1. Structure-function analysis of NPC2 suggests a mechanism of cholesterol transport in the late
endo/lysosomal compartment
Richa Mehta, Leslie McCauliff, Judith Storch
Department of Nutritional Sciences and Rutgers Center for Lipid Research, Rutgers, the State University of New Jersey, 96 Lipman Drive, New Brunswick, NJ 08901
Abstract
Niemann-Pick C disease is a rare, inherited disease
in which cholesterol builds up to toxic levels in the
late endo/lysosomes (LE/LYs). Deficiencies in either
of two lysosomal proteins, NPC1 or NPC2 have
been implicated in the disease. NPC2 is a soluble
intralysosomal protein that binds cholesterol in vitro.
In previous studies shown by our lab NPC2 exhibits
cholesterol transport properties, likely explaining its
role in normal LE/LY cholesterol egress. Cholesterol
transfer was shown to occur via direct interaction of
NPC2 with donor or acceptor membranes, and
transfer rates were markedly enhanced by the
inclusion of the unique lysosomal phospholipid, bis-
monoacylglycerol phosphate (BMP/LBPA). To
elucidate the structural basis of the NPC2
cholesterol transfer function, a series of point
mutations that neutralize charged residues on the
surface of NPC2 are constructed. These mutants
bind cholesterol normally. An examination of the
cholesterol transfer properties of eleven mutants
using fluorescence quenching and resonance
energy transfer assays suggest s that there may be
more than one domain on the NPC2 surface that is
involved in cholesterol trafficking. To test this
hypothesis we will use a turbidity assay which
monitors the aggregation or fusion of large
unilamellar vesicles in the presence of NPC2
protein. Preliminary results from membrane
aggregation studies suggest that NPC2 may in fact
bind greater than one membrane simultaneously,
effectively forming a “bridge” for rapid transport of
cholesterol from internal LE/LY membranes,
eventually leading to efflux of cholesterol from the
LE/LY.
Introduction
Niemen Pick C (NPC) is an inherited lipid storage disorder in
which the intracellular trafficking of cholesterol is disturbed,
resulting in the accumulation of the unesterified cholesterol
and glycolipids in the late endosome/lysosomes. This results
in physiological consequences, almost always including
neurodegeneration, which is thought to secondary to the
specific absence of normal post- lysosomal cholesterol
metabolism and the effects of general lysomal dysfunction.
This dysfunction arises from the build in up of cholesterol and
other lipids in LE/LY. Defects in either NPC1 or NPC2 gene
product causes the accumulation of cholesterol, yet the exact
function of NPC1 and NPC2 in lysosomal cholesterol
trafficking remains unknown. In previous studies, we
examined the transfer of cholesterol from NPC2 protein to
membranes in vitro, using a fluorescence quenching assay.
This experiment showed that NPC2 could be rapidly
transporting cholesterol. To further investigate this
hypothesis we are currently studying the structure-function
relation of NPC2 in the transport of cholesterol in the late
endo/lysosomals.
Acknowledgment
We thank Dr. Matt Scott for providing plasmid for the K32A, D72A and K75A mutants. Also, I would like to
thank every one who helped me in the research and would like to thank my parents for their
support
Funding source
Summary
• A series of NPC2 point mutants were prepared by site directed mutagenesis. Kinetic studies
on these mutants show that certain charged residues on the surface of NPC2 appear
necessary for the sterol transport property of NPC2.
• Turbidity assay suggests that NPC2 may bind to more than one membrane simultaneously.
• It is hypothesized that cholesterol transfer by NPC2 may involve the formation of inter-
membrane bridges that effectively move cholesterol out of the inner endo/lysosomal
membranes for eventual egress from the compartment.
Various Sites on the NPC2 surface Mutated in the study
Wavelength, nm
290 300 310 320 330 340 350 360 370
TrpEmission
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
2 uM NPC2
2 uM NPC2 + 1 uM Chol
2 uM NPC2 + 2 uM Chol
Time (sec)
0 10 20 30 40 50
0.0071
0.0072
0.0073
0.0074
0.0075
0.0076
0.0077
RelativeFluorescence
Wavelength, nm
320 360 400 440 480 520 560
Fluorescenceintensity
0
5000
10000
15000
20000
25000
30000
Asp85
Lys97
Glu99
Lys115
Lys6
Lys97
Lys16
Glu97
Glu70
Asn39
Lys75
Asp72
Arg32
Time, sec
0 200 400 600 800 1000 1200
Tryptophanemission
0.990
0.995
1.000
1.005
1.010 wt NPC2
K97A
K75A
K32A
D72A
D85A
no WT D72AK75AD85AK115AN39AK32AK16AE70A K6A E99AK97A
DHEintramembranetransferrate,s
-1
0.000
0.002
0.004
0.006
0.008
0.010
0.012
[Protein], uM
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
A350
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
Time, sec
0 2 4 6 8 10
A350
0.015
0.016
0.017
0.018
0.019
0.020
These are tryptophan
fluorescence of NPC2 with
different amounts of cholesterol
Cholesterol transfer from NPC2 protein
(to membranes is monitored by
dequenching of tryptophan fluorescence.
DHE is a florescent cholestrol analyse.
A fluorescence resonance energy
transfer (FRET) assay is utilized to
monitor DHE transfer. DHE transfer
from donor to acceptor membranes is
monitored by energy transfer quenching
of DHE by acceptor membrane Dansyl-
phosphatidylethanolamine.
Structure-Function Analysis of NPC2
Effect of specific mutations on DHE
intermembrane transfer rates
Turbidity assay: To determine whether NPC2 can cause
membrane-membrane aggregation
BSA
NPC2NPC2 with LUV
containing LBPA
NPC2 with EPC
LUV
BSA with
EPC and LUV
Point mutants of NPC2 were obtained by
site directed mutagenesis and sterol
transport properties were examined. For this
study, wt and all mutants contained a myc-
6xhis tag
Cholesterol transfer from NPC2
protein (wt and mutants) is very
slow for some of the mutants.
NPC2 causes aggregation of
membranes, as indicated by the
increase of A350.
The results shows NPC2, but not
BSA, caused aggregation/fusion
of LUV, and LBPA in LUV makes
this process much faster.
Methods for quantifying sterol transfer rates
Ara Parseghian Medical Research Foundation
These results suggest that there may be more than one membrane binding domain, implying
that NPC2 could be binding to two membranes at the same time. This is why turbidity
experiments were done, and these results support the hypothesis.