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RED CELLS
Ca⫹⫹-dependent vesicle release from erythrocytes involves stomatin-specific
lipid rafts, synexin (annexin VII), and sorcin
Ulrich Salzer, Peter Hinterdorfer, Ursula Hunger, Cordula Borken, and Rainer Prohaska
Cytosolic Caⴙⴙ induces the shedding of
microvesicles and nanovesicles from
erythrocytes. Atomic force microscopy
was used to determine the sizes of these
vesicles and to resolve the patchy, fine
structure of the microvesicle membrane.
The vesicles are highly enriched in glycosyl phosphatidylinositol–linked proteins,
free of cytoskeletal components, and depleted of the major transmembrane proteins. Both types of vesicles contain 2
as-yet-unrecognized red cell proteins,
synexin and sorcin, which translocate
from the cytosol to the membrane upon
Caⴙⴙ binding. In nanovesicles, synexin
and sorcin are the most abundant proteins
after hemoglobin. In contrast, the microvesicles are highly enriched in stomatin.
The membranes of both microvesicles and
nanovesicles contain lipid rafts. Stomatin is
the major protein of the microvesicular lipid
rafts, whereas synexin and sorcin represent
the major proteins of the nanovesicular rafts
in the presence of Caⴙⴙ. Interestingly, the
raft proteins flotillin-1 and flotillin-2 are not
found in the vesicles but remain in the red
cell membrane. These data indicate the presence of different types of lipid rafts in the
erythrocyte membrane with distinct fates
after Caⴙⴙ entry. Synexin, which is known to
be vital to the process of membrane fusion,
is suggested to be a key component in the
process of vesicle release from erythrocytes. (Blood. 2002;99:2569-2577)
© 2002 by The American Society of Hematology
Introduction
Erythroctes are known to respond to physiological stimuli via a
diversity of receptors and effectors.1 In particular, prostaglandin E2
and lysophosphatidic acid (LPA) induce Ca⫹⫹-dependent processes.2,3 The rise of cytosolic Ca⫹⫹ in erythrocytes triggers a
sequence of biochemical and morphologic changes that finally
result in the release of hemoglobin-containing exovesicles. Two
sorts of vesicles, differing in size, have been described; these have
been named microvesicles (150 nm diameter) and nanovesicles (60
nm diameter).4 Elevated cytosolic Ca⫹⫹ levels cause alterations in
the membrane protein pattern, notably the aggregation of proteins
catalyzed by transaminases and cytoskeletal rearrangements owing
to the proteolysis of protein 4.1.5 Another Ca⫹⫹-mediated effect is
the breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a concomitant increase in 1,2diacylglycerol and phosphatidic acid.6 Moreover, the phospholipidscramblase is activated and the aminophospholipid translocase is
inhibited,7 thereby leading to a randomization of the phospholipid
asymmetry over the 2 membrane leaflets. The Ca⫹⫹-activated
potassium efflux via the Gardos channel causes the loss of cell
water and concomitant shrinkage of the erythrocyte.8 The latter
factors are essential for the release of the vesicles from the
echinocytic erythrocytes.
The physiological importance of the vesiculation process can be
seen in a protection strategy of the red cell against the destruction
by complement.9 An erythrocyte being attacked by the complement
components C5b through C9 faces a local Ca⫹⫹ influx and
responds by a rapid release of vesicles containing the membrane
attack complex. A similar defense mechanism has been described
for platelets,10 neutrophils,11 and oligodendrocytes.12 The clinical
importance of the vesicles themselves has been highlighted by the
finding that, owing to the loss of phospholipid asymmetry, they
provide substrates for the secretory phospholipase A2 to generate
the proinflammatory lipid mediator LPA.13 LPA is an important
second messenger and has been shown to open Ca⫹⫹ channels in
human erythrocytes,3 thereby stimulating further vesiculation.
The vesicles released from human erythrocytes after treatment
with Ca⫹⫹ and ionophore A23187 are free of cytoskeletal components and are specifically enriched in glycosyl phosphatidylinositol
(GPI)–anchored proteins, such as acetylcholinesterase (AChE) and
decay accelerating factor (CD55).14 They are also enriched in the
transmembrane protein complement receptor 1 (CD35).15 Particularly, in nanovesicles, AChE is highly concentrated in relation to
total protein and phospholipid content.4 Similarly, in mechanically
induced vesiculation of erythrocytes, the specific enrichment of
GPI-linked CD59 in the vesicles has been shown.16 Interestingly,
red cells of patients with paroxysmal nocturnal hemoglobinuria,
which lack GPI-linked proteins, have an impaired ability to
vesiculate.17 Extensive studies on different cell types unequivocally show that GPI-linked proteins are specifically located in
cholesterol- and sphingolipid-rich domains of the plasma membrane, also termed lipid rafts.18 In the erythrocyte membrane,
GPI-anchored proteins are similarly organized within lipid rafts,19,20
with stomatin, flotillin-1, and flotillin-2 being present in great
abundance at the cytoplasmic face.20 This finding led us to
investigate whether the Ca⫹⫹-induced vesiculation is a lipid
raft–based process and whether stomatin21-27 and the flotillin
proteins28,29 are enriched within the released vesicles.
From the Institute of Medical Biochemistry, University of Vienna, Vienna
Biocenter, Vienna, Austria, and the Institute for Biophysics, Johannes Kepler
University, Linz, Austria.
Reprints: Rainer Prohaska, Institute of Medical Biochemistry, University of
Vienna, Vienna Biocenter, Dr Bohr-Gasse 9/3, A-1030 Vienna, Austria; e-mail:
[email protected].
Submitted May 9, 2001; accepted November 9, 2001.
Supported by grants P12907 (R.P.) and P12802 (P.H.) from the Austrian
Science Fund.
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
2569
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2570
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
SALZER et al
In this study, we show that stomatin, in contrast to the flotillin
proteins, is enriched within the microvesicles. Moreover, synexin
and sorcin are present in microvesicles and are highly enriched in
nanovesicles. These vesicles contain lipid rafts, with synexin and
sorcin being Ca⫹⫹-dependent raft-associated proteins. Our data
shed new light on the organization and dynamics of red cell lipid
rafts and the generation of exovesicles.
Silver staining
Electrophoresis gels were fixed in 50% methanol, 12% acetic acid, and 0.5
mL/L 37% formaldehyde for 1 hour and then washed with 50% ethanol for
1 hour (2 changes). The gels were pretreated with 0.2 g/L Na2S2O3 for 1
minute, rinsed 3 times with water, and incubated with 2 g/L AgNO3 and
0.75 mL/L 37% formaldehyde for 20 minutes, in the dark. Subsequently, the
gels were rinsed twice in water; developed in a solution containing 60 g
Na2CO3, 4 mg Na2S2O3, 0.5 mL 37% formaldehyde per liter; stopped with
50% methanol and 12% acetic acid; and equilibrated with 30% methanol
and 2% glycerol before drying.
Materials and methods
Measurement of AChE activity
Cells
Whole blood was obtained from healthy donors by venipuncture at the
general hospital of Vienna, collected into heparinized tubes and either used
immediately or stored overnight at 4°C. Erythrocytes were pelleted (at 200g
for 10 minutes) and subsequently washed 5 times with 10 mM Tris-Cl, 150
mM NaCl, pH 7.5 (Tris-buffered saline [TBS]).
Preparation of microvesicles and nanovesicles
Method A. Vesicles were prepared according to Allan et al.4 Briefly,
erythrocytes were resuspended in 3 volumes TBS containing 1 mM CaCl2
and 5 ␮M ionophore A23187 (Sigma, St Louis, MO) and incubated at 37°C
for 30 minutes. After addition of EDTA to a final concentration of 5 mM, the
erythrocytes were pelleted (at 15 000g for 30 seconds), and the supernatant
was centrifuged (at 15 000g, 4°C, for 20 minutes). The resulting pellet of
microvesicles was resuspended in an appropriate volume of TBS and
cleared of contaminating erythrocytes by a short centrifugation as above,
whereas the nanovesicles in the supernatant were pelleted by an ultracentrifugation step (Beckman SW50.1 rotor [Beckman Instruments, Palo Alto,
CA], at 100 000g, 4°C, for 60 minutes) and resuspended in an appropriate
volume of TBS. The vesicles were assayed for AChE activity and either
used immediately for further experiments or stored overnight at 4°C.
Method B. Erythrocytes were resuspended in 9 vol TBS containing 1 mM
CaCl2 and 5 ␮M ionophore A23187 and incubated at 37°C for 30 minutes. After
the addition of EDTA to a final concentration of 5 mM, the erythrocytes were
pelleted (at 15 000g for 30 seconds), and the supernatant was subjected to 4
subsequent centrifugation steps. The first 3 steps were performed in 1.5-mL
plastic tubes at 15 000g, 4°C, for 10, 20, and 30 minutes, respectively, and the
subsequent ultracentrifugation step was performed as above (Beckman SW50.1
rotor, at 100 000g, 4°C, for 60 minutes). Each vesicle pellet was resuspended in
an appropriate volume of TBS, assayed for AChE activity, and used immediately
for further experiments.
Identification of proteins
Protein samples were analyzed by gel electrophoresis and silver staining, as
described below, or by immunoblotting with monoclonal antibodies against
band 3 (Sigma); flotillin-1, flotillin-2, and synexin (Transduction Laboratories, San Diego, CA); sorcin (Zymed Laboratories, San Francisco, CA); and
stomatin22; subsequent detection was by horseradish peroxidase–goatantimouse immunoglobulin G (Promega, Madison, WI) and the SuperSignal chemiluminescence kit (Pierce, Rockford, IL). For mixed peptide
sequencing, proteins were separated on 12% polyacrylamide gels, electroblotted onto Immobilon-P (Millipore, Bedford, MA), and stained by Amido
Black (1 mg/mL in 5% acetic acid, 10% methanol). Selected stained bands
were cut out, and cyanogen bromide (CNBr) cleavage was performed as
described,30 with minor modifications. Briefly, the membrane pieces were
washed in 5% acetic acid, 10% methanol, and twice in water, and then
immersed for a short time in a CNBr solution (500 mg CNBr/mL 70%
formic acid), fixed above the CNBr solution in a sealed plastic vial, and
incubated at 34°C for 2 hours. The membrane pieces were vacuum dried
(SpeedVac, Savant, Fullerton, CA) and directly subjected to automated
Edman degradation (ABI model 476A, Applied Biosystems, Foster City,
CA). The mixed peptide sequence data were matched to the public
databases by means of the FASTF or TFASTF algorithms.
AChE activity was determined according to Ellman‘s method, as described
by Steck and Kant31 with minor modifications. Briefly, an aliquot of the
sample, not exceeding 50 ␮L, was mixed with an equal volume 0.5% Triton
X-100 in TBS and incubated at 37°C for 5 minutes to assure solubilization
of lipid rafts. Then, 100 mM sodium phosphate, pH 7.5, was added, up to
700 ␮L; next, 50 ␮L 10 mM 5,5⬘-dithiobis-(2-nitrobenzoic acid) in 100 mM
sodium phosphate, pH 7.0, and 50 ␮L 12.5 mM acetylthiocholine chloride
(Sigma) were added and mixed, and the reaction was followed spectrophotometrically (U-2000 Hitachi, Tokyo, Japan) at 412 nm at room temperature. The initial increase in absorbance was measured for 60 seconds.
Identification of ganglioside GM1
First, 10-␮L aliquots of the sucrose gradient fractions were dotted onto
nitrocellulose membrane and vacuum dried for 2 hours. The membrane was
blocked by incubation with 5% milk powder and 0.1% Tween-20 in TBS for
1 hour at room temperature and subsequently incubated with cholera toxin
B subunit–peroxidase conjugate (Sigma) diluted 1/1000 in 0.1% Tween-20
in TBS at 4°C overnight. The membrane was washed 3 times for 15 minutes
in 0.1% Tween-20 in TBS, and GM1 was detected by means of the
SuperSignal chemiluminescence kit (Pierce).
Determination of the membrane association of vesicle proteins
Equivalent amounts of microvesicles and nanovesicles (according to their
AChE activity) were mixed, adjusted to 50 ␮L with TBS, and lysed by
incubation with 100 ␮L 0.5% saponin in TBS containing 1.5 mM CaCl2 or
7.5 mM EDTA, for 20 minutes, on ice. The vesicle membranes were
pelleted by centrifugation in a fixed-angle rotor (Beckman, TLA-100.1, at
200 000g, 4°C, for 30 minutes). The pellets were resuspended in lysis buffer
to give an equal volume to the supernatants, and aliquots of the supernatants
and pellet suspensions were analyzed by gel electrophoresis/silver staining
and immunoblotting. Bands of interest were scanned and quantified by
densitometry by means of the ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
Phase separation of vesicle proteins in Triton X-114 solution
The phase separation of membrane proteins in Triton X-114 according to
the difference in hydrophobicity32 was applied with slight modifications. To
purify Triton X-114, the precondensation of the detergent was performed 3
times, as recommended.32 Equivalent amounts of microvesicles and
nanovesicles, according to their AChE activity, were mixed and adjusted to
50 ␮L with TBS. The vesicles were lysed by addition of 50 ␮L cold 1%
Triton X-114 in TBS, containing 2 mM CaCl2 or 10 mM EDTA, and
incubated on ice for 20 minutes. This lysate was placed on top of a 200-␮L
sucrose cushion (6% sucrose, 0.06% Triton X-114 in TBS, containing 2 mM
CaCl2 or 10 mM EDTA) in an Eppendorf vial and incubated at 30°C for 5
minutes for condensation. The tubes were placed in a swinging bucket rotor
(Heraeus Variofuge 3.0, Hanau, Germany), and the detergent phase was
pelleted at room temperature (at 300g for 5 minutes). The upper, aqueous
phase was removed and cooled on ice, and the separation was repeated by
addition of fresh Triton X-114 (0.5% final concentration). After incubation
on ice for 20 minutes, the aqueous phase was placed on top of the
previously used sucrose cushion, incubated at 30°C, and centrifuged as
before. The aqueous phase and the detergent phase (oily droplet) were
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
collected, and TBS or Triton X-114 was added to obtain equal volumes and
composition. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
sample buffer was added, and aliquots of the samples were analyzed by gel
electrophoresis/silver staining and immunoblotting.
Flotation assay
Microvesicles or nanovesicles (50 ␮L) were lysed by the addition of 50 ␮L 1%
Triton X-100 in TBS, containing either 2 mM CaCl2 or 10 mM EDTA, incubated
on ice for 20 minutes, and mixed with 100 ␮L 80% sucrose, 0.5% Triton X-100
in TBS containing 2 mM CaCl2 or 10 mM EDTA. The resulting suspensions
were placed in centrifuge tubes (Beckman, 13 ⫻ 51 mm), and each was overlaid
with 1250 ␮L 30% sucrose in TBS containing 1 mM CaCl2 or 5 mM EDTA, and
333 ␮L 10% sucrose in TBS containing 2 mM CaCl2 or 10 mM EDTA, and
centrifuged in a precooled SW50.1 rotor (Beckman) at 230 000g, 4°C, for 17
hours. Thirteen fractions, 140 ␮L each, were collected from the top, and aliquots
were analyzed by gel electrophoresis/silver staining, immunoblotting, and GM1
dot blotting and were also analyzed for AChE activity and cholesterol content
(Unisys kit, Roche Diagnostics, Mannheim, Germany).
Atomic force microscopy
A Macmode PicoSPM magnetically driven dynamic-force microscope (Molecular Imaging, Phoenix, AZ) was used. The topography images were recorded in
the Magnetic AC (Mac) mode33,34 with MacLevers (Molecular Imaging) of about
RED CELL MICROVESICLES AND NANOVESICLES
2571
1 N/m nominal spring constant in aqueous buffer solutions at room temperature.
Measurements were performed with 5 nm free-tip oscillation amplitude at 40
kHz driving frequency, and the feedback-loop was driven at 20% amplitude
reduction. Amplitude images were recorded simultaneously. Image size was 1
␮m ⫻ 1 ␮m to 30 ␮m ⫻ 30 ␮m at 1 Hz lateral scan rate. Microvesicles and
nanovesicles were specifically bound to wheat germ agglutinin (WGA)–coated
mica surfaces. For this, mica sheets (Gröpl, Tulln, Austria) were first derivatized
with ethanolamine as previously described,35,36 then incubated for 1.5 hours with
a solution of 1 mg/mL ethylene glycol-bis(succinimidylsuccinate) (Pierce) in
chloroform containing 0.5% triethylamine, washed in chloroform, and dried with
N2. Subsequently, a solution of 0.5 mg/mL WGA (Sigma) in 10 mM sodium
phosphate and 150 mM NaCl, pH 7.5 (phosphate-buffered saline), was applied
for 2 hours, and the excess of WGA was removed. Finally, the vesicles were
bound to the mica surface in Hepes-buffered saline (5 mM Hepes, 150 mM NaCl,
pH 7.3) for 15 minutes at appropriate concentrations and imaged in the same
buffer by means of atomic force microscopy (AFM).
Results
Microvesicles and nanovesicles released from erythrocytes after
Ca⫹⫹/A23187 treatment were prepared according to method A and
Figure 1. AFM images of erythrocyte microvesicles and nanovesicles. Microvesicles (panels A, C) and nanovesicles (panel B) were adsorbed to WGA-coated mica
surfaces. (A) (B) Topography images were obtained with the use of the Mac mode in buffered saline. Singly distributed vesicular structures are clearly resolved. Heights are
indicated by a gray scale bar, ranging from 0 nm (black) to 200 nm (white) (panel A) and from 0 nm (black) to 100 nm (white) (panel B). Image size was 7.5 ␮m. (C) The
high-resolution AFM amplitude image reveals distinct, texturelike structures on the microvesicular membranes and flat membrane patches that are occasionally associated
with the microvesicles (arrows). Image size was 1.5 ␮m. (D) Size distribution of microvesicles (n ⫽ 82, asterisks) and nanovesicles (n ⫽ 85, triangles). Maxima are found at
179 nm and 81 nm, respectively.
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2572
SALZER et al
studied by AFM to assess their sizes und structure under physiological conditions. The vesicles were immobilized in solution by
WGA, covalently attached to the mica support, and imaged by
means of dynamic-force microscopy.33,34 In this procedure, a
magnetically oscillated cantilever scans the surface of the sample,
touching it upon every downstroke, thus reducing the oscillation
amplitude. The amplitude is held constant by a feedback loop so
that the cantilever follows the surface topography, yielding the
height image shown in Figure 1. Owing to the small oscillation
amplitudes of only a few nanometers, this mode provides a most
gentle tip-surface interaction, minimizing the compression of the
soft vesicular structures on the surface. Single vesicles with almost
no deterioration or disassembling of the structures during imaging
were clearly resolved (Figure 1). Apparently, the vesicles were
tightly bound to the surface; this was most likely accomplished by
numerous, strong bonds between the WGA molecules on the mica
surface and carbohydrate moieties on the membrane surface.
Microvesicles (Figure 1A) appear considerably larger in both
diameter and height than nanovesicles (Figure 1B). Higher magnification amplitude images (Figure 1C) revealed a patchy, texturelike, fine structure of the microvesicular membrane. As the
microvesicles are essentially free of cytoskeletal components, this
fine structure reflects an inherent organization of the vesicular
membrane itself. Some of the microvesicles (Figure 1C) appear
associated with flat membrane sheets, possibly reflecting a segregation of vesicular membrane domains. In Figure 1D, the size
distribution of nanovesicles and microvesicles is shown. For
nanovesicles, the maximum of distribution is centered around 81
nm (n ⫽ 85), with 62 nm peak width at half maximum; for
microvesicles, the maximum is found at 179 nm (n ⫽ 82), with 92
nm peak width at half maximum. These data are in good agreement
with earlier results obtained by electron microscopy.4 Additionally,
the size distribution indicates the cross-contamination of
nanovesicles with small microvesicles (about 16%) and of microvesicles with large nanovesicles owing to the limited separation
efficiency of method A.
As the GPI-linked protein AChE is abundant in the microvesicles and nanovesicles,4 we were specifically interested
whether other lipid raft proteins, like stomatin and the flotillins, are
similarly enriched within these vesicles. Figure 2A shows the
protein pattern of washed erythrocyte membranes (lane 1) compared with that of whole microvesicles and nanovesicles (lanes 2
and 3), normalized to AChE activity. Both types of vesicles contain
the major cytosolic protein components, hemoglobin (16-kd monomers) and carbonic anhydrase (30 kd). The AChE activity relative
to the hemoglobin content is highly enriched in both types of
vesicles, with roughly 80 times (in microvesicles) and 250 times (in
nanovesicles) enrichment when compared with erythrocytes (data
not shown), as previously reported.4 The vesicles are essentially
free of cytoskeletal proteins, and the relative amounts of the major
integral membrane proteins band 3 and glycophorins are diminished. Stomatin (band 7.2) is the only major erythrocyte membrane
protein that parallels the specific enrichment of the GPI-anchored
protein AChE in microvesicles (Figure 2A). Interestingly, only
trace amounts of the flotillins are found in the vesicles.
In nanovesicles, 2 major proteins can be identified by gel
electrophoresis/silver staining as prominent bands at 47 kd and 22
kd, respectively; they represent the most abundant proteins after
hemoglobin. Similar bands were already described as band 4.5 and
band 8 by Allan et al4; so far, they have remained uncharacterized.
Mixed peptide sequencing identified the 47-kd component as
synexin (annexin VII) and the 22-kd component as sorcin. These
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
Figure 2. Identification of proteins in erythrocyte microvesicles and
nanovesicles. (A) Erythrocytes were treated with Ca⫹⫹/A23187, and microvesicles
and nanovesicles were prepared according to method A. Aliquots of the erythrocyte
membranes (lane 1), microvesicles (lane 3), and nanovesicles (lane 3), normalized to
AChE activity, were analyzed by 12% polyacrylamide gel electrophoresis/silver
staining (upper panel) and immunoblotting (lower panel) as indicated. (B) Vesicles
were prepared according to method B. Aliquots, normalized to AChE activity, of the
erythrocyte membranes (lane 1) and vesicles obtained after low-speed centrifugation
for 10 minutes (lane 2) and 20 minutes (lane 3) and ultracentrifugation (lane 4) were
analyzed as in panel A. CA indicates carbonic anhydrase; Hb, hemoglobin.
results were confirmed by immunoblotting (Figure 2). Synexin is a
member of the large annexin protein family,37 which has important
functions in membrane organization and intracellular traffic. Upon
Ca⫹⫹ binding, synexin translocates to the membrane and acts as a
membrane-fusion protein, for instance in the exocytotic secretion
of chromaffin granules.38 Sorcin, too, translocates to the membrane
in a Ca⫹⫹-dependent manner39,40 and has been shown to bind to
specific membrane proteins and synexin.41
Synexin and sorcin are also found in the microvesicle preparation (Figure 2A, lane 2), and stomatin is present in the nanovesicle
preparation (Figure 2A, lane 3); however, this could be due, at least
partly, to cross-contamination of the respective vesicle pools
(Figure 1D). To address this question, we modified the preparation
method and fractionated the vesicles by extended differential
centrifugation (method B). The 4 centrifugation steps recovered
68% (10 minutes), 15% (20 minutes), 5% (30 minutes), and 12%
(ultracentrifugation) of the total AChE activity in the pellet. After
normalization to the AChE activity, the protein patterns of the
vesicle preparations were determined (Figure 2B). Apparently, the
10- and 20-minute vesicles are very similar and probably represent
different size populations of microvesicles, in accordance with the
size distribution analysis (Figure 1D). The nanovesicles obtained
by method B (Figure 2B, lane 4) completely lack band 3, indicating
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
that there is no cross-contamination with microvesicles. Synexin is
present in both microvesicles and nanovesicles, yet is enriched in
the nanovesicles. Sorcin is highly enriched in nanovesicles compared with microvesicles; in contrast, only trace amounts of
stomatin can be found in nanovesicles. Thus, stomatin and sorcin
can be used as marker proteins for microvesicles and nanovesicles,
respectively.
To address the question of whether synexin and sorcin reside in
the lumen or at the membrane of the vesicles, we permeabilized the
vesicles with saponin in the presence of Ca⫹⫹ or EDTA and
isolated the vesicular membranes (Figure 3A). In the presence of
Ca⫹⫹, synexin and sorcin remained partly associated with the
vesicular membranes, with more than 50% and 25%, respectively,
being present in the pellet. Probably the actual percentage of the
membrane-bound fraction of synexin and sorcin is higher, because
the integral proteins stomatin and band 3 were also partly
solubilized under the given conditions. The unequal distribution of
synexin and sorcin between supernatant and pellet may suggest that
these proteins are partly organized in different protein complexes.
When Ca⫹⫹ was replaced by EDTA, the membrane association of
both proteins was completely abolished.
To investigate whether this membrane association was due to
Ca⫹⫹-dependent binding of membrane components or to a Ca⫹⫹induced change in hydrophobicity of the respective proteins, we
analyzed the distribution of the vesicle proteins after Triton X-114
phase separation (Figure 3B). In the presence of Ca⫹⫹, sorcin
predominantly partitioned into the detergent phase, whereas in the
absence of Ca⫹⫹ it was exclusively present in the aqueous phase.
RED CELL MICROVESICLES AND NANOVESICLES
2573
This gain in hydrophobicity upon Ca⫹⫹ binding has already been
described for recombinant sorcin.39 In contrast, synexin did not
change its partitioning characteristic in the presence of Ca⫹⫹,
reflecting an unaltered hydrophilic behavior. This finding is in
accordance with the idea that Ca⫹⫹ ions mediate the binding of
synexin to the membrane by association with acidic phospholipids
rather than inducing a conformational change.
The patchy, fine structure of the microvesicular membrane
(Figure 1C) and the specific enrichment of the lipid raft components stomatin and AChE in microvesicles suggested the presence
of lipid rafts within the vesicular membrane. Therefore, we
prepared microvesicles by the 10-minute centrifugation according
to method B, lysed them in cold Triton X-100, and analyzed the
solution by density gradient equilibrium centrifugation for the
presence of floating lipid rafts. Each experiment was done in
parallel, with Ca⫹⫹ or EDTA being present during lysis and in the
sucrose gradient. The flotation assays in Figure 4 reveal that most
of stomatin and AChE is present in the low-density region of the
discontinuous density gradients. Moreover, the lipid raft markers
cholesterol and ganglioside GM1 show a parallel distribution in the
low-density region of the gradient. These results clearly indicate
the existence of lipid rafts within the microvesicular membrane.
Interestingly, in the presence of Ca⫹⫹, most of synexin and part of
sorcin comigrated with the lipid raft markers to low densities
(Figure 4A). Replacement of Ca⫹⫹ by EDTA completely abolished
this association of synexin and sorcin with the lipid rafts (Figure
4B). Here we want to mention that some variation concerning the
relative amount of the lipid raft markers in the low-density region
can be observed among different blood samples. However, this is
not specific for vesicular rafts but is true for erythrocyte rafts in
general and is probably related to variations in the serum lipid
content of the donors.
In parallel, we investigated whether lipid rafts are also present
in nanovesicles prepared by method B, which virtually lack the raft
protein stomatin. Again, the lipid raft markers AChE, cholesterol,
and GM1 are partly present at low densities in the discontinuous
density gradient, indicating the existence of lipid rafts in the
nanovesicular membrane (Figure 5). In the presence of Ca⫹⫹, most
of synexin and a part of sorcin are found in the lower-density
fractions of the sucrose gradient. However, the distribution of
synexin and, particularly, of sorcin is not strictly correlated with
that of the lipid raft markers but is shifted toward higher densities
(Figure 5A). When Ca⫹⫹ is replaced by EDTA, neither synexin nor
sorcin shows a floating behavior, but both are found in the bottom
fraction of the gradient (Figure 5B).
Discussion
Figure 3. Caⴙⴙ dependence of the synexin and sorcin membrane association
and change in sorcin hydrophobicity. (A) Mixtures of microvesicles and
nanovesicles were lysed by 0.5% saponin in the presence of Ca⫹⫹ or EDTA. The
vesicle membranes were pelleted, and aliquots of the supernatants (S) and pellets
(P) were analyzed by 12% polyacrylamide gel electrophoresis/silver staining (upper
panel) and immunoblotting (lower panel). (B) Mixtures of microvesicles and
nanovesicles were dissolved in Triton X-114 in the presence of Ca⫹⫹ or EDTA, and
phase separation was performed. Aliquots of the aqueous (aq) and detergent (dt)
phases were analyzed by 12% polyacrylamide gel electrophoresis/silver staining
(upper panel) and immunoblotting (lower panel).
The data presented here show the involvement of stomatin-specific
lipid rafts and the as-yet-unrecognized red cell components synexin
and sorcin in the Ca⫹⫹-induced exovesiculation process of erythrocytes. AFM determination of the sizes of quasi-native microvesicles and nanovesicles (Figure 1A,B) is in good agreement
with the data obtained by electron microscopy.4 The patchy, fine
structure of the microvesicular membrane revealed at higher
magnification (Figure 1C) may reflect various lipid domains and/or
lipid-protein complexes.Whereas the size distribution of
nanovesicles is rather narrow, the microvesicles reveal a higher size
variation (Figure 1D).
The specific enrichment of stomatin in microvesicles and of
synexin and sorcin in microvesicles and nanovesicles (Figure 2)
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2574
SALZER et al
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
Figure 4. Caⴙⴙ-dependent association of synexin and sorcin with microvesicular lipid rafts. Microvesicles prepared according to method B (10-minute low-speed
centrifugation) were lysed with cold 1% Triton X-100 in TBS, in the presence of Ca⫹⫹ or EDTA, and subjected to discontinuous density gradient centrifugation. Thirteen fractions
were collected from the top, and aliquots were analyzed by 12% polyacrylamide gel electrophoresis/silver staining (panel A) and immunoblotting (panel B), as indicated, and for
AChE activity and cholesterol content (percentage of total).
indicates a vital function for these proteins in the vesiculation
process. Stomatin is an oligomeric integral membrane protein,20,26
which is palmitoylated,27 is associated with lipid rafts,20,42 and has
been suggested to play a role as a scaffolding component20,42
similar to the raft protein caveolin. Synexin (annexin VII) was first
isolated from adrenal medullary chromaffin cells and shown to
cause Ca⫹⫹-dependent aggregation of isolated chromaffin granules.38 The 13 members of the annexin protein family share a
common core domain that is composed of 4 highly conserved
repeats that are thought to mediate binding of Ca⫹⫹ ions.37
However, their N-terminal domains are highly variable and function as docking sites for specific interaction partners. Synexin,
which contains an extraordinarily long N-terminus, binds to sorcin
in a Ca⫹⫹-dependent manner.41 Sorcin belongs to the pentaEF-hand family and plays a role in the Ca⫹⫹-mediated signal
transduction by association with specific membrane proteins at
specific subcellular locations.41,43-45 Interestingly, the N-terminal
domains of sorcin and synexin contain similar structural motifs and
mediate the interaction of these proteins.41,46 This interaction,
which has been studied in the adrenal medullary tissue41 and in
differentiating myoblasts,47 interferes with the membrane-aggregation activity of synexin, indicating a regulatory role for sorcin in the
exocytotic process.
Our finding that sorcin and synexin translocate to the erythrocyte membrane upon Ca⫹⫹ entry (Figure 3A) is in good agreement
with the reported characteristics of these proteins in other tissues.
Sorcin is the only red cell vesicular protein that increases its
hydrophobicity in a Ca⫹⫹-dependent manner (Figure 3B). This
gain in hydrophobicity is due to a conformational change accompanying Ca⫹⫹-binding, a property common to all EF-hand proteins,
and suggests that exposed hydrophobic parts of sorcin directly
insert into the membrane. The Ca⫹⫹-dependent membrane association of synexin is based on a different mechanism: synexin-bound
Ca⫹⫹ ions are thought to act like an electrostatic glue that mediates
binding to negatively charged phospholipids.48
We have recently shown that stomatin and the related flotillins
are the major integral proteins of erythrocyte lipid rafts.20 Interestingly, these proteins behave differently in the vesiculation process.
Whereas stomatin is specifically concentrated in the microvesicles,
flotillins are virtually absent from microvesicles and remain in the
red cell membrane (Figure 2). This result suggests that there are at
least 2 distinct sets of lipid rafts in the erythrocyte membrane, with
stomatin and the flotillins being respective marker proteins. In
accordance with this concept is the finding that stomatin and the
flotillins are organized in independent oligomeric complexes.20
Generally, the coexistence of functionally different types of lipid
rafts at the plasma membrane provides an exciting new perspective
in the hypothesis of membrane microdomains.49-51
In microvesicles, the lipid raft components stomatin and AChE
are highly enriched relative to other membrane proteins. We show
that lipid rafts exist within the microvesicular membrane (Figure
4), with most of stomatin and AChE being raft-associated. We
suggest that the patchy, fine structure of the microvesicular
membrane seen by AFM reflects the inherent lipid organization in
raft and nonraft domains (Figure 1C). Interestingly, synexin and, to
a lesser extent, sorcin are also associated with the microvesicular
rafts in a Ca⫹⫹-dependent manner. This is the first report to show
the lipid raft association of synexin. Several other members of the
annexin protein family have already been described as associating
with lipid rafts in various cellular contexts. Annexin II and the
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
RED CELL MICROVESICLES AND NANOVESICLES
2575
Figure 5. Caⴙⴙ-dependent association of synexin and sorcin with nanovesicular lipid rafts. Nanovesicles prepared according to method B (ultracentrifugation) were
lysed with cold 1% Triton X-100 in TBS, in the presence of Ca⫹⫹ or EDTA, and subjected to discontinuous density gradient centrifugation. Thirteen fractions were collected from
the top, and aliquots were analyzed by 12% polyacrylamide gel electrophoresis/silver staining (panel A) and immunoblotting (panel B), as indicated, and for AChE activity and
cholesterol content (percentage of total).
interacting protein p11 are the major lipid raft components in
Madin-Darby canine kidney cells.52 In baby hamster kidney cells,
the membrane association of annexin II is partially Ca⫹⫹ independent but sensitive to cholesterol sequestration,53 thereby proving a
tight interaction of annexin II with lipid rafts. Annexin XIIIb,
which is myristoylated at the N-terminus, associates with lipid rafts
and plays a role in the apical delivery of vesicular carriers.54 In
smooth muscle cells, the concerted action of annexins II, V, and VI
is thought to influence Ca⫹⫹-dependent lipid raft dynamics, with
important consequences for signaling events and Ca⫹⫹ flux.55
In our study of nanovesicles, the flotation experiments in Figure
5A,B show a distinct pool of GM1, cholesterol, and AChE comigrating in the low-density region of the sucrose gradient, indicating the
existence of lipid rafts. As the erythrocyte raft marker proteins,
stomatin and the flotillins, are virtually absent from nanovesicles, it
is conceivable that the nanovesicular rafts represent another type of
microdomain in the erythrocyte membrane. In the presence of
Ca⫹⫹, synexin and sorcin show a floating behavior; however, their
distribution lacks correlation with the lipid raft markers. Our
interpretation of this phenomenon is that there is a certain
reversibility in the calcium-mediated binding of synexin and sorcin
to the nanovesicular lipid rafts, with an equilibrium between
raft-bound and unbound protein. During the centrifugation, these
partitioning states of the respective proteins encounter opposing
forces, with the raft-bound form floating to lower densities and the
unbound form sedimenting to higher densities. This would result in
the segregation and observed difference in the distribution of the
lipid raft markers and synexin and sorcin. In this light, the
difference in the distribution of synexin and sorcin suggests a lower
affinity of sorcin to the nanovesicular rafts as compared with
synexin. Sorcin has not yet been described as a lipid raft protein,
and it remains to be determined whether sorcin interacts directly
with raft lipids or whether it is associated via the N-terminus of
synexin. Moreover, comparison of Figures 4A and 5A suggests that
the calcium-dependent association of synexin and sorcin is stronger
for microvesicular than for nanovesicular rafts. However, further
lipid and proteomic analyses are necessary to define the difference
between microvesicular and nanovesicular rafts and to understand
their roles in the vesiculation process of erythrocytes.
There is increasing evidence that lipid rafts are involved in
membrane budding and vesiculation during diverse biological
processes.56 It is well documented that lipid rafts play a key role in
the vesicle-based exocytic and endocytic transport (for review, see
Ikonen57). Lipid rafts were shown to serve as platforms for the
inclusion of sorting receptors and cargo molecules to the forming
vesicle.58 Moreover, it has been shown that viral assembly takes
place within lipid rafts of the plasma membrane and that the viral
envelope is enriched in lipid raft components.59-61 Our data suggest
that the calcium-induced microvesiculation and nanovesiculation
of erythrocytes are also lipid raft–based processes, with stomatin
and synexin being the major raft proteins, respectively. It remains
to be determined whether the membrane-aggregating38 and fusogenic62,63 activities of synexin play a role in these processes, with
sorcin being a modulator.41 Our finding that lipid rafts specific for
stomatin, flotillin, synexin, and sorcin are segregated during the
vesiculation process introduces a novel aspect of the dynamics and
organization of the erythrocyte membrane.
Acknowledgments
We thank Diethelm Gauster for peptide sequencing, Elisabeth
Legenstein for quantitative cholesterol analyses, and Tim Skern for
critically reading the manuscript.
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2576
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
SALZER et al
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2002 99: 2569-2577
doi:10.1182/blood.V99.7.2569
Ca++-dependent vesicle release from erythrocytes involves stomatin-specific
lipid rafts, synexin (annexin VII), and sorcin
Ulrich Salzer, Peter Hinterdorfer, Ursula Hunger, Cordula Borken and Rainer Prohaska
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