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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
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