Investigating Intestinal Alkaline Investigating Intestinal Alkaline Phosphatase as a Risk Factor for Phosphatase as a Risk Factor for

Inflammatory Bowel DiseaseInflammatory Bowel Disease

Vanessa DanquahVanessa Danquah

Guillemin LabGuillemin Lab

What is Inflammatory What is Inflammatory Bowel Disease ?Bowel Disease ?

Intestinal Alkaline Intestinal Alkaline Phosphatase Detoxifies Phosphatase Detoxifies Lipopolysaccharide in Lipopolysaccharide in

ZebrafishZebrafish

IAP is an enzyme that interacts with the IAP is an enzyme that interacts with the microbiota in the gut. microbiota in the gut.

It was proven that decreased IAP activity It was proven that decreased IAP activity caused inflammation in zebrafish intestine. caused inflammation in zebrafish intestine.

Is there a correlation Is there a correlation between IAP and between IAP and

Inflammatory Bowel Inflammatory Bowel Disease ?Disease ? Is the function of Is the function of

intestinal alkaline intestinal alkaline phosphatase in phosphatase in mediating bacterial-mediating bacterial-host interactions host interactions evolutionarily evolutionarily conserved?conserved?

Do polymorphisms in Do polymorphisms in human IAP alter human IAP alter enzymatic activity enzymatic activity and contribute to and contribute to IBD?IBD?

Measuring activity of IAP in Measuring activity of IAP in Drosophila Drosophila gutgut

Wild Type Gut vs. Carcass

ng

Pi/m

in/m

g t

iss

ue

Wild Type Gut

Wild Type Carcass

Gender and Age Gender and Age ExperimentsExperiments

GenderGender AgeAge

Day 0 -5 Gut Activity

0.00

0.05

0.10

0.15

0.20

0.25

0.30

ng P

i/min

/mg

tissu

e Day 0

Day 1

Day 2

Day 3

Day4

Day 5

Female Gut vs. Male

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

ng P

i/min

/mg

tissu

e

Female Gut

Male Gut

L-Phenylalanine-IAP L-Phenylalanine-IAP InhibitorInhibitor

L-Phenylalanine Inhibitor Threshold

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

ng P

i/min

/mg

tissu

e

Control

10uM

20uM

30uM

40uM

50um

L-Phenylalanine Inhibitor

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

ng

Pi/m

in/m

g t

iss

ue

Control-Gut

Control-Carcass

L-phenyl 50 mM - Gut

L-phenyl 50 mM-Carcass

Searching for the gene or Searching for the gene or genes that encode IAP in genes that encode IAP in

DrosophilaDrosophila

Each deficiency stock deletes Each deficiency stock deletes approximately 50 genes, one of approximately 50 genes, one of which is the candidate for which is the candidate for encoding IAP.encoding IAP.

Female X 2L 2R 3L 3R

4 2L 2R 3L 3R 4 X

Two chromosomal regions Two chromosomal regions encode IAP genesencode IAP genes

Deficiencies 1-4

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

ng

Pi/m

in/m

g t

iss

ue

Wt

Df1

Df2

Df3

Df4

Human IAP Human IAP PolymorphismsPolymorphisms

Different people have different amino acids within their IAP Different people have different amino acids within their IAP proteins at certain locationsproteins at certain locations

We will investigate whether these amino acid changes alter the We will investigate whether these amino acid changes alter the enzyme’s activityenzyme’s activity

P65LR125H

H279L

H279L

How did we create known How did we create known IAP variants?IAP variants?

Make IAP variants via site Make IAP variants via site directed mutagenesisdirected mutagenesis

Transfect and express them in Transfect and express them in Chinese Hamster Ovary Cells Chinese Hamster Ovary Cells (CHO )(CHO )

Assay for activity with pNPP Assay for activity with pNPP substratesubstrate

Clone IAP variants and wild Clone IAP variants and wild type construct into an type construct into an expression vectorexpression vector

Initial results of human IAP Initial results of human IAP enzyme activity enzyme activity in vitroin vitro

L-Phenylalanine Activity of Transfected Cells

1

Abso

rban

ce a

t 405

nm

Untreated

50mM L-phenyl

IAP Activity- Post Cell Transfection

1

Ratio of Transfection Reagent: DNA

Abso

rban

ce a

t 405

nm

3:2 4:2 5:2 6:2 7:2 8:2 UT

24

48

Conclusion and Future Conclusion and Future DirectionsDirections

Further characterize Further characterize the genes within the genes within chromosomal chromosomal deletions that reduce deletions that reduce IAP activityIAP activity

Continue to test IAP Continue to test IAP variants and whether variants and whether they alter activity they alter activity in in vitrovitro

Test whether Test whether expression of human expression of human IAP variants in IAP variants in DrosophilaDrosophila alter alter bacterial-host bacterial-host interaction in fliesinteraction in flies

DrosophilaDrosophila intestines intestines have high alkaline have high alkaline phosphatase activity phosphatase activity that is inhibited by the that is inhibited by the mammalian specific IAP mammalian specific IAP inhibitor L-inhibitor L-PhenylalaninePhenylalanine

We have identified two We have identified two chromosomal regions in chromosomal regions in the the DrosophilaDrosophila genome genome that are likely to encode that are likely to encode DrosophilaDrosophila IAP genes IAP genes

We have established a We have established a cell culture system to cell culture system to study human IAP study human IAP polymorphismspolymorphisms

AcknowledgementsAcknowledgements

Guillemin LabGuillemin Lab

Dr. Karen Dr. Karen GuilleminGuillemin

Ms. Anica Ms. Anica WandlerWandler

Dr. Peter O’DayDr. Peter O’Day

SPUR 2009SPUR 2009