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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1784-1792
In Vitro Incorporation of GPI-Anchored Proteins Into Human
Erythrocytes and Their Fate in the Membrane
By
Gianluca Civenni,
Samuel T. Test,
Urs Brodbeck, and
Peter Bütikofer
From the Institute of Biochemistry and Molecular Biology, the
University of Bern, Bern, Switzerland; and Children's Hospital Oakland
Research Institute, Oakland, CA.
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ABSTRACT |
In many different cells, glycosylphosphatidylinositol (GPI)-anchored
molecules are clustered in membrane microdomains that resist extraction
by detergents at 4°C. In this report, we identified the presence of
such domains in human erythrocytes and examined the ability of
exogenously-added GPI-anchored molecules to colocalize with the
endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three
purified GPI-anchored proteins having different GPI lipid moieties
resulted in their efficient and correct incorporation into the
membrane. The extent of membrane insertion was dependent on the
intactness of the GPI lipid moiety. However, unlike the endogenous
GPI-anchored proteins, the in vitro incorporated GPI molecules were not
resistant to membrane extraction by Triton X-100 at 4°C.
In addition, in contrast to the endogenous GPI-anchored proteins, they
were not preferentially released from erythrocytes during vesiculation
induced by calcium loading of the cells. These results suggest that in
vitro incorporated GPI-linked molecules are excluded from pre-existing
GPI-enriched membrane areas in human erythrocytes and that these
microdomains may represent the sites of membrane vesicle formation.
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INTRODUCTION |
GLYCOSYLPHOSPHATIDYLINOSITOLS (GPIs)
serve as an important alternative mechanism for anchoring proteins to
cell membranes. They are synthesized by all eukaryotic cells examined
to date and anchor a wide variety of functionally diverse proteins to cell surfaces.1,2 In mammalian cells GPI-anchored proteins have been found to be involved in intracellular targeting,3 potocytosis,4 and signal transduction.5,6 All
these functions may relate to the reported preferential association of
GPI-anchored proteins with specialized membrane microdomains. These
microdomains are rich in glycolipids, sphingolipids, and cholesterol,
and resist solubilization by neutral detergents, such as Triton
X-100.7,8 However, whether such specialized
GPI-enriched microdomains involved in transmembrane signaling indeed
exist or are artifacts induced by the extraction procedure has remained
controversial.9-11
A composition reminiscent of the GPI membrane microdomains has been
reported for vesicles released from mammalian cells. Plasma membrane
vesiculation has been described to occur spontaneously in normal and
tumor cells in culture.12-18 The released vesicles seem to
be enriched in sphingomyelin and cholesterol,13,15,18 gangliosides,19 and several proteins such as
5 -nucleotidase13,15 and alkaline
phosphatase,17,18 which in most cells are now known to be
GPI-anchored. Similarly, release of vesicles can also be induced in
normal and pathological human erythrocytes.20-24 Although
in these cases the vesicles have a similar lipid composition as the
erythrocytes, they are highly enriched in the GPI-anchored proteins
acetylcholinesterase (AChE) and CD55 (decay accelerating factor).25
Recent evidence suggests that mammalian and parasite GPI-anchored
molecules may transfer spontaneously from donor to acceptor membranes.
In addition, it has been shown that purified GPI-linked molecules
readily incorporate in vitro into mammalian cells. The transferred
molecules seem to be stably inserted into the outer leaflet of the
plasma membrane via their acyl/alkyl chains on the GPI
moiety.26,27 In some cases, the in vivo transferred or in
vitro inserted GPI-anchored proteins displayed biological activity28-33 indicating that they integrated correctly
into the target cell membrane. Efficient incorporation of purified
GPI-anchored proteins into target membranes was shown to require the
intact GPI lipid moiety.34
The aim of our work was to study if GPI-anchored molecules inserted
into a target cell would colocalize with endogenous GPI-anchored proteins in detergent-insoluble microdomains. For this purpose we
incorporated three purified GPI-anchored proteins, having different GPI
lipid moieties, plus a set of molecules derived from them into human
erythrocytes and studied their fate during extraction with Triton
X-100. In addition, we followed their release from erythrocytes during
vesiculation to study if the in vitro incorporated GPI-anchored
molecules would be enriched in the released vesicles similar to the
endogenous GPI-anchored proteins.
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MATERIALS AND METHODS |
All reagents were of analytical grade and from Boehringer (Mannheim,
Germany), Fluka (Buchs, Switzerland), Sigma (St Louis, MO), or Merck
(Darmstadt, Germany). [1-3H]Ethan-1-ol-2-amine
hydrochloride ([3H]ethanolamine) was purchased from
Amersham (Buckinghamshire, UK) and
[3H]diisopropylfluorophosphate ([3H]DFP)
from Du Pont NEN (Regensdorf, Switzerland). GPI-PLD was purified from
bovine serum as described elsewhere.35
Phosphatidylinositol-specific phospholipase C (PI-PLC) from
Bacillus cereus was from Boehringer.
Erythrocytes.
Concentrated erythrocytes in standard anticoagulant buffer were
obtained from the ZLB Central Laboratory, Swiss Red Cross Blood
Transfusion Service (Bern, Switzerland). Erythrocytes were pelleted by
centrifugation and washed twice with 0.9% (wt/vol) NaCl.
GPI-anchored proteins.
Human erythrocyte AChE was purified by affinity chromatography as
described before.36 The final suspension contained purified AChE in 10 mmol/L Tris-HCl, pH 7.4; 144 mmol/L NaCl; and 0.05% (wt/vol) Triton X-100 (Fluka). The absolute amounts of
AChE in the assays were calculated based on its enzymatic activity with 1 IU equaling 2.5 pmol of protein. The AChE activity was measured according to Ellman et al.37 Purified AChE was then labeled covalently in the active site by incubation in the presence of [3H]DFP for 16 hours at room temperature. Unreacted DFP
was removed by extensive dialysis.
[3H]Ethanolamine-labeled variant surface glycoprotein
(VSG) from Trypanosoma brucei bloodstream forms was prepared
following the procedure of Hereld et al.38 The resulting
suspension contained radiochemically pure VSG in 1% (wt/vol) sodium
dodecyl sulfate (SDS).
[3H]Ethanolamine-labeled procyclin from Trypanosoma
brucei brucei 427 insect forms was prepared exactly as described by
Bütikofer et al.39 The surface of insect form
trypanosomes is covered by an invariant protein coat consisting of
GPI-anchored procyclins. T b brucei 427 cells express two
different forms of procyclin, the so-called EP and GPEET procyclins
consisting either of extensive tandem repeat units of glutamic acid (E)
and proline (P) or internal pentapeptide (GPEET) repeats. Thus, the
[3H]thanolamine-labeled extract from T b brucei
427 cells contains a mixture of radiochemically pure EP and GPEET
procyclin in 9% (vol/vol) butan-1-ol in water.39
All proteins were subsequently purified by octyl-Sepharose
chromatography exactly as described before.39 The resulting
specific radioactivities of the three protein preparations were 66,265 cpm/µg AChE, 737 cpm/µg VSG, and 15,416 cpm/µg procyclin.
Enzymatic and chemical treatment of GPI-anchored proteins.
The purified [3H]-labeled proteins were modified in the
GPI or protein portion by the following procedures: (1) GPI-PLD
treatment: [3H]-labeled AChE, VSG, and procyclin were
incubated with GPI-PLD in 50 mmol/L
2-morpholino-ethanesulfonic acid monohydrate (MES), pH 6.5, containing
0.5 mmol/L CaCl2 and 0.02% (wt/vol) Triton X-100, for 16 to 24 hours at 37°C40; (2) mild base treatment:
[3H]-labeled AChE, VSG, and procyclin were treated with
mild base (50 mmol/L NaOH in 90% [vol/vol] ethanol) for 2 hours at
37°C to remove ester-linked fatty acids41; (3) Pronase
treatment: [3H]-labeled VSG and procyclin were treated
with Pronase (5 mg/mL final concentration) in 50 mmol/L Tris-HCl, pH
7.5, containing 5 mmol/L CaCl2, for 16 hours at 37°C.
The radiolabeled reaction products were repurified by octyl-Sepharose
chromatography as described above. Fractions containing radioactivity
were pooled and dried in a speed vacuum system.
Incorporation of [3H]-labeled AChE, VSG, and procyclin
into human erythrocytes.
Human erythrocytes were washed twice with incubation buffer (10 mmol/L
Tris-HCl, pH 7.4, containing 144 mmol/L NaCl, 0.54 mmol/L adenine, 12.7 mmol/L inosine, and 2 g/L glucose) and resuspended in the same buffer
at a hematocrit level of 16%. Subsequently, [3H]-labeled
AChE (2,000 cpm/mL), VSG (2,000 cpm/mL), procyclin (2,500 cpm/mL), or
the [3H]-labeled products derived from these proteins
(1,000 cpm/mL), were added to the erythrocytes and the suspension was
incubated for 1 hour at 37°C. After centrifugation for 5 minutes at
800g and at 10°C, the erythrocytes were washed
twice with cold incubation buffer and the radioactivity in the
supernatants was counted.
Treatment of erythrocytes with GPI-hydrolyzing phospholipases.
After incorporation of [3H]-labeled GPI-anchored
proteins, erythrocytes were treated with 4.5 IU/mL GPI-PLD or 0.1 IU/mL
PI-PLC for 3 hours at 37°C40 and the release of
radioactivity from erythrocytes was determined in the supernatant after
pelleting the cells.
Preparation of ghosts.
Erythrocyte ghost membranes were prepared according to the method of
Dodge et al.42
Detergent extraction of erythrocyte ghosts.
Erythrocyte ghost membranes (0.5 mL) were incubated with 9.5 mL of
extraction buffer (25 mmol/L HEPES, pH 7.5; 150 mmol/L NaCl; and 1%
[wt/vol] Triton X-100) at 4°C or 37°C for 20 minutes followed by
centrifugation at 12,000 rpm for 10 minutes at 4°C using a Sorvall
SS-34 rotor (Du Pont Instruments, Wilmington, DE). The
supernatant was saved and the pellet was resuspended in 200 µL water
by sonication.
Density gradient centrifugation.
Detergent-insoluble fractions were layered on top of 20% to 40%
(wt/vol) sucrose gradients (12-mL gradients with a 0.5-mL cushion of
60% [wt/vol] sucrose at the bottom) and centrifuged at 36,000 rpm
for 15 hours at 4°C using a Centricon TST 41.14 rotor
(Kontron Ltd, Zurich, Switzerland). After centrifugation, fractions of
0.4 mL were collected from the bottom and aliquots were removed for
determination of protein, AChE activity, and radioactivity.
Vesiculation of human erythrocytes.
Human erythrocytes can be induced to vesiculate by loading the cells
with calcium21 or by depletion of their intracellular adenosine triphosphate (ATP) stores.20 The release of
membrane vesicles from erythrocytes can be monitored by following the
increase in AChE activity in the cell-free supernatant after pelleting the cells.20,21,25 Calcium-induced and ATP
depletion-induced vesiculation of human erythrocytes was performed
exactly as described by Bütikofer et al.25 Briefly,
for calcium loading, washed cells (16% hematocrit) were equilibrated
in buffer (10 mmol/L Tris-HCl, pH 7.4; 144 mmol/L NaCl; and 2 g/L
glucose) containing 1 mmol/L CaCl2 for 3 minutes at 37°C.
Subsequently, the calcium ionophore A23187 was added to the suspension
from a 10 mmol/L stock solution in ethanol to give a final
concentration of 4 µmol/L, and the incubation was carried out for 1 hour at 37°C. Vesicle release was stopped by the addition of EDTA (12 mmol/L, final concentration) to the suspension. ATP depletion of
erythrocytes was performed by incubating washed cells at a hematocrit
level of 20% in buffer (10 mmol/L Tris-HCl, pH 7.4; 144 mmol/L NaCl; 1 mmol/L EDTA; 0.5 mmol/L adenine; 2 × 105 IU/L
penicillin; and 1.5 × 105 IU/L streptomycin) at 37°C.
After 4 to 6 hours of incubation, the erythrocytes were pelleted and
the pH in the supernatant was readjusted to pH 7.4. Then the cells were
resuspended and the incubation was continued for 40 hours under
constant gentle shaking.
Isolation of vesicles.
At the end of the incubation, erythrocytes were pelleted by
centrifugation for 5 minutes at 1,800 rpm and at 10°C
and the vesicle-containing supernatant was collected. Vesicles were
pelleted by centrifugation in a centrifuge (Micro Centaur,
Loughborough, UK) for 20 minutes at 18,000 rpm and at 4°C and washed
three times with large volumes of buffer (10 mmol/L
Tris-HCl, pH 7.5; and 2 mmol/L EDTA).
Purification and iodination of monoclonal anti-human CD59 antibody.
YTH 53.1 rat monoclonal anti-human CD59 hybridoma cells were kindly
provided by Dr Ethan Shevach (National Institutes of Health, Bethesda,
MD). The antibody was purified from tissue culture supernatants by
ammonium sulfate precipitation followed by chromatography on DEAE-Sephacel (Pharmacia Biotech, Uppsala, Sweden). The purified antibody was iodinated with 125I as NaI (Amersham Corp,
Arlington Heights, IL) by using Iodogen (Pierce Chemical Co, Rockford,
IL). The specific activity of the final preparation was 7.25 × 105 cpm/µg and 98.4% of the 125I was
protein-bound as determined by precipitation with 10% trichloroacetic acid.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
electroblotting of detergent-extracted erythrocyte membranes.
SDS-PAGE and electroblotting were performed using a Mini-PROTEAN II
electrophoresis system and Mini Trans-Blot Cell (Bio-Rad Laboratories,
Hercules, CA). Aliquots of detergent-extracted erythrocyte membranes
containing 20 µg of protein were run on 12% SDS-PAGE gels under
nonreducing conditions and transferred electrophoretically to
polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories) at 100 V (0.25 A limit) for 1 hour in transfer buffer (25 mmol/L Tris, pH 8.5;
192 mmol/L glycine; and 20% [vol/vol] methanol). Completeness of transfer was assessed by silver staining the gels. No
bands were visible at the predicted apparent molecular mass of CD59 (18 to 20 kD).
Quantitation of CD59 on electroblots of detergent-extracted
erythrocyte membranes.
After transfer of protein onto PVDF membranes, the blots
were incubated twice with blotting buffer (25 mmol/L Tris, pH 7.5; 137 mmol/L NaCl; 2.7 mmol/L KCl; 0.05% [vol/vol] Tween-20) containing 5% instant nonfat milk powder for 1 hour at room temperature. The blot
then was incubated with 100 µg of 125I-labeled anti-CD59
in 8 mL of blotting buffer for 1 hour at room temperature, washed six
times with the same buffer, and air dried. 125I bound to
the blot was quantitated by exposing a storage phosphor screen
(Molecular Dynamics, Sunnyvale, CA) to the blot for 16 hours at room
temperature, imaging the exposed screen with a PhosphorImager SF
(Molecular Dynamics), and analyzing the CD59 bands on the image of the
blot using Molecular Dynamics ImageQuant software. To determine the
linearity of transfer of CD59, aliquots of untreated erythrocyte membranes were run on gels simultaneously with the detergent-extracted membranes. Transfer of CD59 (as determined by probing blots with 125I-labeled anti-CD59) was linear for membrane aliquots
containing from 1.25 to 40 µg of protein.
Protein determination.
Protein was determined using the BCA reagent kit (Pierce Chemical Co)
with bovine serum albumin as standard. Because erythrocyte-derived vesicles contain high amounts of hemoglobin and are resistant to
hypotonic lysis, the amount of membrane protein in vesicles was
determined by subtracting the amount of hemoglobin43 from the total amount of protein.
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RESULTS |
Preparation of GPI-anchored proteins and derivatives.
To study the incorporation of GPI-anchored proteins into human
erythrocytes, we purified several GPI-anchored proteins from different
sources and modified their GPI lipid moieties and protein parts by
enzymatic and chemical treatment (Fig 1).[3H]DFP-labeled AChE from human erythrocytes, and
[3H]ethanolamine-labeled VSG and procyclin from T
brucei bloodstream and insect forms, respectively, were treated
with GPI-PLD to remove the glycerol-bound lipid components from the GPI
anchors. The GPI-PLD reaction products were separated from unreacted
material by octyl-Sepharose chromatography. Intact GPI-anchored AChE,
VSG, and procyclin bound to the column material and eluted after
increasing the 1-propanol concentration from 5% to 40%
(vol/vol)39,44 (Fig 2A). In
contrast, the GPI-PLD-treated proteins no longer bound to the
octyl-Sepharose and were recovered in the flow-through of the column
(Fig 2A). The removal of the glycerol-bound lipid components by GPI-PLD
treatment renders the GPI anchor of VSG completely hydrophilic, whereas
the GPI moieties of AChE and procyclin still contain a fatty acid
attached to the inositol (Fig 1). Treatment of GPI-anchored proteins
with mild base results in the removal of ester-bound fatty acids from
the GPI moiety.41 In the case of VSG and procyclin, base
treatment removed all hydrophobic components from the GPI structures,
whereas in the case of AChE, the long chain alkyl group remained
attached to the glycerol (Fig 1). To generate radiolabeled GPI
structures containing a small peptide or a single amino acid attached
to an intact GPI moiety, purified [3H]ethanolamine-labeled VSG and procyclin were treated
with large amounts of Pronase to digest the protein portion attached to
the GPI anchor. We found that treatment of procyclin with Pronase decreased the apparent molecular masses of the EP and GPEET forms of
procyclin from 42 kD and 22 to 32 kD, respectively, to a single broad
band of 17 to 22 kD (Fig 2B). This band has been shown to represent the procyclin GPI anchor with only a single amino acid attached to it.39 Similarly, treatment of VSG with Pronase
resulted in a significant reduction of its apparent molecular mass from 55 kD to < 6 kD (Fig 2B), indicating that most of the protein part
was removed. Because of the limited supply of material, the exact
extent of degradation by Pronase (ie, the N-terminal amino acid of the
VSG fragment after Pronase treatment) was not determined. The
[3H]-labeled material obtained after Pronase treatment of
VSG and procyclin was rechromatographed on an octyl-Sepharose column. The GPI-linked structures were found to bind to the column material and
eluted after increasing the 1-propanol concentration from 5% to a
concentration of 25% to 30% (vol/vol) (results not
shown).39,44
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| Fig 1.
Schematic representation of the GPI anchors of AChE from
human erythrocytes, VSG from T brucei bloodstream forms,
and procyclin from T brucei insect forms. The purified proteins
were incorporated into human erythrocytes with their intact GPI
anchors, or after (partial) removal of the lipid moieties by GPI-PLD or
NaOH treatment.
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| Fig 2.
Purification and characterization of GPI-anchored
proteins. (A) Delipidated SDS extracts from bloodstream form
trypanosomes containing [3H]-labeled VSG before ( ) and
after ( ) treatment with GPI-PLD were applied to an octyl-Sepharose
column (2 mL bed volume) previously equilibrated in 100 mmol/L ammonium
acetate containing 5% (vol/vol) 1-propanol. The column was washed with
5 mL of the same buffer (fractions 1 through 10) followed by 5 mL of
100 mmol/L ammonium acetate containing 20% (vol/vol) 1-propanol
(fractions 11 through 20), and was eluted with a linear gradient of
20% to 40% (vol/vol) 1-propanol in 100 mmol/L ammonium acetate
(fractions 21 through 40). Fractions of 0.5 mL were collected and
aliquots were counted for radioactivity. Intact VSG eluted at 31%
1-propanol. After treatment with GPI-PLD, VSG eluted in the column
flow-through. Intact procyclin (P) and AChE (A) eluted at 25% and 35%
1-propanol, respectively (as indicated by the arrows), whereas
GPI-PLD-treated procyclin and AChE eluted in the same fractions as
GPI-PLD-treated VSG. The actual 1-propanol concentration in an
individual fraction was determined by measuring the refractive index.
(B) Purified radiolabeled VSG and procyclin were treated with large
amounts of Pronase to completely digest the protein portions of the
molecules followed by repurification by octyl-Sepharose chromatography. Control untreated VSG (lane a) and procyclin (lane c) and the corresponding Pronase reaction products (lanes b and d, respectively) were analyzed by SDS-PAGE and autoradiography.
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Incorporation of GPI-anchored proteins and derivatives into human
erythrocytes.
Several purified GPI-anchored proteins have been shown to spontaneously
incorporate in vitro into mammalian cells. However, their rate and
extent of incorporation differs considerably depending on the system
used.26,27 In addition, there is inconsistency on whether
the exogenously added proteins become functional within the target cell
membrane.33,45 In the present work, the availability of
three different purified GPI-anchored proteins plus a set of well-defined structures derived from them, allowed us to systematically study the influence of different GPI anchor and protein components on
the incorporation of GPI-anchored proteins into erythrocytes.
In a first set of experiments, erythrocytes were incubated in the
presence of the intact [3H]-labeled GPI-anchored proteins
AChE, procyclin, and VSG. Our results showed that after 1 hour of
incubation at 37°C, 44% ± 14% of AChE, 68% ± 5% of procyclin,
and 67% ± 3% of VSG added to the suspension could not be removed
from erythrocytes by repetitive washing (Fig 3A, B, and
C). In contrast, when the
[3H]-labeled GPI-anchored proteins were incubated with
the cells at 4°C, less than 4% of radioactivity was incorporated.
Longer incubation times did not result in increased amounts of
radioactivity in the erythrocyte pellets (results not shown). When the
washed [3H]-labeled cells were subsequently incubated for
7 hours at 37°C, less than 2% of radioactivity was released into the
cell-free supernatant after pelleting the cells, indicating that
the radiolabeled proteins were stably associated with the erythrocytes.
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| Fig 3.
Incorporation of exogenously added GPI-anchored proteins
into human erythrocytes. Erythrocytes were incubated for 1 hour at 37°C in the presence of purified [3H]-labeled AChE (A),
procyclin (B), and VSG (C), and thoroughly washed to remove
nonincorporated material. The total radiolabel in the wash supernatants
was counted and used to calculate the amounts of label incorporated
into erythrocytes. The figure shows the relative incorporation of
intact (bars a), GPI-PLD-treated (bars b), mild base-treated (bars c),
and Pronase-treated (bars d) GPI-anchored proteins. The numbers
represent the mean values ± SD from n independent experiments with
the number of experiments shown in parentheses. *, Not determined.
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When we treated the GPI-anchored proteins with GPI-PLD before their
addition to erythrocytes, we found that they no longer incorporated
into the cells (Fig 3A, B, and C). This result indicates that the
removal of the glycerol moiety with one (procyclin) or two (AChE and
VSG) fatty acyl or alcohol chains prevents incorporation of the protein
into erythrocytes. Furthermore, the results show that the presence of a
single fatty acid attached to the inositol (AChE and procyclin) is not
sufficient to stably incorporate the protein into the cells. Similarly,
we found that when all hydrophobic components of the GPI anchors of
procyclin and VSG were removed by mild base treatment, the proteins no
longer incorporated into erythrocytes (Fig 3B and C). In contrast, mild
base-treated AChE still bound to the cells, although much less
efficiently than the intact protein (Fig 3A). Because the ether-bound
alkyl chain of AChE is not removed by base treatment, this finding
indicates that a single long chain alkyl substituent on the glycerol
moiety is sufficient to mediate incorporation of the protein into
cells. When the protein parts of procyclin and VSG were removed by
extensive proteolysis, the residual GPI-anchored structures were found
to readily incorporate into erythrocytes (Fig 3B and C). However, while
the extent of incorporation of the procyclin GPI anchor was only
slightly decreased compared with the intact protein, incorporation of
the VSG anchor was reduced by greater than 85%. It is possible that
the different levels of incorporation of the two GPI anchor structures
may relate to the different carbohydrate substituents present on the
conserved GPI glycan backbones. VSG GPI anchors have been shown to
contain very few extra carbohydrates attached to the GPI
tetrasaccharide core46 and thus may form micellar
structures when added to aqueous media and not readily incorporate into
acceptor membranes. In contrast, the procyclin GPI anchors contain very
large and complex glycosylated side chains44,47 that may
prevent self-association and thus facilitate insertion into membranes.
Treatment of erythrocytes with GPI-hydrolyzing phospholipases.
After incorporation of [3H]-labeled GPI-anchored
proteins, when the erythrocytes were treated with GPI-PLD for 3 hours
at 37°C, less than 3% of incorporated radioactivity was released
from the cells irrespective of the type of incorporated protein.
Similarly, erythrocytes loaded with [3H]-labeled AChE or
procyclin showed no release of radioactivity after treatment with
bacterial PI-PLC. In contrast, PI-PLC treatment of erythrocytes loaded
with [3H]-labeled VSG resulted in the release of greater
than 50% of radioactivity after 3 hours of incubation at 37°C,
whereas mock-treated erythrocytes released less than 2% of
radioactivity. Hemolysis under these conditions was less than 1%.
Isolation of Triton X-100-insoluble fractions from human
erythrocytes and partitioning of endogenous proteins.
In many different cell types GPI-anchored proteins have been found to
partition preferentially into Triton X-100-insoluble fractions.8 To determine if detergent-resistant complexes
are also present in human erythrocytes and if endogenous GPI-anchored proteins are enriched in these fractions, we analyzed the Triton X-100-insoluble fractions from erythrocyte ghosts for the presence of
the endogenous GPI-anchored proteins AChE and CD59 (also known as
membrane inhibitor of reactive lysis). We found that 47.2% ± 2.5%
of total membrane protein (mean ± SD from six independent experiments) resisted extraction by Triton X-100 at 4°C. This number
is in good agreement with a previous report showing that after
extraction of human erythrocyte ghost membranes with cold 0.1% Triton
X-100, approximately 45% of total protein was recovered in the
detergent-insoluble residue.48 Protein analysis by SDS-PAGE showed that the Triton X-100-insoluble fraction from erythrocytes contained most of the major skeletal proteins, ie, spectrin, ankyrin, protein 4.1, and actin; whereas band 3, ie, the major integral membrane
protein, together with protein 4.2 and band 6, were preferentially present in the soluble fraction (results not shown).48 In
addition, we found that the endogenous GPI-anchored proteins, AChE and
CD59, were enriched 1.69- and 1.55-fold, respectively, in the Triton X-100-insoluble fractions relative to bulk protein (Table
1). The relative enrichment of AChE in the
Triton X-100-insoluble residue was unchanged after purification by
sucrose density gradient centrifugation, after which the material was
found to band at 31% (wt/vol) sucrose (result not shown)
as compared with the detergent-insoluble material from other membranes
which has been found to band at 15% to 25% (wt/vol)
sucrose.6,7 In contrast, when the detergent extraction was
carried out at 37°C, very little AChE and CD59 were recovered in the
Triton X-100-insoluble residue which represented 20.3% ± 2.8% of
total membrane protein (mean ± SD from three independent experiments) (Table 1).
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Table 1.
Partitioning of Endogenous and Exogenous GPI-Anchored
Proteins in Triton X-100-Insoluble Fractions of Erythrocyte Ghost
Membranes
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Partitioning of in vitro incorporated GPI-anchored proteins in Triton
X-100-insoluble fractions.
To study the partitioning of exogenously added GPI-anchored proteins in
detergent-resistant fractions, [3H]-labeled AChE,
procyclin and VSG, as well as the procyclin and VSG GPI anchor
structures, were incorporated into erythrocytes and their distribution
in Triton X-100-resistant fractions at 4°C was measured. Our results
show that the specific radioactivity of all exogenously added
radiolabeled GPI-anchored proteins in the detergent-resistant complexes
was decreased compared with ghost membranes (Table 1). Thus, in
contrast to endogenous AChE and CD59, the incorporated GPI-anchored
structures were depleted from Triton X-100-insoluble complexes.
Interestingly, the procyclin and VSG GPI anchors were depleted more
severely (fivefold) from the detergent-resistant complexes than the
intact proteins (<1.7-fold). Because it has been reported that
exogenous GPI-anchored proteins may redistribute into
detergent-resistant fractions after equilibration in the
membrane,33 we incubated erythrocytes preloaded with radiolabeled procyclin or procyclin GPI anchor for 24 hours at 37°C
and determined the distribution of radioactivity in the Triton X-100-insoluble fractions. Our results showed that the specific radioactivity in the detergent-resistant fractions from these equilibrated erythrocytes was unchanged compared with extracts from
freshly labeled cells (Table 1).
Release of GPI-anchored proteins during vesiculation of erythrocytes.
It has been shown before that the endogenous GPI-anchored proteins,
AChE, CD55, and CD59, are preferentially released from human
erythrocytes by vesiculation.20,21,25 As a result of this
process, GPI-anchored proteins are depleted in the remnant cells and
highly enriched in the released vesicles. To study whether GPI-anchored
proteins incorporated in vitro would behave similarly, we followed the
release of incorporated [3H]-labeled proteins from
erythrocytes during Ca2+-induced vesiculation. Our results
showed that during 1 hour of Ca2+-loading, the in vitro
incorporated VSG was progressively released from erythrocytes together
with the endogenous AChE (Fig 4). However, while we typically observed a release of 30% to 40% of total
endogenous AChE, only 10% to 15% of incorporated VSG was shed from
erythrocytes. A similar release was also observed for incorporated AChE
and procyclin (results not shown). When vesiculation was induced 2 hours or 7 hours after incorporation of GPI-anchored proteins into
erythrocytes, the amounts of radiolabel released into the cell-free
supernatant after pelleting the cells were similar (results not shown).
In contrast, less than 1% of the radiolabel was released from
erythrocytes loaded with the radiolabeled GPI anchors of VSG (Fig 4)
and procyclin (result not shown). Our observation that greater than
94% of radioactivity and greater than 96% of AChE activity in the
vesicle-containing supernatants could be pelleted by high speed
centrifugation shows that the GPI-anchored proteins were firmly
associated with the vesicles.
View larger version (20K):
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| Fig 4.
Release of endogenous AChE and in vitro incorporated VSG
from human erythrocytes during Ca2+-induced vesiculation.
[3H]-labeled GPI-anchored molecules were incorporated
into erythrocytes as described in Fig 3 and the release of membrane
vesicles was induced by loading the cells with calcium using the
ionophore A23187. At designated times, erythrocytes were pelleted and
the AChE activity () and the radioactivity ( ,  ) were measured
in the vesicle-containing supernatants of cells labeled with in vitro incorporated VSG () and Pronase-treated VSG (). The values
represent single determinations from a typical experiment;
corresponding results were obtained with in vitro incorporated AChE,
procyclin, and Pronase-treated procyclin.
|
|
Furthermore, in agreement with a previous report,25 we
found that the endogenous AChE was highly enriched (4.9-fold relative to bulk membrane protein) in the released vesicles compared with untreated cells (Table 2). In contrast, the
relative enrichment of [3H]-labeled AChE, VSG, and
procyclin in the vesicles was 0.9- to 1.7-fold compared with untreated
ghost membranes (Table 2). Similar results were obtained when the
release of vesicles was induced by ATP-depletion of erythrocytes
(results not shown). These findings show that in contrast to endogenous
AChE and CD55,25 and CD59,49 the exogenously
added GPI-anchored proteins are not preferentially released from
erythrocytes during vesiculation.
| |
DISCUSSION |
Incorporation of purified GPI-anchored molecules into plasma membranes
in vitro provides a general means for modifying cell surfaces with
exogenously added proteins.26,27 It has been reported that
GPI-anchored molecules may incorporate directly into GPI-enriched
microdomains50 or alternatively, insert randomly and
diffuse laterally in the plane of the plasma membrane until they reach
microdomains rich in GPI-linked structures.33 The reported
biological activity of some of the incorporated GPI-linked molecules is
taken as evidence for their correct integration into the target cell
membrane. However, it is not clear if the correct localization and
functionality of an inserted GPI-anchored molecule in the membrane is
typical for certain proteins or if different exogenously added
GPI-anchored proteins would colocalize in the same microdomains.
In the present work we incorporated the GPI-anchored proteins AChE,
VSG, and procyclin into human erythrocytes and studied their fate in
the membrane. The use of a series of structures derived from these
proteins having different GPI lipid moieties allowed us to study the
roles of the lipid and protein parts of the molecules during
incorporation into target cells. We found that efficient incorporation
of GPI-anchored proteins into human erythrocytes only occurred when the
GPI lipid moiety was intact. These results are in good agreement with a
previous study using modified forms of CD55 incorporated into sheep
erythrocytes.34 The reasons for the considerably higher
extent of incorporation of GPI-anchored proteins into erythrocytes
observed in our work (40% to 60%) compared with the earlier study
(1% to 3%)34 are not known. In addition, our results
showed that the incorporated proteins could not be released from the
cells by GPI-PLD treatment which is consistent with the reported
inability of the enzyme to cleave membrane-bound GPI-anchored
proteins.51 Similarly, because PI-PLC is known to be
inactive on inositol-acylated GPI-linked structures,52
incorporated AChE and procyclin could not be released from the
erythrocytes by PI-PLC treatment, whereas the in vitro incorporated VSG
with its PI-PLC-sensitive GPI anchor was readily released. Together
with the observation that the incorporated proteins remained associated
with the cells during prolonged incubation at 37°C, these results
show that the exogenously added GPI-anchored proteins inserted stably
and in a correct orientation into erythrocyte membranes. The view that
membrane insertion is mediated primarily by the GPI lipid moiety is
further supported by our finding that the isolated procyclin and VSG
GPI anchors (obtained by Pronase treatment of procyclin and VSG,
respectively) also readily incorporated into erythrocytes. The
observation that the free GPI anchors were less efficiently inserted
into the membrane than the intact proteins suggests that the protein
portions may play an important role in facilitating incorporation of
GPI-linked molecules into membranes, possibly by decreasing their
ability to form micellar structures.
Interestingly, we found that a single fatty acyl chain attached to the
inositol was unable to insert the protein into the target cell
membrane, whereas a single alkyl chain on the glycerol was sufficient
to mediate incorporation. This finding contradicts an earlier report
showing that inositol-acylated CD55 after GPI-PLD treatment was able to
insert into sheep erythrocytes, although to a much smaller extent than
the intact protein.34 This apparent discrepancy may be
explained by the purity of the protein preparation. While in our study
the GPI-PLD-treated proteins were separated from nonreacted material
by octyl-Sepharose chromatography, the GPI-PLD-treated CD55 in the
earlier study may have been contaminated with residual amounts of
intact protein.
Treatment of human erythrocytes with Triton X-100 at 4°C has been
reported to result in the solubilization of most of the major integral
and cell surface proteins.48 In contrast, most skeletal
proteins (ie, >40% to 50% of total protein) together with greater
than 80% of sphingomyelin were retained in the detergent-insoluble residue. In the present report we found that under the same conditions the majority of the endogenous GPI-anchored proteins, AChE (>75%) and CD59 (>55%), also resisted extraction by Triton X-100. The two
proteins were enriched 1.5- to 1.7-fold relative to bulk protein in the
detergent-resistant complexes compared with untreated ghost membranes.
In contrast, when erythrocytes loaded with the exogenously added
radiolabeled AChE, VSG, or procyclin were extracted with Triton X-100
at 4°C, the incorporated proteins were found not to be enriched in
the detergent-resistant complexes. In fact, the relative specific
radioactivities of the incorporated proteins in Triton X-100-insoluble
complexes were lower than in intact membranes showing that, in contrast
to the endogenous GPI-anchored proteins, the inserted proteins were
mostly solubilized by Triton X-100 at 4°C. In addition, the
solubility in Triton X-100 of the incorporated GPI-anchored molecules
did not change during incubation of erythrocytes at 37°C, indicating
that the proteins did not undergo time-dependent redistribution into
detergent-insoluble domains, as has been shown to occur for CD59
incorporated into U937 monocytic cells.33
Vesicles released from human erythrocytes have been found to be
enriched in several endogenous GPI-anchored proteins, as we and
others20-22,25 have shown. Although the
process of membrane vesiculation has been shown to involve the
detachment of the skeletal protein network from the
membrane,23,53 the exact mechanism leading to a release of
GPI-enriched vesicles has not been elucidated. It has been proposed
that the reported increased lateral mobility of some GPI-anchored
proteins compared with transmembrane proteins54-56 may
allow them to (rapidly) move into the membrane spicules that are formed
before vesicle release.53 However, we now found that the
incorporated [3H]-labeled GPI-anchored proteins, AChE,
VSG, and procyclin, were not enriched in the released vesicles,
indicating that an increased lateral mobility of GPI-anchored proteins
per se cannot account for their enrichment in the vesicles.
Alternatively, our results suggest that GPI-enriched microdomains may
pre-exist in the erythrocyte membrane and represent the sites of
membrane vesicle formation from which exogenously added GPI-anchored
proteins are excluded. Similar to the situation in other
cells,8 such GPI-enriched microdomains in human
erythrocytes may also contain other non-GPI-linked membrane proteins.
One of these proteins may be the complement receptor 1 (C3b receptor,
CD35), a transmembrane glycoprotein, which has been shown to be
preferentially lost from human erythrocytes during vesiculation induced
by ATP-depletion.57 The unique composition of the
microdomains released from human erythrocytes during vesiculation may
have important physiological consequences because vesiculation has been
reported to be involved in the aging process of human erythrocytes both
in vivo and in vitro.23,58-60
Interestingly, when we incorporated the VSG and procyclin GPI anchors
instead of the intact proteins into erythrocytes, we found that they
were almost completely absent from the detergent-resistant complexes.
This indicates that the described insolubility of membrane-bound GPI-anchored proteins in Triton X-100 at 4°C results from
interactions of the GPI-linked molecules with detergent-insoluble
membrane components and not from a (partial) insolubility of the GPI
anchor. In addition, we found that the incorporated VSG and procyclin GPI anchors were not released from erythrocytes during vesiculation, indicating that the GPI anchor by itself does not incorporate, or
relocate, into the GPI-rich membrane microdomains that are involved in
vesiculation.
In summary, our results show that purified GPI-anchored proteins added
to human erythrocytes readily incorporate into the plasma membrane in a
correct orientation. However, unlike the endogenous GPI-anchored
proteins, the exogenously added GPI molecules are readily solubilized
by Triton X-100 at 4°C. In addition, they are not released from
erythrocytes together with the endogenous GPI-anchored proteins during
vesiculation. Thus, although in some cases exogenously added GPI-linked
molecules have been shown to be biologically functional within the
acceptor cell membrane,28,33,61 they may not necessarily
colocalize with the endogenous GPI-anchored proteins in specific
microdomains. Alternatively, it is possible that only a small fraction
of incorporated GPI-anchored molecules partitions into the pre-existing
GPI-rich domains but that this fraction may be sufficient to infer a
biological activity.
The recent development of novel methods to study the lateral mobility
of membrane proteins has led to a revision of the fluid mosaic model
initially proposed by Singer and Nicolson.62
The `transient interaction model'45,63 and the
`membrane-skeleton fence model'64 propose that the
observed confinement of a protein in the plane of a membrane may result
from it being transiently trapped by the membrane-apposed skeletal
protein network or by direct interactions with proteins bound to the
skeleton. In addition, the lateral mobility of a GPI-anchored protein
may be restricted by its association with specific lipid
microdomains.63-65 Our observation that exogenously added
GPI-anchored molecules are unable to redistribute into pre-existing GPI-rich membrane microdomains may be explained by either model. Of
course, these models are not mutually exclusive and it is likely that
combinations of them exist. In addition, these models also offer a
possible mechanism for the shedding of vesicles from erythrocytes. Because the released vesicles are enriched in GPI-anchored proteins and
devoid of skeletal proteins, vesicle formation likely occurs at
specific sites in the membrane. Such a proposed initiation point for
vesiculation may be identical with the above mentioned `fenced' areas
rich in GPI-anchored proteins and explain why exogenously added
GPI-anchored molecules are not enriched in the released vesicles.
| |
FOOTNOTES |
Submitted July 28, 1997;
accepted October 20, 1997.
Supported by Swiss National Science Foundation Grants No. 31-039209.93 and 31-049458.96 and by National Institutes of Health Grant No. HL
20985.
Address reprint requests to Peter Bütikofer, PhD, Institute of
Biochemistry and Molecular Biology, University of Bern,
Bühlstrasse 28, CH-3012 Bern, Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank the ZLB Central Laboratory, Swiss Red Cross Blood Transfusion
Service (Bern, Switzerland) for supplying fresh human erythrocytes. We
also thank Joyce Mitsuyoshi and Monika Boschung for excellent technical
assistance during parts of the study and Tracy Chapman for stimulation.
| |
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Abstract
Introduction
Abstract