From the Lipid Cell Biology Section and
§ Cell Biochemistry Section, Laboratory of Cell Biochemistry
and Biology, NIDDK, and ¶ The Developmental and Metabolic
Neurology Branch, NINDS, National Institutes of Health, Bethesda,
Maryland 20892, the
Neurology Research Laboratory, Veterans
Affairs Medical Center, Newington, Connecticut 06111, and the
** Center for Research on Reproduction and Women's Health,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, June 20, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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The Niemann-Pick C1 (NPC1) protein and
endocytosed low density lipoprotein (LDL)-derived cholesterol were
shown to enrich separate subsets of vesicles containing lysosomal
associated membrane protein 2. Localization of Rab7 in the
NPC1-containing vesicles and enrichment of lysosomal hydrolases in the
cholesterol-containing vesicles confirmed that these organelles were
late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a
lipid marker of the late endosomal pathway, was found in the
cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7
compartments was stimulated by cellular uptake of cholesterol. The NPC1
compartment was shown to be enriched in glycolipids, and
internalization of
GalNAc The generation of unesterified cholesterol from endocytically
derived cholesteryl esters in lysosomes does not itself initiate metabolic responses in cells. This lysosomal pool of cholesterol remains metabolically inert until it has been delivered to other cellular organelles. The metabolic significance and mechanisms of
lysosomal cholesterol trafficking have begun to be revealed through the
study of Niemann-Pick C
(NP-C)1 disease (1). The
prominent cellular feature of this metabolic disorder is the extensive
sequestration and accumulation of LDL cholesterol in lysosomes
resulting from a defect in the translocation of this sterol pool to
other cellular membranes (2-4). The recognition of this genetically
induced sterol-trafficking defect introduced the concept of specific
protein-mediated egress of cholesterol from lysosomes. This notion was
further supported by the subsequent recognition of two separate
defective gene loci for NP-C disease (5). The gene most commonly
mutated in this disorder (NPC1) was recently cloned, and its
sequence predicts a unique multiple membrane-spanning protein of 1278 amino acids (6, 7). Analysis of the primary sequence suggests that the
NPC1 protein has several different domains likely to have functional
significance. In addition to a signal peptide for endoplasmic reticulum
insertion (amino acid residues 1-22), the N terminus of NPC1 contains
a region (amino acid residues 55-165) that has been termed the "NPC1
domain" because of its high degree of conservation in a wide array of existing NPC1 orthologs. Downstream of the NPC1 domain are five predicted transmembrane domains (at amino acid residues 615-797) in a
region with strong homology to the putative sterol sensing domains of
several other proteins, including the sterol response element binding
protein cleavage-activating protein, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and the Hedgehog signaling protein, Patched. The C terminus of NPC1 contains a dileucine
motif suggested to be required for the targeting of proteins to
lysosomes. The functional significance of these domains was recently
confirmed in studies using site-directed mutagenic mapping strategies
(8, 9).
Earlier studies demonstrated that the NPC1 protein is located in late
endosomes and that the clearance of endocytosed
[14C]sucrose as well as cholesterol was defective in NP-C
cells (10). These findings suggested that general retroendocytic
trafficking and mobilization of multiple lysosomal cargo are defective
at a late endosomal trafficking step. This concept provided an
explanation for the accumulation of multiple lipids in NP-C cells and
tissues (1).
The present study characterizes the NPC1 compartment with respect to
glycolipid content using antibodies to glycolipids. The glycolipid
profile of the NPC1 compartment was found to be modulated by
cholesterol. This report also documents an unusual mode of tubular
membrane trafficking for the NPC1 compartment that appears to be linked
to the NPC1 protein. Based on these results we discuss the concept of a
multiple functional repertoire for the NPC1 compartment that includes
the sorting of glycolipids and cholesterol.
Materials--
Fetal bovine serum (FBS) was obtained from
HyClone Laboratories, Inc., Logan, UT. Lipoprotein-deficient bovine
serum (LPDS) and human low-density lipoprotein (LDL) were prepared by
Intracel Corp., Rockville, MD. Glass and plastic chamber slides
(Lab-Tek) were purchased from Thomas Scientific. Rabbit polyclonal
antiserum to human NPC1 generated with a conjugated synthetic peptide
of residues 1256-1274 was employed as described previously (10). Mouse
anti-human LAMP2 and LIMP1 (CD63) antibodies, developed by Dr. J. T. August, were obtained from the Developmental Studies Hybridoma Bank
maintained by the University of Iowa (Iowa City, IA). Monoclonal
anti- Tissue Culture--
Normal and NP-C fibroblasts were derived
from volunteers and confirmed patients of the Developmental and
Metabolic Neurology Branch under the guidelines approved by the NINDS
Intramural Review Board. Five different normal cell lines (ENZ123,
ENZ125, ENZ143, GM5565, and GM1652) and three NP-C cell lines (GM3123,
DMN92.31, and DMN87.57) were studied. Two null mutant NP-C cell lines,
DMN98.16 (10) and DMN93.41,2
which do not express NPC1 protein were also used for immunocytochemical studies. Fibroblasts were cultured in Eagle's minimal essential medium
supplemented with 10% FBS, 1% nonessential amino acids, 2 mM glutamine, and 100 units of penicillin/streptomycin/ml
in humidified 95% air and 5% CO2 at 37 °C. For
immunocytochemical analyses, fibroblasts were seeded at a density of
20,000 cells/well in McCoy's/5% LPDS medium in 9.5-cm glass chamber
slides (Nunc, Inc., Naperville, IL) coated with human fibronectin.
Immunocytochemical Analyses--
Cells in glass chamber slides
were washed in phosphate-buffered saline and fixed in 3%
paraformaldehyde for 30 min. Cells were immunocytochemically
labeled using an indirect procedure in which all incubations
(quench, primary and secondary antibodies, and washes) were performed
in blocking solution containing filipin (0.05%) and IgG (2.5 mg/ml) of
the secondary antibody species. Primary antibody solutions were used at
the following dilutions; NPC1 (1:1000), LAMP2 (1:50), LBPA (1:50), apoD
(1:100), M6PR (1:500), Rab7 (1:100), GM2 (1:50), O1
anti-Gal/Lac-Cer (1:100), CTH (1:50), and GD3 (1:50).
Secondary FITC Cy5 and LRSC-labeled antibodies were used at 1:100
dilution. Fluorescence was viewed with a Zeiss 410 laser confocal
scanning microscope using an Omnichrome model krypton-argon laser
(American Laser Corp., Salt lake City, UT) with excitation wavelengths
of 488, 633 and 568 nm, for FITC Cy5 and LRSC, respectively. Filipin
fluorescence was viewed using an Enterprise model argon laser
(Coherent, Santa Clara, CA) with an excitation wavelength of 360 nm.
Ganglioside Metabolism--
Normal (ENZ143) and NP-C
fibroblasts expressing mutated (GM3123) or no detectable (DMNB98.16)
NPC1 protein were seeded at a density of 500,000 cells in plastic
100-mm dishes (Costar, Cambridge, MA) and incubated in Eagle's minimum
essential medium (EMEM) supplemented with 10% fetal bovine medium, 1%
nonessential amino acids, 2 mM glutamine, and 100 units of
penicillin/streptomycin/ml in humidified 95% air and
5%CO2 at 37 °C. Cells were cultured for 72 h in
medium containing 10 µCi of
N-acetyl-D-[3H]mannosamine.
Gangliosides were isolated according to the method of Fishman (19) and
separated on high-performance thin-layer chromatography plates
developed in chloroform/methanol/0.25% KCl (v/v/v). The radioactivity
of gangliosides identified using authentic standards was determined by
liquid scintillation counting.
Plasmid Construction--
The mammalian expression vector, pNNE,
was constructed by inserting the NaeI/BglII
fragment (4057 base pairs) from plasmid 1-1 (6) encoding NPC1
into pEGFP-C1 (CLONTECH, Palo Alto, CA) between the
BspEI (filled with dNTP by T4 DNA polymerase) and BglII sites in-frame. The plasmid containing GFP-NPC1
cDNA was sequenced to confirm insertion at the appropriate sites.
Another mammalian expression vector, pNES-6, was constructed in
multiple steps. Briefly, the fragment encoding NPC1 was obtained by
restriction digestion of plasmid 1-1 with EcoRI and was
inserted into pSV-SPORT1 (Life Technologies, Gaithersburg, MD) at the
EcoRI site. Directionality of this construct, pSN, was
confirmed by restriction digestion. A linker encoding 6 histidine
residues and a BamHI site was inserted at the 3'-end of NPC1
to replace the stop codon of NPC1. Finally, GFP from pEGFP-N1
(CLONTECH, Palo Alto, CA) was inserted in-frame into the BamHI site in the linker in pSN to create the
plasmid pNES-6 encoding an NPC1-GFP fusion protein.
Expression of NPC1 in CHO (CT-60) Cells or Null NPC1 Human NP-C
Fibroblasts--
Functional expression of the plasmids pNNE and pNES-6
was confirmed by complementation of the NP-C phenotype in both
CT-60 NP-C mutant CHO cells (20) or null NPC1 human NP-C
fibroblasts. CT-60 cells and 93.41 primary skin fibroblasts from
an NP-C patient were cultured to ~70% confluence. Cells were
transfected with pNNE, pNES-6, or the control plasmid pEGFP-C1, using
LipofectAMINE 2000 (Life Technologies) according to manufacturer's
instructions. 48-72 h after transfection and incubation in 10% FBS,
cells were fixed with 3% paraformaldehyde for 30 min and stained with
filipin to assess cholesterol clearance (6, 8). The percentage of cells
that expressed the GFP (from pNNE) or NPC1-GFP chimera (from pNES-6)
and complemented the NP-C phenotype was determined (9). Expression of NPC1 was confirmed by indirect immunofluorescence using
polyclonal antibodies directed against a C-terminal peptide of NPC1
(10).
Cytochemical Identification of the NPC1 Compartment--
Earlier
immunocytochemical studies showed that the NPC1 protein is located in a
unique set of LAMP2-containing vesicles that do not contain mannose
6-phosphate receptor (M6PR) or LDL-derived cholesterol (10). We have
now further defined the intracellular distribution of the NPC1 protein
in cultured fibroblasts with additional markers of the late endosomal
pathway. The data are summarized in Table
I and are collected from fibroblasts
incubated with LDL for 24 h. As previously reported, NPC1 and
cholesterol are located in separate vesicles (Fig.
1, A and B), but
both are positive for LAMPs 2 and 3 and LIMP (Table I). The NPC1
protein-containing vesicle contains the late endosomal marker Rab7
(Fig. 1, C and D, and Table I). The NPC1
compartment is not enriched in cathepsin D (Fig. 1, E and
F), and NPC1 sparsely localizes as well with
In fibroblasts, LDL-cholesterol-enriched lysosomes contain also
apolipoprotein D (Table I) and lysobisphosphatidic acid, a presumptive
marker for the late endosomal compartments (12) (Fig.
2, A and B, and
Table I), whereas neither is present in the NPC1 compartment. Adaptins
1 and 2 are not present in either the NPC1 vesicle or the
cholesterol-enriched vesicle (Table I). Cellular uptake of the fluid
phase marker, fluorescent dextran, by fibroblasts (Table I), and
Chinese hamster ovary cells (CHO) enriched both the NPC1 vesicle (Fig.
1, G and H) and the cholesterol-containing (Fig.
2, C and D) compartment. DiI-LDL, an endocytosed
marker for lysosomes (22), colocalized with cholesterol-enriched
vesicles in both fibroblasts (Table I) and CHO cells (Fig. 2,
E and F).
Sterol Modulation in the Cellular Distribution of NPC1--
In
cholesterol-depleted normal fibroblasts, NPC1 has a diffuse cellular
distribution with little discernible vesicular staining (Fig.
3A). During a subsequent
period of cellular cholesterol enrichment with LDL, prominent detection
of NPC1 protein in endocytic vesicles is now noted (Fig.
3B). This marked alteration in the cytochemical detection of
NPC1 appears to reflect changes in the organization and distribution of
the protein, because Western blot comparisons of cell extracts from
cholesterol-depleted and -enriched cultures did not show any
differences in absolute levels (data not shown). Because the NPC1
protein has transmembrane domains, it is likely the diffuse cellular
immunofluorescence under conditions of cholesterol depletion represents
NPC1's diffuse distribution in an extensive membranous compartment,
perhaps the endoplasmic reticulum. A similar effect of cellular
cholesterol enrichment on NPC1 protein distribution was reported in an
independent study (23). It should be noted that in these
cholesterol-enriched cells, the NPC1 protein and cholesterol remain in
separate and distinct LAMP-containing vesicles.
The NPC1 Compartment Contains Glycolipids--
Glycolipids as well
as cholesterol accumulate in NP-C disease (1). Both acidic glycolipids
such as the ganglioside
GalNAc
These glycolipid pools are modulated by LDL-cholesterol. In
cholesterol-depleted fibroblasts that were enriched with cholesterol by
co-culture with LDL for 24 h instead of maintenance in fetal bovine serum, GM2 (Fig. 5,
A-C) and Gal-Cer/Lac-Cer (Table I) remained in
NPC1-positive vesicles. However, GD3 (Fig. 5D)
and CTH (Table I) now translocated to cholesterol-enriched lysosomal vesicles.
In stark contrast, NP-C fibroblasts lacking NPC1 expression (Fig.
6A) did not endocytically
store GM2 (Fig. 6B) and Gal-Cer/Lac-Cer (data
not shown). These lipids appear to remain at the plasma membrane. Other
glycolipids such as CTH and GD3 are internalized into
cholesterol-loaded lysosomes in these mutant cell lines (data not
shown). The null mutant NPC1 cells were shown to synthesize normal
levels of GM2 (Table II).
Transfection of these cells with NPC1 cDNA re-established lysosomal
cholesterol trafficking (data not shown) and re-established
GM2 localization in endocytic vesicles that contained NPC1
(Fig. 6, C and D).
Further studies of glycolipid transfer through the NPC1 compartment
were carried out with NP-C CHO cells (CT-60) that have been shown to
carry an early translational termination mutation in the
NPC1 gene (20). When a population of CT-60 cells are transfected with wild type NPC1-GFP, some of the cell population appears in an early phase of fluorescent protein expression with the
majority of NPC1-GFP still residing at the periphery of lysosomes that
remain filled with cholesterol (Fig.
7A). These particular cells
reveal no endocytic GM2 staining (Fig. 7B). In
other cells in which the NPC1-GFP protein resides in the core of
endocytic vesicles (Fig. 7C), GM2 reappears in
NPC1-containing vesicles (Fig. 6D) that are emptied of
cholesterol (data not shown).
The cellular profile of GD3 distribution in these
transfected CT-60 cells differs entirely (Fig.
8). In cells that were not transfected
(Fig. 8A), GD3 was found in lysosomes filled
with cholesterol (Fig. 8B). In cells in which NPC1-GFP
protein still largely resides in the limiting membranes of lysosomes
(Fig. 8C), GD3 remains in the cholesterol-filled
lysosomes (Fig. 8, D-F). In transfected CT-60 CHO cells
cleared of cholesterol, where NPC1-GFP protein was distributed into the
interior of endocytic vesicles (Fig. 8G), GD3
has now been cleared from the vesicles (Fig. 8H). The
extensions associated with NPC1-GFP vesicles, noted in Fig. 8G have been monitored in viable cells by time-lapse
confocal microscopy at enhanced detection and have been found to
represent flexible and kinetically mobile tubules that extend from
the GFP-positive vesicles (data not shown).
The NPC1 protein has proven to be a valuable biological tool in
the study of intracellular lipid trafficking. The current report
represents an extenuated immunocytochemical characterization of the
positioning of NPC1 within the compartments of the late endosomal
pathway as it relates to its role in sterol and glycolipid transport
(see Table I for summary).
Cellular Cholesterol Uptake Enriches the NPC1 Content of Late
Endosomes--
Defective retroendocytic clearance of endocytosed
[14C]sucrose from NP-C cells predicted a trafficking
lesion in an endocytic compartment with the size and turnover rate of
late endosomes (10). The present studies confirm the late endosomal
nature of the NPC1-enriched compartment. In sterol-deprived cells, the NPC1 protein appears in a cellular dispersed state (Fig.
3A). When cells are enriched with lipoprotein-derived
cholesterol, dispersed cellular NPC1 protein relocates to an enriched
vacuolar state (Fig. 3B). These NPC1 vesicles contain Rab7
(Fig. 1F) a marker protein for late endosomes said to
function in vesicular trafficking to lysosomes (24). Rab7 was not
present in cholesterol-laden vesicles (Table I). Rab proteins,
belonging to a superfamily of low molecular weight GTPase, are known to
be crucial for vesicular transport and are found on the membranes of
the pairs of organelles that interact during the transport cycle (25).
Rabs are recruited onto the donor vesicle membrane where they are
thought to direct targeting, docking, and fusion of those vesicles to
the recipient organelle. Rab9, which has been suggested to regulate
endosomal traffic to the trans-Golgi network (26), has also
been reported to be associated with the NPC1-containing vesicle (27).
The location of both Rab7 and Rab9 in NPC1-enriched vesicles suggests this compartment is a late endosome having the potential to shuttle cholesterol and perhaps other endosomal products to lysosomes and Golgi
complex. In this regard, it has been shown that NPC1 mutations disrupt
the flow of LDL-derived cholesterol through both lysosomes and the
trans-Golgi network (28-29). The mechanism of the
NPC1-mediated transport between these organelles remains to be elucidated.
Mannose-6-phosphate receptor (M6PR) has been described in many cell
types as a marker protein for prelysosomal and late-endocytic vesicles
(30). M6PR's deliver newly synthesized acid hydrolases to the
endocytic pathway and direct their return to the Golgi (11). However,
in human fibroblasts we did not find M6PR in the NPC1-containing
vesicles that we consider to be late endosomes (10). In other cells,
including Hep-2 cells, M6PR was also not visualized in late endosomes
at steady states and most of the receptor was found within the
trans-Golgi network and in vacuolar structures in the
peripheral cytoplasm (31). We found comparable locations for M6PR in
normal fibroblasts (Fig. 4 in Ref. 10), suggesting that the M6PR does
not become enriched in the late endocytic pathway. Lack of accumulation
of the M6PR in late endosomes could reflect a rapid dissociation and
egress of the receptor from the late endosomes. Thus, a stable
M6PR-rich compartment, equivalent to the late endosome stage of the
endocytic pathway, may not exist in human fibroblasts under the
employed culture conditions thus reflecting the finding that the NPC1
vesicles are not M6PR-positive.
LDL Uptake Enriches the Cholesterol Content of Lysosomes--
We
examined the cytochemical distribution of the lysosomal enzymes,
cathepsin D (24) and
These cholesterol-enriched lysosomes contain lysobisphosphatidic acid
(LBPA) (Fig. 2A). This particular marker is not located in
NPC1 vesicles. LBPA was reported in baby hamster kidney (BHK) cells to
be a lipid marker of late endosomal vesicles displaying the morphology
of multivesicular bodies, which contain Rab7 (32). LBPA was later shown
to reside in the cholesterol-laden vesicles in a particular NP-C mutant
fibroblast cell line and in normal cells treated with a drug that
blocks lysosomal cholesterol transport (12). Because these vesicles
also contained M6PR, they were considered to represent late endosomes
(12). In contrast, the LBPA/cholesterol-containing vesicles we
identified were not of the multivesicular form as described previously
(32) but had typical lysosomal multilamellar structure (33, 34). Based on their enrichment with the two lysosomal markers, cathepsin D and
We have found that treatment of normal fibroblasts with drugs that
block lysosomal cholesterol transport (progesterone or hydrophobic
amines) produces a hybrid organelle having the characteristics of both
the NPC1/Rab7-positive late endosomes and the cholesterol-laden lysosomes (Ref. 10 and current data not shown). These are very reminiscent of the vesicular structure reported previously (32). When
these pharmacologic agents are washed out of the cells, the two
separate vesicles are regenerated, each with their distinct marker
components (data not shown). In many of the NP-C cell lines we have
examined, the differential intracellular distribution of LBPA and
NPC1/Rab7 into cholesterol enriched and cholesterol poor compartments,
respectively, is maintained. Uptake of LDL by these mutant NPC1 cells
produces massive intracellular accumulation of unesterified cholesterol
in LAMP- and LBPA-containing vesicles, whereas all other
noncholesterol-containing LAMP-positive vesicles contain NPC1 and Rab7.
We have, however, found in certain specific NP-C cell lines that the
normally separate NPC1 and cholesterol vesicles now are merged into
single vesicles (data not shown) reminiscent of the hybrid organelle
found with drug treatment (10, 12). It thus appears that certain NPC1
genotypes mimic the vesicular trafficking defects induced by drugs. One
may entertain the possibility that the formation of such aberrant
vesicular structures is due to genetic or chemically induced blocks in
the disassociation of a normally transient late endosomal/lysosomal interaction. Presumably, delays in normal dissociation of the two types
of vesicles leads to impaired lysosomal retroendocytosis.
The cholesterol-enriched lysosomes were immunocytochemically shown to
also contain apolipoprotein D (Table I). Apolipoprotein D is a member
of the lipocalin family of transport proteins whose roles are thought
to include the binding and transport of small hydrophobic ligands such
as progesterone and cholesterol (35). Although not previously shown to
be a marker of cholesterol-laden lysosomes, apolipoprotein D levels and
its metabolism are severely affected in NP-C disease (14).
The NPC1 Compartment Is a Sterol-sensitive Sorting Vesicle in the
Endocytic Trafficking of Glycolipids--
When fibroblasts are
cultured in fetal bovine serum, NPC1-enriched vesicles were found to
contain numerous glycolipids. Four different types of glycolipids (CTH,
Gal-Cer/Lac-Cer, GM2, and GD3) were shown
immunocytochemically to colocalize in NPC1/LAMP/Rab7-positive vesicles
when normal fibroblasts are maintained in complete fetal bovine serum
(Fig. 4). When cells are induced to endocytically process a large bolus
of LDL-derived cholesterol, GM2 (Fig. 5) and
Gal-Cer/Lac-Cer (data not shown) remain in the NPC1-positive vesicles
but GD3 (Fig. 5) and CTH (data not shown) are redistributed to cholesterol-enriched lysosomes.
In those NP-C lines where NPC1 and cholesterol remain segregated in
their respective LAMP-positive vesicles, LDL-induced vesicular glycolipid sorting was comparable to that noted in normal cells (data
not shown). However, in human mutant NPC1 fibroblast cells expressing
no NPC1 protein, endocytic accumulation of GM2 (Fig. 6) or
Gal-Cer/Lac-Cer (data not shown) is absent. These null mutant NPC1
cells were not defective in their ability to synthesize such glycolipids (Table I), but the distribution of these lipids was predominately limited to the plasma membrane (Fig. 6). In these cells
LDL uptake induced GD3 and CTH to internalize into
cholesterol-laden lysosomes (data not shown). Transfection of the null
mutant human fibroblasts with wild type NPC1 cDNA not only corrects
the sterol transport lesion, but the defect in glycolipid sorting as
well evidenced by the reappearance of GM2 in
NPC1-containing vesicles (Fig. 6).
Similarly, in CT-60 NP-C CHO cells, GM2 does not accumulate
endocytically and GD3 comes to be stored in
cholesterol-loaded lysosomes (Figs. 7 and 8). Transfection of these
cells with NPC1 cDNA also clears lysosomal sterol and promotes
internalization of GM2 into NPC1-positive late endosomes
and clears GD3 from lysosomes. Taken together, these
studies reveal that normal endocytic trafficking and metabolism of
glycolipids depends upon a functional NPC1 late endosomal compartment.
Tubulation as a Mechanism of NPC1 Vesicular Membrane
Exchange--
The budding, fission, and fusion of limiting membranes
has defined the major mechanism by which vesicular trafficking occurs within the endocytic pathway (21). Unexpectedly, an additional mode of
membrane communication was observed when CT-60 NP-C cells were
biologically corrected by transfection with NPC1-GFP cDNA. Relatively short, flexible, and mobile NPC1-GFP(+) tubules were noted
to emanate from and retract to NPC1-enriched vesicles (Fig. 8G). The potential biological importance of this mechanism
in membrane/lipid transfer is underscored by the finding that
drug-induced blocks in lysosomal cholesterol egress as well as targeted
mutational alterations of NPC1 function suppress the morphology and
kinetic activity of these tubules (data not shown). The physiological relevance of these tubular extensions in the transfer of cholesterol and glycolipids by the NPC1 protein is currently under active investigation.
Conclusion--
Based on the results of the current studies we
propose that the NPC1 compartment serves as a sorting station in the
endocytic trafficking of both cholesterol and glycolipids. We suggest
that enriching the cholesterol content of lysosomes recruits the NPC1 protein into endocytic vesicles containing glycolipids that are in transit from the plasma membrane. Further
characterization of the topological relations of the glycolipid and
cholesterol pools at the plasma membrane will need to be carried out in
future studies. In the presence of elevated cholesterol levels, certain glycolipids (galactolipids and GM2, but not CTH and
GD3) are restricted from entering the lysosomal compartment
for degradation and are efficiently recycled in NPC1-sorting vesicles
to the plasma membrane accompanied by cholesterol leaving the
lysosomes. This NPC1 compartment-mediated recapture of glycolipids
could serve to stabilize the ratio of glycolipids/cholesterol
concentrations in the plasma membrane during LDL processing.
Niemann-Pick C disease could, therefore, be envisioned as primarily a
vesicular trafficking defect producing a disruption of glycolipid as
well as cholesterol trafficking. In this regard, it is interesting to
note that glycolipids that accumulate in NP-C cells and tissues, such
as galactolipids and GM2 (1) are those sorted through the
NPC1 compartment, whereas nonaccumulating glycolipids such as CTH and
GD3 are shown to traffic on to the lysosomes for probable
degradation. Cholesterol-mediated sorting of the NPC1 compartment may
suggest that GM2 and galactolipids have higher affinity for
membranes containing NPC1, whereas CTH and GD3 have greater
affinity for membranes enriched in cholesterol.
However, the specific manner in which the NPC1 protein affects
glycolipid trafficking remains to be established. The possibilities of
either a direct NPC1 interaction with these lipids, or alternatively, an indirect intervention related to the ability of this protein to
modulate cholesterol trafficking through the late endosomal compartments are currently attractive.
1-4[NeuAc
2-3]Gal
1-4Glc
1-1'-ceramide (GM2) into endocytic vesicles depends on the presence
of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green
fluorescent protein revealed rapidly moving and flexible tubular
extensions emanating from the NPC1-containing vesicles. We conclude
that the NPC1 compartment is a dynamic, sterol-modulated sorting
organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adaptin (AP-1) and anti-
-adaptin (AP-2) antibodies were
obtained from Sigma Chemical Co. (St. Louis, MO), antibodies to the
300-kDa cation-independent mannose-6-phosphate receptor were a gift of
Dr. Suzanne Pfeffer (11), Stanford University, Stanford, CA. Mouse
anti-human cathepsin D and mouse anti-Rab7 antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Goat
anti-lysobisphosphatidic acid (IgG), was a gift of Drs. Kobayashi and
Gruenberg (12), Department of Biochemistry, Geneva, Switzerland. Goat
anti-
-hexosaminidase and anti-
-hexosaminidase (IgG) were obtained
from Dr. Richard Proia (13), Genetics and Biochemistry Branch, NIDDK,
National Institutes of Health. Rabbit anti-apolipoprotein D (14) was
obtained from Dr. Shutish Patel, Veterans Affairs Medical Center,
Newington, CT. Mouse monoclonal antibody O1 was obtained from Dr. R. Bansal, University of Connecticut Health Center, Farmington, CT. The O1
antibody (15) has been shown to recognize lactosylceramide (16),
galactosylceramide, and other galactolipids (15). Mouse monoclonal
antibodies against GalNAc
1-4[NeuAc
2-3]Gal
1-4Glc
1-1'-ceramide
(GM2) (17) and anti-Gal
1-4Gal
1-4Glc
1-1'-ceramide (ceramide trihexoside or CTH) (18) were generously provided by Dr. Tadashi Tai, Tokyo Metropolitan Institute of Medical Science. Mouse
anti-NeuAc
2-8NeuAc
2-3Gal
1-4Glc
1-1'-ceramide (GD3) was from Matreya (Pleasant Gap, PA). FITC- and
LRSC-labeled secondary antibodies were obtained from Jackson
ImmunoResearch (West Grove, PA). DiI-LDL and fluorescent dextran
(70,000 mr lysine fixable) were obtained from
Molecular Probes (Eugene, OR). Filipin was obtained from Polysciences
(Warrington, PA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
-hexosaminidase (Table I). However, both cathepsin D and
- and
-hexosaminidase strongly colocalize with cholesterol-enriched LAMP-positive vesicles (Table I). The distribution of markers among the
separate NPC1 and cholesterol-enriched vesicles confirms that the NPC1
protein is normally localized in a late endosomal compartment apart
from cholesterol-enriched lysosomes. These two separate subsets of
LAMP-positive vesicles of the endocytic pathway, NPC1-positive late
endosomes and LDL-cholesterol enriched lysosomes, are present in all
normal fibroblasts and most NP-C fibroblast cell lines studied. We
have, however, found two NPC cell lines in which both NPC1 and
LDL-derived cholesterol locate together in the same LAMP-positive
vesicle. Although a single gene is affected by the NPC1 mutation, the
protein has multiple domains each of which may be affected and produce
a different phenotype.
Comparison of the NPC1 containing vesicles with the cholesterol
enriched vesicles: immunocytochemical and endocytic marker
characterization of fibroblasts incubated with LDL for 24 h
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Fig. 1.
Cytochemical characterization of the
NPC1-containing late endosomal compartment. Normal human
fibroblasts (A-F) were incubated in McCoy's/5%
LPDS medium at 37 °C for 4 days and then incubated in fresh medium
containing LDL (50 µg/ml) for 24 h to enrich cellular membranes
with sterol. Fibroblasts were immunostained for NPC1 (A,
C, E; red), stained with filipin for
cholesterol (B; blue), and immunostained for Rab7
(D; green) and cathepsin D (F;
green). Confocal microscopy revealed that the NPC1 vesicles
(A; arrows) are not enriched with endocytosed
cholesterol (B; open arrowheads), and conversely,
cholesterol-laden vesicles (B, arrows) do not contain NPC1
protein (A, open arrowheads). NPC1 vesicles
(C; red) contain Rab-7 (D;
green) a marker for late endosomes. Arrows
highlight vesicles that show clear colocalization between NPC1 and Rab7
in C and D. NPC1 vesicles (E;
arrows) do not contain cathepsin D (F; open
arrowheads), and conversely, cathepsin D-containing vesicles
(F, arrows) do not contain NPC1 protein
(E, open arrowheads). NP-C mutant CT-60 Chinese
hamster ovary cells (G, H) transfected with human
NPC1-GFP cDNA express fluorescent NPC1-GFP (G;
green). These cells were incubated with fluorescent dextran
(H; red), and confocal microscopy revealed that
the fluorescent NPC1-GFP (G, arrows)is in the
same compartment containing endocytosed dextran (H,
arrows). Bars for A-H = 5 µm.
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Fig. 2.
Cytochemical characterization of the
cholesterol-containing lysosomal compartment. Normal human
fibroblasts (A, B) were incubated in McCoy's/5%
LPDS medium at 37 °C for 4 days and then incubated in fresh medium
containing LDL (50 µg/ml) for 24 h to enrich cellular membranes
with sterol. Fibroblasts were immunostained for lysobisphosphatidic
acid (LBPA) (A; green) and cytochemically stained
with filipin (B; blue) to reveal the cellular
distribution of endocytosed cholesterol. Arrows highlight
vesicles that show clear colocalization between LBPA and cholesterol.
CT-60 Chinese hamster ovary cells (C-F) were incubated with
fluorescent dextran (C; red) and fluorescent
DiI-LDL (E; red) and stained for
cholesterol-loaded lysosomes (D, F;
blue). Arrows highlight vesicles in which
endocytosed fluorescent dextran or DiI-LDL colocalize with
cholesterol-loaded lysosomes. Bars for A-F = 5 µm.
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Fig. 3.
Uptake of LDL induces NPC1 protein
accumulation in vesicles. Normal human fibroblasts grown in lipid
depleted serum (A) or serum supplemented with LDL for
24 h (B). Cells were immunostained for NPC1 protein.
Incubation with LDL increases the amount of NPC1 protein (B;
red) sequestered in late endosomes. Bar = 25 µm.
1-4[NeuAc
2-3]Gal
1-4Glc
1-1'-ceramide (GM2) and neutral glycolipids such as
Gal
1-4Glc
1-1'-ceramide (Lac-Cer) are stored in NP-C tissues and
cells. Because earlier studies had suggested that the NPC1 compartment
functions in vesicular trafficking of multiple membrane components
(10), movement of glycolipids through the NPC1 compartment was
evaluated. We immunocytochemically examined with established mouse
monoclonal anti-glycolipid antibodies the intracellular location of the
gangliosides GM2 and GD3
(NeuAc
2-8NeuAc
2-3Gal
1-4Glc
1-1'-ceramide) and the
neutral glycolipids, Gal
1-4Gal
1-4Glc
1-ceramide (ceramide trihexoside or CTH) and (Gal-Cer/Lac-Cer) in both normal and mutant NP-C fibroblasts. In normal fibroblasts cultured with fetal bovine serum, all four internalized glycolipid pools localized to the NPC1
compartment (Fig. 4).
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Fig. 4.
Glycolipids are present in NPC1-containing
vesicles in fibroblasts. Normal human fibroblasts grown in bovine
serum were immunostained for NPC1 protein (A, C,
E, G; red panels) and
glycolipids (B, D, F, H;
green panels). Glycolipids and NPC1 colocalize.
Arrows highlight vesicles that show clear colocalization
between NPC1 and GM2 (A and B), NPC1
and Gal-Cer/Lac-Cer (C and D), NPC1 and
GD3 (E and F), and NPC1 and CTH
(G and H). Bars = 5 µm.
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Fig. 5.
Cellular uptake of LDL-cholesterol modulates
the glycolipid profile of the NPC1 compartment.
Cholesterol-depleted normal human fibroblasts grown in LDL (50 µg/ml)
for 24 h were immunostained for NPC1 (A;
red), gangliosides GM2 (B,
C; green), and GD3 (D;
green) and stained with filipin (C, D;
blue) for cholesterol localization. Arrows
highlight clear colocalization of NPC1 (A; red)
with GM2 (B; green). The merged image
(C) shows that GM2 (green) does not
colocalize with cholesterol (blue). The color bar
shows green, aqua, and blue
(aqua is a merge of the green and
blue, indicating colocalization). The merged image
(D) shows that GD3 colocalizes with cholesterol
as indicated by the aqua color. Bars = 5 µm.
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Fig. 6.
NPC1 cDNA transfection of NPC1
null-mutant fibroblasts restores GM2 to endocytic
vesicles. NPC null mutant human fibroblasts, either nontransfected
or transfected with GFP-NPC1 cDNA, were immunostained for NPC1
(A, C; red) and GM2
(B, D; green). Nontransfected, NPC1
null mutant fibroblasts contained no specific staining for NPC1
(A; lack of red), and staining for
GM2 (B; green) appeared limited to
the cell surface. Transfected cells showed clear colocalization between
immunostained NPC1 vesicles (C; red) and
internalized GM2 (D; green),
highlighted by arrows. Expression of NPC1 in cells was
determined by the presence of GFP fluorescence in the cytosol and
nucleus. Bars = 5 µm.
Ganglioside synthesis in normal and NP-C fibroblasts
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Fig. 7.
NPC1 cDNA transfection of CT-60 mutant
NP-C CHO cells restores GM2 in endocytic vesicles.
CT-60 CHO cells were transfected with NPC1-GFP cDNA as described
under "Experimental Procedures." In transfected cell that have not
yet cleared of cholesterol (A, and inset),
NPC1-GFP appears as rings (A and inset;
green), at the surface of cholesterol laden lysosomes
(inset; blue). These cholesterol-laden cells do
not contain intracellular GM2 (B; no
red immunostaining). In transfected cells that have cleared
cholesterol from lysosomes, NPC1-GFP is in the lumen (C;
green) of vesicles that now also contain GM2
(D; red). Arrows highlight vesicles
that show clear colocalization between NPC1 and GM2
(C and D). Bars = 5 µm.
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Fig. 8.
NPC1 cDNA transfection of CT-60 mutant
NP-C CHO cells causes clearing of cholesterol and GD3 from
lysosomes. CT-60 CHO cells were transfected with NPC1-GFP
cDNA. Nontransfected cells (A; lack of green
fluorescence) contain intracellular GD3 (B;
red). In transfected CT-60 CHO cells that have not yet
cleared of cholesterol, NPC1-GFP appears as rings (C;
green) at the surface of lysosomes and intracellular
GD3 (D; red) is present in the
lysosomes. A highly magnified, merged image (E) shows
NPC1-GFP present at the periphery of cholesterol-laden lysosomes (NPC1
appears as green rings around a blue
filipin-stained core). A second, highly magnified merged image
(F) shows the relationship between NPC1-GFP (F;
green) and GD3 (F; red).
NPC1-GFP present at the periphery of lysosomes appears as
green rings around a core containing red
immunostained GD3. Transfected CT-60 CHO cells
(G and H), which have been cleared of lysosomal
cholesterol, contain vesicular and tubular NPC1-GFP compartments
(G; green) and no intracellular GD3.
Arrowheads in G and H mark the
boundary of the transfected cell. Bars = 2.5 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-hexosaminidases (13), with specific
reference to the distinct LAMP/NPC1-positive and LAMP/cholesterol-containing compartments. Cathepsin D is considered to
reside primarily in lysosomes, although precursor enzyme travels from
the Golgi to late endosomes en route to lysosomes (24). Cathepsin D was
found infrequently in NPC1-containing vesicles (Fig. 1) but was
consistently associated with LAMP/cholesterol-containing vesicles
(Table I). In addition,
- and
-hexosaminidases were also
infrequently associated with NPC1-positive vesicles and were consistently found in LAMP/cholesterol-containing vesicles (Table I).
These distribution patterns of marker enzymes support previous reports
(10, 27) that the endosomal compartment enriched with cholesterol
during active LDL uptake is primarily lysosomes.
-hexosaminidase, we consider these vesicles lysosomes. These
cholesterol/LBPA-positive and Rab7-negative vesicles are the prevalent
LAMP-containing vesicles found in the cytoplasm of our cultured
fibroblasts. The only other LAMP-positive vesicles noted are the
NPC1/Rab7-containing late endosomes.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Lipid Cell Biology
Section, Bldg. 8, Rm. 427, NINDK, National Institutes of Health, 8 Center Drive, MSC 0850, Bethesda, MD 20892. Tel.: 301-496-2050; Fax:
301-402-0723; E-mail: joanbm@bdg8.niddk.nih.gov.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M005393200
2 M. Zhang and S. Patel, unpublished data..
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ABBREVIATIONS |
---|
The abbreviations used are:
NP-C, Niemann-Pick C
disease;
NPC1, Niemann-Pick C1 protein;
GM2, GalNAc1-4[NeuAc
2-3]Gal
1-4Glc
1-1'-ceramide;
GD3, NeuAc2-8NeuAc2-3Gal
1-4Glc
1-1'-ceramide;
CTH, Gal
1-4Gal
1-4Glc
1-ceramide;
LDL, low density lipoprotein;
LPDS, lipoprotein-deficient bovine serum;
GFP, green fluorescent
protein;
LAMP, lysosomal associated membrane protein;
LIMP, lysosomal
integral membrane protein;
LBPA, lysobisphosphatidic acid;
M6PR, mannose-6-phosphate receptor;
apoD, apolipoprotein D;
CHO cells, Chinese hamster ovary cells;
FITC, fluorescein isothiocyanate;
LRSC, lissamine rhodamine B sulfonyl chloride;
DiI-LDL, octadecyl (C18)
indocarbocyanine;
GFP, green fluorescence protein;
Cy5, indodicarbocyanine.
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