Homology modeling and functional testing of an abca1

1. HOMOLOGY MODELING AND
FUNCTIONAL TESTING OF AN ABCA1
MUTATION CAUSING TANGIER
DISEASE
Rachel J. Suetania, Brie Sorrensona, Joel D.A. Tyndallb, Michael J.A.
Williamsc, Sally P.A. McCormicka
a Department of Biochemistry, University of Otago, Dunedin, New
Zealand
b School of Pharmacy, University of Otago, Dunedin, New Zealand
c Department of Medical and Surgical Sciences, University of Otago,
Dunedin, New Zealand

2. CONTENTS
• ABSTRACT
• INTRODUCTION
• MATERIALS AND METHODS
• RESULTS
• DISCUSSION
• CONCLUSION

3. INTRODUCTION

4. • TANGIER DISEASE:
Tangier disease (TD) is a rare, autosomal recessive
disease.
The disease is caused by mutations in ABCA1, an
extracellular membrane bound protein crucial for
HDL production.
ABCA1 activity is required for initial efflux of cholesterol
and phospholipids from cells to circulating apoA-1 to
form pre-beta HDL.

5. • SYMPTOMS:
The lack of lipid efflux can cause orange
tonsils, peripheral neuropathy.

6. • TESTS:
1. Homology model was constructed for the
human ABCA1 protein in order to better predict
the effect of p.R1068H mutation.
2. Direct testing of the functional impact of the
p.R1068h mutation on ABCA1 protein.

7. MATERIALS AND METHODS

8. • COMPUTATIONAL MODELING:
p.R1068H substitution was
predicted with PANTHER and
SIFT.
Using Modeler a model of NBD of
the human ABCA1 was constructed
based on 2.5 crystal structure of
HLY ABC transporter from E.coli
R1068 residue was located and
potential interactions with nearby
amino acids were identified.

9. • STUDY SUBJECTS:
The TD proband, 3 Heterozygous carriers of the
mutation and 4 wildtype family members were
recruited for skin biopsy.

10. • FIBROBLAST CELL CULTURE
ESTABLISHMENT:
Fibroblasts were obtained using the
explant growth method
Primary human fibroblasts were grown
in Advanced DMEM supplemented
with 10% fetal bovine serum.
Cultured fibroblasts were used for
cholesterol assays.

11. • ABCA1 cDNA EXPRESSION VECTORS:
This vector contains the human ABCA1 cDNA
sequence.

12. • TRANSFECTION OF HEK293 CELLS WITH
cDNA EXPRESSION VECTORS:
HEK293 cells were cultured in high glucose
DMEM with the same supplementation as the
primary fibroblast cell culture medium.
Cells were seeded in either 12-well plates or on
coverslips in 6-well plates.
After 12 h, cells were transiently transfected with
the ABCA1 cDNA vectors.

13. • WESTERN BLOTTING OF ABCA1 PROTEIN:
Western blots were performed on cell lysates from
primary fibroblasts and transfected HEK293 cells
to confirm expression of ABCA1.
• CHOLESTEROL EFFLUX ASSAYS:
Cholesterol efflux assays were performed on primary
fibroblast and transfected HEK293 cells.
• CONFOCAL MICROSCOPY:
Transfected HEK293 cells were GFP-tagged and they
were observed under confocal microscopy.

14. RESULTS

15. COMPUTATIONAL MODELING OF ABCA1
Sequence alignment of the human ABCA1 NBD-1 sequence with
the HlyB NBD-1 sequence. Dark grey shading represents
completely conserved residues and light grey shading represents
semi-conserved residues. Residues are numbered according to the
human ABCA1 protein sequence. The R1068 residue is indicated
by an asterisk.

16. A homology model of the NBDs of human ABCA1 with ATP
bound was constructed based on the crystal structure of the E.
Coli ABC transporter HlyB. NBD-1 is shown in green and NBD-2
in yellow. The ATP and side chains of the R1068, D1092 and
E1093 residues are in stick representation.
The Walker B motif is highlighted in purple.

17. Alignment of a partial sequence of the ABCA1 NBD-1 sequence
from various species. Dark grey shading represents completely
conserved residues and light grey shading represents semi-
conserved residues. Residues are numbered according to the
human ABCA1 protein sequence. The R1068, D1092 and E1093
residues are indicated by an asterisk. The human sequence
(NP005493.2) was aligned with mouse (NP038482.3),
chimpanzee (XP001138040.1), chicken (NP989476.1), and
zebrafish (NP001139161.1).

18. FUNCTIONAL ACTIVITY OF p.R1068H
PROTEIN
A. Cultured primary human fibroblasts from members of a Tangier disease
(TD) family were equilibrated with [3H]-cholesterol and incubated in
serum-free medium with or without human apoA-I (10gmL−1). Cholesterol
efflux was measured as the percentage of labeled cholesterol present in the
medium after 8 h. Numbers in brackets indicate number of individuals of
each genotype.
B. Cholesterol efflux assays repeated after exposure to 2M of the LXR
agonist, to increase ABCA1 expression.

19. C. Western blot analysis of ABCA1 from unstimulated fibroblasts
using -actin as a loading control.
D. Western blot analysis of ABCA1 from LXR-stimulated
fibroblasts using -actin as a loading control.

20. E. Quantification of ABCA1 protein levels in unstimulated fibroblasts
F. Quantification of ABCA1 protein levels in stimulated fibroblasts. The
ABCA1 band intensities from Western blots were quantified by
densitometry and normalised to actin levels.

21. Cholesterol efflux activity in HEK293 cells. Cultured HEK293 cells
were transiently transfected with a GFP-tagged ABCA1 cDNA
expression vector for either wildtype or p.R1068H.
A. ABCA1 expression was analysed by Western blot. Actin was
used as a loading control.NT is nontransfected HEK293 cells.

22. B. Cells were equilibrated with [3H]-cholesterol and incubated in
serum-free medium with or without human apoA-I (10gmL−1).
Cholesterol efflux was measured as the percentage of labeled
cholesterol present in the medium after 12 h.

23. SUB-CELLULAR LOCALISATION OF
ABCA1 PROTEIN IN VITRO

24. HEK293 cells were transiently transfected with GFP-tagged
ABCA1 cDNA expression vectors for either wildtype or
p.R1068H. Cells were counter-stained with AlexaFluor 594-
conjugated Wheat Germ agglutinin to mark cell membranes.
Cells were imaged with a 63× objective lens on a Zeiss LSM
510 confocal microscope with Argon (488nm excitation),
and HeNe (633nm excitation) lasers. Images are presented
as single channel and merged images representative of three
separate experiments.

25. DISCUSSION
• PANTHER and SIFT predicts any residue
substitution at position 1068 to be tolerated.
• The 3D model indicates potential ionic
interaction between R1068 and two carboxylic
acid containing residues, D1092 and E1093.
• The R1068H mutation does not appear to affect
expression or stability of ABCA1 protein as
shown by Western blotting analysis of both
primary human fibroblasts and transfected
HEK293 cells.

26. • Localisation studies carried out using GFP-
tagged ABCA1 expression vectors showed
trafficking of the mutant protein to the plasma
membrane to be defective.
• According to this paper, it has been proposed
that the R1068H mutation disrupts the structure
of ABCA1 monomer around the ATP-binding site
altering the conformation of NBD-1.

27. CONCLUSION
• Functional studies confirm that the p.R1068H
mutation produces a dysfunctional ABCA1
protein due to defective trafficking.
• Homology modeling of the NBDs of human
ABCA1 predicts the R1068 residue to make ionic
interactions crucial to the conformation of ATP-
binding site.

28. PRESENTED BY:
PRAM PRIYANCA S
A50210716003
M.TECH (BT) 3RD SEM
AIB-AUH