthy
individuals in the United States
Can appear at anytime
throughout life!
No known cure
No wheat, rye, or barley allowed!!
Image: http://www.reallygreatfoods.com/
What are the classic symptoms of CD?
•
Malnutrition
•
Abdominal cramping
•
Intestinal gas and
stomach bloating
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Chronic diarrhea
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Weight loss or weight gain
•
Dermatitis Herpetiformis
Images: http://healthhype.com; http://www.ehow.com/about_5141982_pediatric-celiac-disease-symptoms.html
HLA-DQA1 protein structure
•cellular
interaction
•cell-cell
recognition
•cell surface receptors
•immune system
How is HLA-DQA1 associated with CD?
Peptide
T-Cell
T-cell
Receptor
Peptide
DQA1
Cell Surface
Antigen
Presenting
Cell
Images: http://en.wikipedia.org/wiki/Gluten_immunochemistry
What is still unknown?
What mechanism causes the
toxic immune system response
in Celiac Disease?
Image: http://jaymecarleton.com/secondnature/green-news/2011/04/toxic-toys-r-us/
What do we know about gliadin?
• Gliadin= a protein
derived from gluten and
the toxic factor involved
in Celiac Disease!
Question: How does
gliadin act??
Experimental Approach
1. Chemical Genetics
How does gliadin affect HLADQA1 expression levels?
2. Phenotype
How does gliadin affect the
phenotype of the small
intestine?
3. Interactions
How is gliadin acting at the
molecular level?
Experimental Approach
1. Chemical Genetics
How does gliadin affect HLADQA1 expression levels?
2. Phenotype
How does gliadin affect the
phenotype of the small
intestine?
3. Interactions
How is gliadin acting at the
molecular level?
Experimental Approach
1. Chemical Genetics
How does gliadin affect HLADQA1 expression levels?
2. Phenotype
How does gliadin affect the
phenotype of the small
intestine?
3. Interactions
How is gliadin acting at the
molecular level?
(1) How does gliadin affect HLA-DQA1
expression levels?
Method: Inject mouse intestinal cells with
gliadin (+ other varieties) and measure
HLA-DQA1 expression levels with
microarray
Why use a mouse model?
Mouse protein domains
H2-Aa
Human protein domains
HLA-DQA1
71.8% conserved
protein structure
Images: From SMART. HLA-DQA1 sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.pl;
SMART. H2-Aa sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.pl
Microarray data
WT patient
expression
levels
Celiac patient
expression
levels
http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726
gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1
(1) Hypothesis
• The addition of gliadin to
mouse intestinal cells should
show decreased HLA-DQA1
expression.
(1) Expected Data
WT
WT + gliadin
Celiac
patient
-
-
Celiac patient + Celiac patient +
HLA-DQA1
Gliadin
Mouse
Intestinal
Cells
HLA-DQA1
expression
+
+/-
= High HLA-DQA1 expression (+)
= Low HLA-DQA1 expression (-)
-
(2) How does gliadin affect small intestine
phenotype?
Method: Observe phenotype of
mouse intestinal cells following
gliadin treatment
What do we know about intestinal cells?
Microvilli line intestinal cell
membrane; involved in
absorption.
Small intestine
Images: http://en.wikipedia.org/wiki/Small_intestine; http://en.wikipedia.org/wiki/Microvillus
CD and the small intestine...
Image: Children’s Hospital Boston. 2009.
http://www.youngmenshealthsite.org/gluten_free_diet.ht
(2) Hypothesis
The addition of gliadin to mouse
intestinal cells should cause
physical damage to the microvilli.
In addition, the cells that showed
low HLA-DQA1 expression should
show increased intestinal damage.
(2) Expected Data
WT
WT + gliadin
Celiac
patient
Celiac patient + Celiac patient +
HLA-DQA1
gliadin
HLA-DQA1
Expression
Levels
Mouse
intestinal
cells
Images: http://health.uml.edu/thc/HealthIssues/Meghan_Donovan/Meghans_Homepage.html
(3) How is gliadin acting at the molecular
level?
Method: TAP-tag
Label gliadin and determine
how it is affecting HLA-DQA1
Gliadin may function in this way...
Gliadin
peptide?
T-Cell
Gliadin
peptide?
T-cell
Receptor
DQA1
Cell Surface
Antigen
Presenting
Cell
HLA-DQA1 protein interactions
Question: Is gliadin
regulating/binding
HLA-DQA1?
Image: From STRING 8.3. HLA-DQA1 evidence map. Retrieved from:
http://stringdb.org/newstring_cgi/show_network_section.pltaskId=8jlOaZSDt
oSa&interactive=yes&advanced_menu=_unassigned&network_flavor=evide
nc
What role do the predicted functional
partners play?
Question: Is gliadin regulating/binding any of
these interacting proteins?
(3) Hypothesis
Using Tandem Affinity
Purification, gliadin
should be found to
regulate HLA-DQA1
protein at the
molecular level.
Image: http://www.nature.com/nrm/journal/v4/n1/fig_tab/nrm1007_F1.html
(3) Expected Data
Mass spectrometry to
analyze the proteinprotein interactions
Gliadin binds HLA-DQA1
Gliadin binds interacting
proteins
Image:
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethod
s/DrugChemicalResiduesMethodology/ucm113209.htm
Future Directions
MUDPIT- Multidimensional Protein Identification
Technology
•
•Hypothesis:
HLA-DQA1 protein observed in WT patient
tissue samples, not observed in celiac samples.
•Experiment:
Take intestinal tissue samples from WT and
celiac patients and observe specific protein identifications
from the two samples.
Future Directions
Microarray primary HLA-DQA1 interacting proteinslook at expression levels in WT vs. Celiac Disease
patients. Are their levels expressed in similar patterns
as HLA-DQA1?
•
THE END
Image: http://www.herbalgranny.com/2009/09/06/herbal-treatment-for-celiac-disease/
References
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University of Chicago Celiac Disease Center. Celiac Disease Facts and Figures. Print.
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SMART- http://smart.embl-heidelberg.de/
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GeneBee- http://www.genebee.msu.su/genebee.html
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http://knol.google.com/k/celiac‐disease#
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Bizzaro, N., R. Tozzoli, D. Villalta, M. Fabris, and E. Tonutti. "Cutting‐Edge Issues in Celiac Disease and in Gluten
Intolerance." Clinical Reviews in Allergy and Immunology 15.1 (1997): n. pag. Web. 7 Feb
2011http://www.springerlink.com.ezproxy.library.wisc.edu/content/j17wu631711u1027/fulltext.pdf.
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http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001280
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PubChem- http://pubchem.ncbi.nlm.nih.gov/
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STRING- http://string.embl.de/
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Homologenehttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=AlignmentScores&list_uids=123820
Microarrayshttp://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gma
x=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1