HLA-DQA1 and Celiac Disease

Presented by Cassie Bac

Image: http://www.scientificamerican.com/article.cfm?id=celiac-disease-insights

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

Image: http://www.reallygreatfoods.com/

No wheat, rye, or barley allowed!!

What are the classic symptoms of CD?

• Malnutrition

• Abdominal cramping

• Intestinal gas and stomach bloating

• Chronic diarrhea

• 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

•cell-cell recognition•cell surface

receptors•immune system

•cellular interaction

How is HLA-DQA1 associated with CD?

Peptide

Cell Surface

Images: http://en.wikipedia.org/wiki/Gluten_immunochemistry

T-Cell

T-cell Receptor

DQA1

Antigen Presenting

Cell

Peptide

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 HLA-DQA1 expression

levels?

2. PhenotypeHow 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 HLA-DQA1 expression

levels?

2. PhenotypeHow does gliadin affect the phenotype of the

small intestine?

3. Interactions

How is gliadin acting at the molecular level?

Experimental ApproachHow does gliadin affect HLA-DQA1 expression

levels?

2. PhenotypeHow does gliadin affect the phenotype of the

small intestine?

3. Interactions

How is gliadin acting at the molecular level?

1. Chemical Genetics

(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-AaHuman 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

http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1

WT patient

expression levels

Celiac patientexpression

levels

(1) Hypothesis

• The addition of gliadin to mouse intestinal cells should show decreased HLA-DQA1 expression.

(1) Expected Data

Mouse Intestinal

Cells

HLA-DQA1 expression + +/-

WT WT + gliadin Celiac patient

Celiac patient + HLA-DQA1

Celiac patient + Gliadin

- - -

= 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?

Images: http://en.wikipedia.org/wiki/Small_intestine; http://en.wikipedia.org/wiki/Microvillus

Microvilli line intestinal cell membrane; involved in absorption.

Small intestine

CD and the small intestine...

Image: Children’s Hospital Boston. 2009. http://www.youngmenshealthsite.org/gluten_free_diet.html

(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

Mouse intestinal

cells

WT WT + gliadin Celiac patient

Celiac patient + HLA-DQA1

Celiac patient + gliadin

Images: http://health.uml.edu/thc/HealthIssues/Meghan_Donovan/Meghans_Homepage.html

HLA-DQA1Expression

Levels

(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?

Cell Surface

T-Cell

T-cell Receptor

DQA1

Antigen Presenting

Cell

Gliadin peptide?

HLA-DQA1 protein interactions

Image: From STRING 8.3. HLA-DQA1 evidence map. Retrieved from: http://stringdb.org/newstring_cgi/show_network_section.pltaskId=8jlOaZSDtoSa&interactive=yes&advanced_menu=_unassigned&network_flavor=evidenc

Question: Is gliadin regulating/binding

HLA-DQA1?

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 protein-protein

interactions

Gliadin binds HLA-DQA1

Gliadin binds interacting proteins

Image: http://www.fda.gov/Food/ScienceResearch/Laboratory

Methods/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 proteins- look 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

• University of Chicago Celiac Disease Center. Celiac Disease Facts and Figures. Print.

• SMART- http://smart.embl-heidelberg.de/

• GeneBee- http://www.genebee.msu.su/genebee.html

• http://knol.google.com/k/celiac‐disease#

• 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.

• http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001280

• PubChem- http://pubchem.ncbi.nlm.nih.gov/

• STRING- http://string.embl.de/

• Homologene- http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=AlignmentScores&list_uids=123820

• Microarrays- http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1