From the Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
Received for publication, November 4, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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Lipid rafts
(glycosphingolipid/cholesterol-enriched membrane microdomains) have
been isolated as low temperature, detergent-resistant membranes from
many cell types, but despite their presumed importance as lateral
sorting and signaling platforms, fundamental questions persist
concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at
physiological temperature to compare with rafts, obtained by
"conventional" extraction using Triton X-100 at low temperature.
Microvillar rafts prepared by the two protocols were morphologically
different but had essentially similar profiles of protein- and lipid
components, showing that raft microdomains do exist at 37 °C and are
not "low temperature artifacts." We also employed a novel method of
sequential detergent extraction at increasing temperature to define a
fraction of highly detergent-resistant "superrafts." These were
enriched in galectin-4, a Lipid rafts are ordered (lo phase) membrane
microdomains consisting mainly of glycosphingolipids and cholesterol in
the outer leaflet of the bilayer that serve as lateral platforms for
many types of proteins in the cell membrane (1-3). The lipid raft hypothesis was originally proposed to explain how lipids and proteins are sorted to the apical surface of polarized cells (4), but in recent
years the raft concept has expanded into other areas of biology,
including signal transduction (5-7), pathogen invasion (8-10),
cholesterol homeostasis and transport (11, 12), drug targeting (13),
and biomedicine (14). However, despite their popularity, fundamental
questions concerning the functional relevance of lipid rafts in
vivo persist (15, 16). To complicate matters further, recent work
has made it increasingly clear that the most commonly used experimental
criterion for lipid rafts, detergent insolubility at low temperature
combined with flotation in a density gradient (17), may define
different subsets of rafts, depending on the type of detergent used
(18-21). A common current view considers lipid rafts as small, dynamic
assemblies of lipids and proteins with the ability to cluster into
larger ordered platforms that may serve in specialized, cell
type-dependent functions, such as nutrient absorption,
cell-cell communication, or endocytosis (2, 14, 22, 23). These
developments have turned the raft concept increasingly complex, and
underline the fact that much is still unknown about lipid rafts
concerning their molecular organization, dynamics of
assembly/disassembly, and mechanism of function.
The brush border membrane of small intestinal enterocytes is a highly
specialized cell surface involved in a multitude of digestive and
absorptive functions (24). The microvilli of this membrane are
particularly rich in glycosphingolipids and cholesterol (25), and
several proteins, including some of the major digestive enzymes, such
as aminopeptidase N and sucrase-isomaltase (26-28), and galectin-4
(29) have been shown to reside in Triton X-100-insoluble lipid rafts.
Microvillar rafts may well be unique in their molecular organization,
because unlike most other types of lipid raft membranes, such as
caveolae (30), those prepared from microvilli are
cholesterol-independent, and caveolin-1, a commonly used lipid raft
marker in other cell types, is essentially excluded from microvillar
rafts (31).
In the present work, we aimed first to answer the fundamental question
whether microvillar rafts are purely a "low temperature" phenomenon, created by the conditions normally used for detergent extraction. To tackle this problem, microvillar rafts were prepared by
using Brij 98, a detergent recently described for isolation of the T
cell receptor signaling machinery at a physiological temperature (20).
Second, we studied in closer detail the molecular organization of lipid
rafts prepared from microvillar membrane vesicles by sequential
extraction with Triton X-100 at increasing temperature. Here, a
fraction of "superrafts" was isolated and characterized with
galectin-4 as the predominant protein. Altogether, our data show that
microvillar rafts do exist also at a physiological temperature. In
addition, the results underline the importance of galectin-4
functioning as a central organizer/stabilizer of lipid rafts in the
microvillar membrane with other classes of membrane proteins depending,
at least partially, on this lectin for their association with these microdomains.
Materials--
Rabbit polyclonal antibodies to galectin-4,
lactase, and aminopeptidase N were those described previously (29, 32,
33). A rabbit antibody to alkaline phosphatase was from Biogenesis (Poole, UK), and a mouse monoclonal antibody to annexin 2 was obtained
from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-coupled swine anti-rabbit IgG and rabbit anti-mouse IgG were
from DAKO (Glostrup, Denmark). Gold-labeled goat anti-rabbit immunoglobulin was from Amersham Biosciences. Methyl-
Pig small intestines were kindly given by Letty Klarskov and Mette
Olesen (Department of Experimental Medicine, The Panum Institute,
Copenhagen, Denmark).
Preparation of Microvillar Membranes--
Right-side-out
microvillar vesicles were prepared from small intestinal mucosa by the
divalent cation precipitation technique (34). Briefly, mucosal
scrapings were homogenized in 2 mM Tris-HCl, 50 mM mannitol, pH 7.1, containing 10 µg/ml aprotinin and
leupeptin, using a manually operated Potter-Elvehjem homogenizer. The
homogenate was cleared by centrifugation at 500 × g
for 10 min, and MgCl2 was added to a final concentration of
10 mM. After incubation at 15 min on ice, the preparation
was centrifuged at 1500 × g for 10 min to pellet
intracellular and basolateral membranes. Finally, the supernatant was
centrifuged at 48,000 × g for 1 h to obtain a
pellet of microvillar membrane vesicles. One common preparation was
used in all subsequent comparative lipid raft experiments with the two
nonionic detergents Brij 98 and Triton X-100 as well as in preparation
of superrafts.
Lipid Raft Analysis--
A lipid raft analysis by detergent
extraction followed by sucrose gradient centrifugation (17) was
performed essentially as previously described (26), with the
modification that the extract was placed in a cushion of 60% sucrose
with a gradient of 50-25% sucrose on top. Extraction with Triton
X-100 and with Brij 98 was carried out on ice and at 37 °C,
respectively. Solutions of each detergent (10% (w/v) dissolved in
distilled water) were added to the microvillar membrane vesicles to a
final detergent concentration of 1% (w/v). After centrifugation
overnight, the gradients were collected in 12 1-ml fractions for
subsequent analysis by SDS-PAGE and Western blotting. Alternatively,
the floating fractions were carefully collected by a pipette, diluted
five times with 25 mM HEPES-HCl, 150 mM NaCl,
pH 7.1, and centrifuged at 48,000 × g, 1 h, to
obtain a pellet of lipid rafts for further biochemical and electron
microscopic analyses.
Preparation of Superrafts--
Lipid rafts were prepared
by extraction with Triton X-100 followed by sucrose density gradient
centrifugation as described above. Rafts contained in the upper six
fractions of the gradient were harvested; resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1; and
extracted with 1% Triton X-100 for 10 min at room temperature. The
extract was centrifuged at 20,000 × g for 30 min, and
the resulting pellet was resuspended in the same buffer and
re-extracted with 1% Triton X-100 for 10 min at 37 °C. After
centrifugation at 20,000 × g for 30 min, the resulting
pellet, termed "superrafts," was collected.
Lipid Analysis--
For lipid analysis of membranes, microvillar
vesicles and lipid rafts were extracted with chloroform/methanol (25,
35) as previously described (31). Aliquots of the lipid extracts (120-130 mg of lipid) were subjected to thin layer chromatography analysis together with appropriate lipid standards on 0.25-mm silica
gel 60 plates (Merck). Cholesterol was separated from other neutral
lipids in petroleumsether/diethylether/acetic acid (50:50:1). Glycolipids and phospholipids were separated in
chloroform/methanol/water (64:24:4). After chromatography, cholesterol
was detected with a CuSO4/H3PO4
reagent and glycolipids with a Gel Electrophoresis and Western Blotting--
SDS-PAGE
electrophoresis in 10% polyacrylamide gels was performed according to
Laemmli (36). After electrophoresis and transfer of proteins onto
ImmobilonTM, Western blotting was performed with antibodies
to galectin-4 (29), lactase (33), aminopeptidase N, alkaline
phosphatase, and annexin 2. Blots were developed by an
electrochemoluminescence detection reagent kit according to the
protocol supplied by the manufacturer (Amersham Biosciences).
Immunoisolation of Lipid Rafts--
Superrafts and lipid rafts,
prepared by extraction either with Triton X-100 on ice or Brij 98 at
37 °C, were isolated on magnetic beads, coated with antibodies to
galectin-4 and aminopeptidase N, by a previously described
procedure (31).
Electron Microscopy--
Lipid rafts, isolated from microvillar
membrane vesicles using either Triton X-100 or Brij 98, were fixed in
2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH
7.2, for 2 h at 4 °C. After a wash in 0.1 M sodium
phosphate buffer, the lipid rafts were treated with osmium tetroxide in
0.1 M sodium phosphate buffer for 1 h at 4 °C,
dehydrated in graded concentrations of acetone, and finally embedded in
Epon. The superrafts and lipid rafts isolated on magnetic beads were
fixed in glutaraldehyde, treated with osmium tetroxide, dehydrated, and
embedded in Epon as described above, with the modification that the
entire fixation and embedding procedure was performed in Eppendorf
tubes. Ultrathin sections were cut on an LKB Ultrotome III
ultramicrotome, stained in 1% uranyl acetate in water and lead
citrate, and finally examined in a Zeiss EM 900 electron microscope
equipped with a Mega View camera system.
Immunoelectron Microscopy--
Postembedding immunogold labeling
using an antibody against aminopeptidase N was performed on ultrathin
Epon sections of lipid rafts and superrafts, as previously described
(37).
Lipid Rafts from Microvillar Membranes Exist at Physiological
Temperature--
The brush border of small intestinal enterocytes is
well suited for studies on the molecular organization of a specialized cell membrane. Upon tissue homogenization, microvilli spontaneously form a homogeneous population of right-side-out vesicles that are
easily isolated from other cell membranes by the divalent cation
precipitation technique (34). We have previously used flotation in a
density gradient after Triton X-100 extraction on ice for isolation of
lipid rafts and observed that several microvillar proteins partition
differently into the rafts. Thus, the
glycosylphosphatidylinositol-anchored alkaline phosphatase is
predominantly (about 90%) and the transmembrane aminopeptidase N and
sucrase-isomaltase partially (40-50%) raft-associated, whereas lactase is essentially excluded from microvillar rafts (26). In
addition, galectin-4, a member of the galectin family of
In the present work, these microvillar proteins were used as probes for
lipid rafts, prepared using either Triton X-100 or Brij 98. Fig.
1 shows a comparison of the flotation
properties in a sucrose density gradient of microvillar membrane
proteins extracted by Triton X-100 on ice or Brij 98 at 37 °C. For
both detergents, distinct subsets of proteins floated into the upper fractions, indicative of their association with lipid rafts. An overall
comparison of the profiles of lipid raft-associated proteins isolated
with the two detergents showed many common prominent bands, but some
minor quantitative differences in the relative intensity of several
bands were observed (Fig. 1). In addition, the densities of the rafts
differed. Thus, rafts extracted with Triton X-100 were relatively
homogeneous and of low density, whereas those extracted with Brij 98 were more heterogeneous in density.
The specific distribution in the density gradient of the four
microvillar proteins described above was revealed by Western blotting
(Fig. 2). As shown in Fig. 2A,
galectin-4 was exclusively present in the raft fractions of Triton
X-100-extracted microvillar membranes and also predominantly
raft-associated in the Brij 98 gradient. Contrary to galectin-4,
essentially all lactase was seen in the heavy fractions of the gradient
that contains solubilized proteins, irrespective of the detergent used
for the membrane extraction (Fig. 2B), confirming lactase
being a "non-raft" marker among microvillar proteins. The
glycosylphosphatidylinositol-anchored alkaline phosphatase was
exclusively present in the floating lipid raft fractions with both
detergents (Fig. 2C), whereas the transmembrane aminopeptidase N was only partially raft-associated with both detergents (Fig. 2D).
The lipids of microvillar rafts, isolated after extraction with the two
detergents, were analyzed by thin layer
chromatography (Fig. 3). Previously, rafts, prepared using Triton
X-100, have been shown to contain cholesterol and to be particularly
enriched in glycosphingolipids (31). When approximately equal amounts of raft lipids were analyzed in parallel by thin layer chromatography, the lipid profiles of those isolated by use of Brij 98 and Triton X-100
were essentially similar, indicating that the overall
lipid-solubilizing properties of the two detergents were alike (Fig.
3). However, rafts obtained with Brij 98 contained relatively
higher amounts of phospholipids.
Taken together, the above experiments with Brij 98 demonstrate that
microvillar lipid raft microdomains isolated at a physiological temperature were quite similar to those prepared using Triton X-100 at
low temperature. Importantly, it can therefore be concluded that
microvillar rafts are not "low temperature artifacts."
Morphology of Lipid Rafts--
As shown in Fig.
4, lipid rafts, prepared as described
above, had a strikingly different morphology, depending on the type of
detergent used for extraction. Those isolated after treatment with
Triton X-100 appeared as a homogeneous population of closed, spherical
vesicle-like structures with an average diameter in the range of
200-300 nm. In addition, some more complex conglomerate structures of
fused vesicles were seen. By immunogold electron microscopy,
aminopeptidase N was predominantly seen lining the membranes. This
localization was also observed in intact microvilli as well as
microvillar membrane vesicles (31), but in some of the more complex
multivesicular structures, labeling was also observed along the
interior membranes. Single raft structures, immunocaptured onto
magnetic beads revealed a distinct bilayer composition. Lipid rafts
isolated using Brij 98 as detergent were seen predominantly as
nonvesiculated, pleiomorphic membrane sheets with an approximate length
of 200-300 nm, but some vesicle-like structures were also present
(Fig. 4). Aminopeptidase N labeling was observed both in membranes
of the vesicles and the sheets.
In conclusion, Triton X-100 extraction on ice generally resulted in
preservation of vesicle-like structures, whereas extraction with Brij
98 at 37 °C largely broke up the microvillar vesicles into open
membrane fragments. The more heterogeneous morphology of the latter
parallel the heterogeneity in lipid raft density indicated by Figs. 1
and 2. Taken together, the results suggest that raft morphology is
quite sensitive to the rather small differences observed with regard to
protein and lipid composition.
Microvillar Rafts Are Resistant to Cholesterol Depletion--
We
have previously reported that microvillar rafts, isolated by extraction
with Triton X-100, are cholesterol-independent, because unlike, for
instance, caveolae in other cell types, they are resistant to treating
the membranes with methyl- Superrafts, Lipid Rafts Insoluble by Triton X-100 at
37 °C--
For preparation of lipid rafts using Triton X-100, it is
essential to perform the extraction at low temperature to preserve the
lipid-lipid and protein-lipid interactions defining these microdomains,
because at higher temperatures, Triton X-100 solubilizes completely
both lipids and proteins of microvillar membrane vesicles (data not
shown). However, when microvillar lipid rafts, prepared by Triton X-100
extraction on ice, were subsequently re-extracted with the same
detergent, first at 20 °C and then at 37 °C, a fraction of the
original lipid raft membranes remained insoluble in Triton X-100 even
at the latter temperature (Fig. 6). A
comparison of these high temperature Triton X-100-insoluble membranes
(termed "superrafts") with the "normal" lipid raft from which
they were derived showed that superrafts are relatively enriched in a
small subset of proteins. A further analysis by Western blotting
revealed a hierarchy of the different microvillar lipid raft proteins
with regard to their partition into the superrafts (Fig. 6). First, the
most prominent protein of the superrafts was galectin-4, which completely resisted extraction with Triton X-100, both at 20 and 37 °C. Second, the glycosylphosphatidylinositol-anchored alkaline phosphatase was partially solubilized at 20 and 37 °C but
predominantly resisted detergent extraction. Third, the major part of
the transmembrane protein aminopeptidase N was solubilized, but a
significant fraction of this abundant microvillar protein remained
associated with the superrafts. Finally, most of the lipid
raft-associated annexin 2, a peripheral membrane protein, was largely
solubilized by Triton X-100 at 20 °C, and only small amounts were
present in the superraft fraction.
The overall lipid composition of superrafts showed a profile
qualitatively similar to native microvillar membranes and rafts, isolated by Triton X-100 or Brij 98 (Fig. 3). However, the enrichment of glycolipids and, to a lesser degree, cholesterol, relative to
phospholipids, was larger than for the other two raft fractions. Native
microvillar membranes from pig enterocytes are rich in glycosphingolipids (>30% of total membrane lipid), indicating that
they constitute more than half the amount of lipid in the exoplasmic
leaflet (25). The relative glycolipid enrichment seen for the
superrafts thus suggests that very little phospholipid remained in the
outer leaflet of the membranes after the sequential extractions at
increasing temperature. The phospholipids remaining in the superraft
fraction most likely constitute the cytoplasmic leaflet of the core
structure of these lipid rafts.
By electron microscopy, superrafts had a surprisingly well preserved
membrane morphology, consisting of spherical vesicle-like structures of
about 150-200 nm in diameter (Fig. 7).
Multilamellar structures were visible in the preparation, and
immunogold labeling with an anti-aminopeptidase N antibody revealed
that the enzyme lined both exterior and interior membranes. In
comparison with lipid rafts isolated by extraction with Triton X-100
(Fig. 4, A-D), the immunogold labeling of superrafts was
more sparse, in agreement with the observation that a substantial
fraction of aminopeptidase N was released from the membranes
during preparation of the superrafts (Fig. 6).
Although the superrafts described above must be considered membrane
artifacts created by the sequential detergent extraction at increasing
temperature, the protocol used for their preparation represents a
method for probing the relative strength of the molecular interactions
that exist between the various lipid and protein constituents that
define lipid rafts. Along this line, the protein composition of
superrafts suggests that galectin-4 is the major raft stabilizer among
the proteins of the microvillar membrane. To test this hypothesis,
superrafts were re-extracted with Triton X-100 at 37 °C after a
preincubation in the absence or presence of lactose, a soluble ligand
for galectin-4. As shown in Fig. 8, the
presence of lactose caused the release of some, but not all, galectin-4
from the superrafts, indicating that at least some of this lectin is
raft-associated via its cognate ligand. However, in addition to
galectin-4, markedly increased amounts of alkaline phosphatase and
aminopeptidase N were also released by lactose, indicating that their
raft association is at least partially
galectin-4-dependent. Lactose also released galectin-4, alkaline phosphatase, and aminopeptidase N from lipid rafts, isolated by Brij 98-extraction and even from microvillar membranes, untreated with detergent (Fig. 8), showing that the association between these
microvillar proteins is not entirely induced by detergent extraction.
Taken together, the results obtained with the superrafts imply that
galectin-4 can be characterized as an organizer/stabilizer within
microvillar lipid rafts. If the lectin thus defines the core of a raft,
the glycosylphosphatidylinositol-anchored alkaline phosphatase and
the transmembrane aminopeptidase N may then represent more
peripheral raft proteins with decreasing affinity for these microdomains.
Despite the rapid proliferation of the lipid raft concept into
many diverse fields of biology, fundamental questions concerning the
functional relevance of these lipid-based microdomains in vivo persist. To a large extent, this has to do with the most frequently used biochemical criterion for raft association, detergent insolubility at low (0-4 °C) temperature. Thus, given the
temperature-dependent phase behavior of membrane lipids
(2), lipid rafts could well be a phenomenon entirely related to the
commonly used method for their isolation. Addressing this problem, Brij
98, a member of the polyoxyethylene ether (Brij) series of nonionic
detergents, was recently used for preparation of lipid rafts at
37 °C from T cells (20). These rafts had a lipid composition
expected of normal lipid rafts and harbored the components of a
functional T cell receptor machinery. In the present work, four
microvillar proteins were used as markers, each of which had a
characteristic lipid raft profile, using Triton X-100 and differing
with regard to type of membrane attachment. Brij 98 extraction at
37 °C defined microvillar rafts having a marker protein composition
essentially similar to lipid rafts, isolated by use of Triton X-100 at
low temperature. The fact that Brij 98 at 37 °C generates authentic lipid rafts from two so very different membrane systems as T cell membranes and microvillar membrane vesicles lends strong credibility to
the bona fide existence and functional relevance of these
lipid-based microdomains.
Admittedly, the Triton X-100-resistant superrafts characterized in this
work are membranes artifactually created by the sequential detergent
extraction at increasing temperature, but importantly, they revealed
the potential of galectin-4 to act as a strong organizer/stabilizer of
these membrane microdomains. Like other members of the galectin family,
galectin-4 is a cytosolic protein synthesized without a signal for
membrane translocation (29, 39). This implies that galectin-4 is
secreted by a "nonclassical" pathway. -galactoside-recognizing lectin residing
on the extracellular side of the membrane. Superrafts also harbored the
glycosylphosphatidylinositol-linked alkaline phosphatase and the
transmembrane aminopeptidase N, whereas the peripheral lipid raft
protein annexin 2 was essentially absent. In conclusion, in the
microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane
microdomain systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin, Brij 98 (polyoxyethylene 20 oleyl ether), and lipid standards were
purchased from Sigma, and DynabeadsTM M-500 Subcellular was
from Dynal (Oslo, Norway).
-naphtol spray reagent.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside-binding proteins (38, 39), has been identified as a
microvillar protein that resides exclusively in rafts and forms
clusters with some of the brush border enzymes (29).
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Fig. 1.
Comparison of microvillar rafts using Triton
X-100 and Brij 98. A, microvillar membrane vesicles
prepared by the divalent cation precipitation method were resuspended
in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and
0.9-ml samples of membranes were solubilized for 10 min by 1% Triton
X-100 on ice or 1% Brij 98 at 37 °C, respectively, before isolation
of lipid rafts by sucrose gradient centrifugation in the same rotor as
described under "Experimental Procedures." After centrifugation,
the gradients were fractionated, and samples of equal volume of the
fractions were subjected to SDS-PAGE. After electrophoresis and
electrotransfer onto ImmobilonTM, total protein was
visualized by staining with Coomassie Brilliant Blue. Molecular mass
values (kDa) are indicated, and fractions containing soluble proteins
and lipid rafts are indicated by the arrows. B,
densitometric scanning of fraction 10 from the two gradients shown in
A. Molecular mass values (kDa) are indicated.
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Fig. 2.
Distribution of microvillar raft
markers. The density gradient fractions, shown in Fig.
1A were analyzed by Western blotting for the distribution of
different microvillar proteins. A, galectin-4; B,
lactase; C, alkaline phosphatase; D,
aminopeptidase N. Fractions containing soluble proteins and lipid rafts
are indicated by the arrows.
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Fig. 3.
Lipid analysis of microvillar rafts. The
lipid composition of native microvillar membrane vesicles
(1), rafts isolated with Triton X-100 (2), rafts
isolated with Brij 98 (3), and superrafts (4) was
analyzed by thin layer chromatography as described under
"Experimental Procedures." The experiment shown is a representative
of five independently performed analyses. Cholesterol was analyzed
separately in a petroleumsether/diethylether/acetic acid solvent system
and glycolipids and phospholipids in a chloroform/methanol/water
solvent system. Lipid markers (M) were chromatographed in
parallel. Chol, cholesterol; Gal-cer,
galactocerebrosides; PE, phosphatidylethanolamine;
PC, phosphatidylcholine; PS, phosphatidylserine;
asialo-GM1, asialoganglioside-GM1
(Cer-Glc-Gal(NANA)-GalNAc-Gal). The percentage values show the amounts
of cholesterol, phosphatidylethanolamine, and
asialoganglioside-GM1 in the raft and superraft fractions
relative to the amounts of the respective lipids in the native
microvillar membrane vesicles, as determined by densitometric scanning.
*, an unidentified lipid that was heavily stained by the -naphtol
reagent, indicating that it is a glycolipid. It was enriched in the
raft and superraft fractions relative to the amount in the native
microvillar membrane vesicles.
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Fig. 4.
Morphology and immunogold labeling of lipid
rafts. Lipid rafts, isolated by extraction with either Triton
X-100 (A-D) or Brij 98 (E-H). A and
E, sections of pellets of lipid rafts; B,
C, F, and G, similar sections,
immunogold-labeled for aminopeptidase N. D and H,
single lipid raft structures, immunocaptured on magnetic beads coated
with antibodies to aminopeptidase N and galectin-4. Bars,
0.5 µm (A, B, E, and F),
0.1 µm (C and G), and 0.05 µm (D
and H).
-cyclodextrin prior to the addition of
detergent (31). To test whether this characteristic property was
temperature- and/or detergent-related, microvillar membrane vesicles
were cholesterol-depleted by treatment with methyl-
-cyclodextrin
(31, 40) before extraction with Brij 98 at 37 °C. About 70% of the
microvillar cholesterol is removed by this treatment (31), but as shown
in Fig. 5, a profile of polypeptides in
the floating fractions was obtained almost identical to that
observed with Brij 98 without methyl-
-cyclodextrin treatment. Only
small amounts of aminopeptidase N, alkaline phosphatase, and galectin-4
were shifted to the heavy fractions containing soluble protein. Thus,
it can be concluded that the cholesterol independence of microvillar
rafts reflects a bona fide membrane microdomain organization
that exists at physiological temperature and is not a phenomenon that
can be attributed to low temperature extraction with Triton X-100.
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Fig. 5.
Cholesterol depletion with
methyl- -cyclodextrin.
Methyl-
-cyclodextrin was added (1% final concentration) to
microvillar membrane vesicles, resuspended in 25 mM
HEPES-HCl, 150 mM NaCl, pH 7.1. After incubation for 30 min
at 37 °C, the vesicles were extracted with Brij 98 and analyzed as
described in the legend to Fig. 1A. Molecular mass values
(kDa) are indicated, and a densitometric scanning of fraction 10 is
shown. ApN, aminopeptidase N; Alk P, alkaline
phosphatase; Gal-4, galectin-4.
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Fig. 6.
Isolation of superrafts. Lipid rafts
were prepared from microvillar membrane vesicles by extraction with 1%
Triton X-100 on ice and sucrose gradient centrifugation as described in
the legend to Fig. 1. After centrifugation, the lipid rafts were
carefully collected from the gradient by a pipette, diluted five times
in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, and
pelleted by centrifugation at 48,000 × g for 1 h.
After resuspension in the above buffer, the lipid rafts were
re-extracted with 1% Triton X-100, first at room temperature and then
at 37 °C, as described under "Experimental Procedures." Samples
of lipid rafts (R), proteins solubilized at room temperature
(S1), proteins solubilized at 37 °C (S2), and
superrafts (SR) were subjected to SDS-PAGE. After
electrophoresis and electrotransfer onto ImmobilonTM, total
protein was visualized by staining with Coomassie Brilliant Blue.
Molecular mass values (kDa) are indicated. Aminopeptidase N (160 kDa),
alkaline phosphatase (67 kDa), galectin-4 (36 kDa), and annexin 2 (36 kDa) were detected by Western blotting.
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Fig. 7.
Morphology and immunogold labeling of
superrafts. A and C, sections of pellets of
superrafts. B, D, and E, similar
sections immunogold-labeled for aminopeptidase N. Bars, 0.5 µm (A and B) and 0.1 µm
(C-E).
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Fig. 8.
Release of proteins from superrafts, Brij 98 rafts, and microvillar membranes. Superrafts, Brij 98 rafts, and
microvillar membranes, each prepared as described under "Experimental
Procedures," were resuspended in 25 mM HEPES-HCl, 150 mM NaCl, pH 7.1, in the absence ( ) or presence (+) of 100 mM lactose. After incubation for 10 min at room
temperature, Triton X-100 was added (final concentration 1%), and
incubation at room temperature continued for 10 min before
centrifugation at 20,000 × g for 30 min (the addition
of Triton X-100 was omitted in the experiment with the microvillar
membranes). The pellet (P) and supernatant (S)
fractions were collected and analyzed by SDS-PAGE, followed by Western
blotting. ApN, aminopeptidase N; Alk P, alkaline
phosphatase; Gal-4, galectin-4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Galactosyl residues
present on microvillar membrane lipids as well as glycoproteins are
likely to serve as ligands for this bivalent lectin and enable it to
form detergent-resistant cross-linked lattices. The resulting dependence of microvillar raft stability on
-galactosyl residues may
explain their characteristic resistance to cholesterol depletion. Such
lattices have been described in the clustering and separation of
distinct glycoprotein receptors on the surface of T cells by galectin-1, leading to apoptosis (41). At the microvillar surface, galectin-4-driven clustering of digestive enzymes may help to preserve
these at the cell surface by preventing them from release into the
lumen of the gut and maybe also from undergoing endocytosis. This
function may be physiologically important, because the intestinal lumen
is a harsh working environment, and many microvillar enzymes are prone
to release from the membrane by the action of pancreatic enzymes. We
imagine these galectin-4-stabilized microvillar rafts as being
relatively stable and large microdomains (31), contrary to the current
view that lipid rafts are typically transient and small (23). In a
recently proposed model, the molecular address for proteins targeted to
rafts is a so-called lipid shell (22). Theoretically, these shells have
an estimated diameter of 7 nm and contain about 80 lipid molecules that
act as a local solvent in the lipid bilayer for the single protein they
harbor. Larger raft structures, such as caveolae, form in a subsequent
step by fusion of numerous shells. However appealing and applicable to other, more dynamic raft-based microdomains, such as assembly of signal
transduction complexes, the lipid shell hypothesis hardly describes the
relatively stable molecular organization of microvillar rafts. We
conclude that the lipid raft concept needs to be further developed to
accommodate the great variety of membrane functions that rely on
lipid-based microdomains. In this process, we believe the protocol for
making superrafts should be applicable to other raft membrane systems
and might be a useful approach for probing the strength of molecular
interactions within these microdomains.
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FOOTNOTES |
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* This work was supported by grants from the Danish Medical Research Council and the Novo-Nordic Foundation and was part of the Danish Biotechnology program.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.
G. H. H. and E. M. D. dedicate this work to the memory of Ove Norén, mentor and longtime friend, who died on December 17, 2001.
Recipient of a Socrates/Erasmus travel stipend from the European
Union. Present address: Universitá Di Perugia, Dipartimento di
Scienze e Biotecnologie Molecolari, Sezione di Patologia Generale, Policlinico Monteluce, 06100 Perugia, Italy.
§ To whom correspondence should be addressed. Tel.: 45-3532-7786; Fax: 45-3536-7980; E-mail: midan@imbg.ku.dk.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211228200
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