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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2573-2580
By
From the Hôpital St Eloi, Laboratoire Central
d'hématologie; INRA, Physiologie Animale; and UMR 5539, Université Montpellier II, Montpellier, France.
Exosomes are membrane vesicles released by reticulocytes during
their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or
disappear from the cell surface during maturation, eg,
acetylcholinesterase (AChE) and transferrin receptor (TfR),
respectively. To better understand the molecular events leading to
protein sorting in exosomes, we analyzed the expression of
glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome
surface through a technique involving bead coupling and flow cytometry
immunodetection. The presence of AChE, decay-accelerating factor (DAF),
membrane inhibitor of reactive lysis (MIRL), and lymphocyte
function-associated antigen 3 (LFA-3) on the surface of exosomes
obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH)
reticulocytes, suggests that (1) the GPI anchor is efficiently sorted
during exosome formation, (2) exosome release could account for the
observed discrepancy in GPI-protein expression between reticulocytes
and erythrocytes from PNH patients, and (3) exosomes could have another
physiologic function related to controlling membrane attack complex
formation.
DURING RETICULOCYTE maturation, some
membrane activities are lost through their release in the extracellular
medium, as they are associated with small lipid vesicles called
exosomes. The major membrane protein lost by this physiologic process
is the transferrin receptor (TfR), which completely disappears from the red blood cell (RBC) surface during
maturation.1 Other membrane proteins such as
acetylcholinesterase (AChE) and transporters of nucleosides, glucose,
and amino acids have been shown to be sorted in exosomes.1
The signals involved in this sorting process are still unknown.
Nevertheless, we recently showed that antibody-induced molecule
clustering increases TfR and AChE release within exosomes,2 suggesting that the aggregation of some membrane constituents may be
involved in this physiologic process. Different forms of AChE have been
described for various cells. RBC AChE was shown to be membrane-bound
through a glycosylphosphatidylinositol (GPI) anchor.3,4
AChE detected in exosomes therefore probably does not span the
membrane, excluding a direct interaction with a cytosolic protein (eg,
hsc 70) suggested to be involved in TfR sorting.5 On the
other hand, GPI-anchored proteins are known to be sorted into specific
lipid domains in different cell compartments such as the trans-Golgi
network or caveolae.6,7 Glycosphingolipids such as the
ganglioside GM1 were shown to be enriched in these lipid
domains.8
Other GPI-anchored proteins are present on the surface of RBCs. These
include the decay-accelerating factor (DAF, CD55) and membrane
inhibitor of reactive lysis (MIRL, CD59), two proteins that inhibit
formation of the membrane attack complex on autologous cell
surfaces.9,10
In hematologic disorder, paroxysmal nocturnal hemoglobinuria (PNH),
impaired transfer of N-acetylglucosamine to phosphatidylinositol leads
to a synthetic defect in GPI and thus to a deficiency in the surface
expression of all GPI-anchored proteins.11 Analysis of many
patients with PNH has shown that somatic mutation in the X-linked gene
PIG-A is responsible for the GPI-anchor deficiency in
PNH.12 Blood cells of PNH patients are affected
differently,13,14 but are generally more susceptible to
complement-mediated attack. Hemolysis of RBCs is the major clinical
indication of the disease, although the lack of DAF and/or MIRL
on the cell surface can vary markedly between patients. Moreover, there
is often a difference in the extent of erythrocytes and reticulocytes
affected in the RBC population.15
In the present study, we showed that the GPI-anchored proteins AChE,
DAF, MIRL, and LFA-3 were released in exosomes by reticulocyte during
in vitro maturation into erythrocytes. The extent of GPI-anchored proteins released within exosomes was not highly modified when reticulocytes of PNH patients were matured in vitro, indicating that
the GPI-anchor was efficiently sorted during vesicle formation. Exosome
release could thus account for the observed discrepancy in GPI-protein
expression between reticulocytes and erythrocytes from PNH
patients.15 Moreover, the presence of DAF and MIRL within
the exosomal membrane could provide vesicles released in the blood
circulation with another physiologic function related to the regulation
of membrane attack complex formation.
Donors.
The two patients with PNH had marked deficiencies of CD55 and CD59
antigens. In particular, the presence of partially CD59 deficient (type
II) and completely deficient (type III) RBCs was detected for case 1 (see Fig 5). The reticulocyte count was determined by flow cytometric
analysis with thiazole orange labeling of RNA. Controls involved
patients with a high percentage of reticulocytes (always >6%) due to
other pathologic states, such as hemolytic anemia.
Reagents.
Magnetic beads with covalently bound affinity purified sheep antimouse
IgG (Dynabeads M-450*SAM) or antihuman TfR (Dynabeads M-450*CD71) were
from Dynal France S.A. (Compiègne, France). Monoclonal antibodies
(MoAb) against human and rat transferrin receptors were obtained from
Monosan (Uden, The Netherlands) and Chemicon (Temecula, CA),
respectively. Culture supernatants of OKT9 cells (American Type Culture
Collection, Rockville, MD) were used to detect human TfR by Western
blot. MoAbs specific to CD55, CD58, and CD59 antigens were from Cymbus
Biotechnology Ltd (Hants, UK), Chemicon and Monosan, respectively.
Fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG
(GAM-FITC) and goat antirabbit IgG (GAR-FITC), FITC-conjugated cholera
toxin B subunit (FITC-CTB), and wheat germ agglutinin (FITC-WGA) were
purchased from Sigma (St Louis, MO). Antibodies to rat
acetylcholinesterase16 were a generous gift of Jean
Massoulié (E.N.S., Paris, France). Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus was obtained from Boehringer Mannheim (Meylan, France).
Exosome preparation.
Peripheral blood was collected from phenylhydrazine-treated rats
(Sprague-Dawley) by cardiac puncture as previously
described.17 After removing the buffy coat, RBCs
(reticulocyte count >70%) were washed three times with
phosphate-buffered saline (PBS) and subcultured (3% suspension) for 24 hours at 37°C in maturation medium (RPMI supplemented with 5 mmol/L
glutamine, 5 mmol/L adenosine, 10 mmol/L inosine). After cell
pelleting, the culture supernatant was centrifuged (20,000g for
20 minutes) to remove cellular debris. Exosomes were separated from the
supernatant by centrifugation (100,000g for 90 minutes). The
pellet (vesicular fraction) was washed once by centrifugation and
resuspended in 250 mmol/L sucrose, 10 mmol/L Tris-HCl (pH 7.4).
Immunobinding of exosomes to magnetic beads.
A total of 100 µL of Dynabeads M-450 coupled with SAM (4 × 108 beads/mL) were washed twice with 4 mL of PBS and
resuspended in the same volume. The magnetic beads were then incubated
with 10 µL of MoAbs (antihuman or antirat) TfR overnight at 4°C
with gentle rotation. After three washes in PBS, the beads were
incubated with the different exosome preparations. A total of 100 µL
of exosomes, containing 2 to 5 mg protein/mL, were added for at least 2 hours at 4°C. Exosome-bead complexes, in some cases blocked with
rabbit antimouse IgG, were then washed twice using a magnet, with 4 mL
of PBS containing 1% fetal calf serum (PBS-FCS).
Electron microscopy.
Exosome-bead conjugates were fixed in glutaraldehyde 2% for 1 hour at
room temperature. Exosomes immunologically bound to magnetic beads were
fixed in suspension with 2% glutaraldehyde in PBS for 1 hour at room
temperature and then centrifuged for 10 minutes at 13,000g and
postfixed in osmium. The pellet was stained in bloc with magnesium
uranyl acetate, dehydrated in graded ethanol, and embedded in epon.
Flow cytometric analysis.
A total of 10 µL of exosome-bead complexes in 100 µL PBS-FCS were
incubated with the different primary antibodies for 30 minutes at room
temperature, washed three times using a magnet with PBS-FCS, and
incubated with corresponding FITC-labeled secondary antibodies for 30 minutes at room temperature. In the indicated experiments, fluorescent
lectins (FITC-WGA and FITC-CTB) were incubated with the exosome-bead
complexes for 1 hour at 37°C. After washing, the samples were
analyzed using an Epics XL flow cytometer (Coulter, Hialeah, FL)
equipped with an ion Argon LASER (488 nm). Beads or exosome-bead
complexes were assessed from the dot plot representation of forward
scatter (size) versus side scatter (granularity), which were set at
logarithmic gain. Only single beads or complexes were gated for
fluorescence analysis (logarithmic scale). Twenty thousand events were
analyzed for each sample.
PI-PLC sensitivity of AChE.
Exosomes (approximately 2 mg protein/mL) were extracted with 1% Triton
X-100, 10 mmol/L Tris/HCl pH 7.4, 1 mmol/L EGTA, 5 mmol/L EDTA, and
proteinase inhibitors (Boehringer Mannheim): aprotinin (2 µg/mL),
leupeptin (0.5 µg/mL), and pepstatin (0.7 µg/mL) for 15 minutes at
4°C with intermittent stirring. After a 15-minute centrifugation at
10,000g, AChE was recovered in the supernatant. This exosome
extract was used as control or treated for 1 hour at 37°C with
PI-PLC (5 U/mL). Nondenaturing polyacrylamide gel electrophoresis was
performed as described previously.18 Briefly, gels and
running buffers contained 50 mmol/L Tris/glycine (pH 8.9) and 0.5%
Triton X-100. Samples (15 µL) were run for 3 hours at 10 V/cm. Gels
were rinsed in distilled water and stained for AChE activity according
to Karnovsky and Roots.19
Exosomal AChE is anchored through a GPI.
PI-PLC treatment of rat exosomes induced a more mobile form of AChE in
nondenaturing polyacrylamide gel electrophoresis
(Fig 1A). This is typical of partial
conversion of the amphiphilic dimer (G2a) into the hydrophilic dimer
(G2h) form of RBC AChE.18 This was confirmed by sucrose
gradient centrifugation analysis (Fig 1B). Using an untreated detergent
extract of rat exosomes, a single AChE activity peak was obtained,
which sedimented at 6 Svedberg (S) in the presence of 1% Triton X-100.
When the detergent was omitted in the gradient, AChE activity was
recovered as a broad peak around 12 S, indicative of the formation of
micellar aggregates. However, AChE activity sedimented in the same
gradient as a well-defined peak shifted to 6.8 S after incubation of
exosomal detergent extract with PI-PLC. The amount of AChE activity
recovered in this 6.8S peak, typical of the hydrophilic dimer of AChE,
indicated that conversion of amphiphilic AChE by PI-PLC was complete.
Taken together, these results showed that AChE was secreted in the
extracellular medium, associated with the exosome membrane through a
GPI anchor.
Exosome-bead complex construction.
Coupling exosomes to beads was dictated by requirement of antibody
binding/washing steps during the immunophenotypic labeling. TfR was
ideally suited to conjugate the exosomes to beads, because of its high
amount on the vesicle surface and specificity as an exosome marker. We
set up the conditions, using highly-available exosomes derived from rat
reticulocytes and magnetic beads coupled with SAM, and a mouse
monoclonal antirat TfR as a secondary antibody. Coupling the two types
of particles was very efficient and homogeneous, as assessed by
electron microscopy (Fig 2A). It also
confirmed the size homogeneity of exosomes (diameter around 60 nm) (Fig 2B). Another effect of coupling, apart from simplifying washing of the
complex, was that the exosomal signal could be distinguished from the
background detection (nonexosomal vesicles, small particles, etc)
during flow cytometry analysis because of the larger size of the beads
(4.5 µm) as compared with exosomes (Fig
3A). As the beads were precisely calibrated, populations that were
double or triple the size of the beads could be detected, probably
because of exosomal cross-linking (Fig 3A). In subsequent experiments, only single beads or complexes were gated for fluorescence analysis. FITC-labeled wheat germ agglutinin (WGA), a lectin binding
N-acetyl-glucosamine-containing glycoproteins and glycoconjugates, was
used as a control to nonspecifically stain the exosome-bead complexes.
The addition of detergent diminished the WGA-FITC labeling of
exosomes-bead complexes to the fluorescent level obtained by WGA-FITC
labeling of anti-TfR antibody-conjugated beads, showing the exosomal
origin of the staining (Fig 3B). Finally, an antirat
acetylcholinesterase16 was used to test the detection potential of the specific antigen on our construction. The complexes were AChE-positive, as shown by a shift in the fluorescence peak (Fig
3C).
DAF, MIRL, and LFA-3 are present on the exosome surface.
The presence of three other GPI-anchored proteins, DAF (CD55), MIRL
(CD59), and LFA-3 (CD58) within exosomes was investigated. For this
immunophenotypic analysis, human reticulocytes were incubated in vitro
in maturation conditions, and exosomes released in the medium were
collected and coupled to either SAM-magnetic beads, using mouse MoAb
antihuman TfR or directly to Dynabeads M-450*CD71. As shown in
Fig 4A, exosomes were labeled with
anti-CD55, anti-CD59, and anti-CD58 antibodies. Note that, in the three
cases, the fluorescence peaks were very sharp and markedly shifted as
compared with control (GAM-FITC alone), indicating a very homogenous
phenotype. Staining also showed a negative population only with LFA-3.
By using the cross-reactivity of antirat AChE with the human
protein,16 we analyzed the staining of these human exosomes
for AChE (Fig 4B). As was the case with rat exosomes, the fluorescence
peak obtained was higher than for the other GPI-anchored proteins. Note
also that the fluorescence intensity of GAM-FITC staining was higher than with GAR-FITC alone, probably due to the labeling of anti-TfR mouse antibody on the magnetic bead surface.
Sorting of DAF and MIRL is very efficient in exosomes.
We used blood from PNH patients to further analyze the sorting of these
GPI-anchored molecules in exosomes. Figure
5 shows the results of the flow cytometry analysis of DAF and MIRL on the surface of RBCs from a PNH (case 1) and a control patient. As
compared with normal RBC, which had uniform, positive staining with
anti-CD59, there was high heterogeneity of MIRL expression in RBC of
this PNH patient. Approximately 40% of the circulating RBCs showed
negative expression (type III), around 50% showed intermediate
expression (type II), and 10% showed normal expression (type I).
Interestingly, the deficiency of DAF expression was less marked than
for the other GPI-anchored protein. RBCs from this PNH patient and from
an anemic patient (reticulocyte count, 12.5% and 6%, respectively)
were cultured in vitro, and the released vesicles were analyzed for the
presence of TfR by Western blot. As shown in
Fig 6, the amount of TfR associated with
exosomes was very similar. As previously described,2,20 the
dimeric form of TfR could be detected, even after electrophoresis in
reducing conditions. Similarly, a lower molecular weight band was
observed, probably resulting from TfR proteolysis.21,22
Moreover, the protein and phospholipid (PL) contents of the released
vesicles were similar (4.5 mg protein/mL and 1.2 mmol/L PL v
5.4 mg protein/mL and 1 mmol/L PL, for the PNH patient v
control, respectively). In contrast, as expected, AChE activity was
reduced (fivefold) in exosomes collected from PNH cells (0.028 U/mg
protein v 0.147 U/mg protein). When analyzed by flow cytometry,
this low-AChE phenotype was observed with PNH exosomes as compared with
exosomes from control patients (Fig 7).
Surprisingly, expression of other GPI-anchored proteins on the surface
of exosomes from PNH or control patients was very similar (DAF) or
identical (MIRL). To further analyze these results, we performed a
dose-response experiment using increasing exosome/bead ratios during
the coupling reaction. The amount of exosomes bound was quantified
biochemically using AChE activity and found to correspond perfectly to
the ratios used for the coupling reaction
(Fig 8). When the complexes were analyzed
by flow cytometry labeling with anti-CD59, increasing amounts of CD59
(shift of fluorescence peak) were detected with higher exosome/bead
ratios, although the relationship between these ratios and the
fluorescence peak shift was not linear.
GM1 is present in rat reticulocyte exosomes.
GM1 is not present in human RBCs, but is found in rat
erythrocytes.23 We thus analyzed the binding of
FITC-conjugated cholera toxin B subunit to exosomes. Vesicles from
human or rat reticulocytes were labeled with increasing amounts of
FITC-CTB (Fig 9). The shift in the
fluorescence peak was plotted as a function of the amount of added
FITC-CTB. As expected, human exosomes used as negative control did not
bind significant amounts of FITC-CTB. Conversely, a clear titration
curve was obtained with rat exosomes. GM1 was thus released in rat
reticulocyte exosomes, together with the TfR and GPI-anchored proteins.
Exosomes are mainly known as vesicles involved in the clearance of
obsolete proteins from the erythrocyte surface, such as the TfR. Other
transmembrane proteins are also sorted in these vesicles, but the
molecule sorting mechanism is still unknown. We have shown that the
sorting step could involve parameters such as the aggregation state of
molecules on the membrane surface, and also the nature of lipid
components of the bilayer. Indeed, we showed that molecule (TfR, AChE)
aggregation reroutes molecules towards exosomal release, instead of
recycling to the plasma membrane.2 Moreover, N-(lissamine
rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE), after
insertion into the plasma membrane of reticulocytes, was shown to be
selectively sorted from other lipid and accumulated in exosomes. We
proposed that such sorting is triggered by the presence of the
fluorescent analog in the membrane as small molecular clusters. As
natural lipids such as glycosphingolipids are also able to interact by
hydrogen bonding, the fate of N-Rh-PE in reticulocytes may represent
amplification of the physiologic route of specific membrane
lipids.24 Moreover, glycosphingolipids are known to be
molecular partners of lipid domains found in the trans-Golgi network or
caveolae,6,7 where proteins are specifically sorted. It has
been shown that GPI-anchored proteins are enriched in these domains.7 AChE is sorted in exosomes and as shown here,
sensitive to PI-PLC treatment, demonstrating its GPI-anchoring to the
exosomal membrane. We propose that the GPI-anchor could be a sorting
signal for AChE, and thus that other GPI-anchored proteins must also be
sorted in reticulocyte exosomes.
Submitted May 12, 1997;
accepted November 14, 1997.
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This article has been cited by other articles:
| ||||||||||
Abstract
Introduction
Abstract
) or not (
, 
) by
PI-PLC, were layered on 5% to 20% sucrose gradients containing (
-galactosidase (16S) and alkaline
phosphatase (6.1S).
).
4
