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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1784-1792
By
From the Institute of Biochemistry and Molecular Biology, the
University of Bern, Bern, Switzerland; and Children's Hospital Oakland
Research Institute, Oakland, CA.
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.
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 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.
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.
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.
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.
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
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.
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).
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.
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.
Submitted July 28, 1997;
accepted October 20, 1997.
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.
1.
Englund PT:
The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.
Annu Rev Biochem
62:121,
1993[Medline]
[Order article via Infotrieve]
2.
McConville MJ,
Ferguson MAJ:
The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes.
Biochem J
294:305,
1993
3.
Rodriguez-Boulan E,
Powell SK:
Polarity of epithelial and neuronal cells.
Annu Rev Cell Biol
8:395,
1992
4.
Anderson RGW,
Kamen BA,
Rothberg KG,
Lacey SW:
Potocytosis: Sequestration and transport of small molecules by caveolae.
Science
255:410,
1992
5.
Robinson PJ:
Phosphatidylinositol membrane anchors and T-cell activation.
Immunol Today
12:35,
1991[Medline]
[Order article via Infotrieve]
6.
Lisanti MP,
Scherer PE,
Tang ZL,
Sargiacomo M:
Caveolae, caveolin and caveolin-rich membrane domains: A signalling hypothesis.
Trends Cell Biol
4:231,
1994 [Medline]
[Order article via Infotrieve]
7.
Brown DA,
Rose JK:
Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.
Cell
68:533,
1992[Medline]
[Order article via Infotrieve]
8.
Simons K,
Ikonen E:
Functional rafts in cell membranes.
Nature
387:569,
1997[Medline]
[Order article via Infotrieve]
9.
Mayor S,
Rothberg KG,
Maxfield FR:
Sequestration of GPI-anchor proteins in caveolae triggered by cross-linking.
Science
264:1948,
1994
10.
Schnitzer JE,
McIntosh DP,
Dvorak AM,
Liu J,
Oh P:
Separation of caveolae from associated microdomains of GPI-anchored proteins.
Science
269:1435,
1995
11.
Liu J,
Oh P,
Horner T,
Rogers RA,
Schnitzer JE:
Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains.
J Biol Chem
272:7211,
1997
12.
Ruoslahti E,
Vaheri A:
Novel human serum protein from fibroblast plasma membrane.
Nature
248:789,
1974[Medline]
[Order article via Infotrieve]
13.
Scott RE:
Plasma membrane vesiculation: A new technique for isolation of plasma membrane.
Science
194:743,
1976
14.
Anglin JH,
Lerner MP,
Nordquist RE:
Blood group-like activity released by human mammary carcinoma cells in culture.
Nature
269:254,
1977[Medline]
[Order article via Infotrieve]
15.
Scott RE,
Perkins RG,
Zschunke MA,
Hoerl BJ,
Maercklein PB:
Plasma membrane vesiculation in 3T3 and SV3T3 cells.
J Cell Sci
35:229,
1979[Abstract]
16.
Black PN:
Shedding from normal and cancer-cell surfaces.
N Engl J Med
303:1415,
1980[Medline]
[Order article via Infotrieve]
17.
Lerner MP,
Lucid SW,
Wen GJ,
Nordquist RE:
Selected area membrane shedding by tumor cells.
Cancer Lett
20:125,
1983[Medline]
[Order article via Infotrieve]
18.
Masella R,
Cantafora A,
Guidoni L,
Luciani AM,
Mariutti G,
Rosi A,
Viti V:
Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells.
FEBS Lett
246:25,
1989[Medline]
[Order article via Infotrieve]
19.
Li R,
Ladisch S:
Shedding of human neuroblastoma gangliosides.
Biochim Biophys Acta
1083:57,
1991[Medline]
[Order article via Infotrieve]
20.
Lutz HU,
Liu SC,
Palek J:
Release of spectrin-free vesicles from human erythrocytes during ATP depletion.
J Cell Biol
73:548,
1977
21.
Allan D,
Thomas P,
Limbrick AR:
The isolation and characterization of 60 nm vesicles (`nanovesicles') produced during ionophore A23187-induced budding of human erythrocytes.
Biochem J
188:881,
1980[Medline]
[Order article via Infotrieve]
22.
Ott P,
Hope MJ,
Verkleij AJ,
Roelofsen B,
Brodbeck U,
van Deenen LLM:
Effect of dimyristoyl phosphatidylcholine on intact erythrocytes: Release of spectrin-free vesicles without ATP depletion.
Biochim Biophys Acta
641:79,
1981[Medline]
[Order article via Infotrieve]
23.
Wagner GM,
Chiu DTY,
Yee MC,
Lubin BH:
Red cell vesiculation
24.
Bütikofer P,
Chiu DTY,
Lubin B,
Ott P:
Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells.
Biochim Biophys Acta
855:286,
1986[Medline]
[Order article via Infotrieve]
25.
Bütikofer P,
Kuypers FA,
Xu CM,
Chiu DTY,
Lubin B:
Enrichment of two glycosyl-phosphatidylinositol-anchored proteins, acetylcholinesterase and decay accelerating factor, in vesicles released from human red blood cells.
Blood
74:1481,
1989
26. Medof ME, Nagarajan S, Tykocinski ML: Cell-surface engineering
with GPI-anchored proteins. FASEB J 10:574, 1996 |
Abstract
Introduction
Abstract
-nucleotidase
) 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.
, 
) were measured
in the vesicle-containing supernatants of cells labeled with in vitro incorporated VSG (
A common membrane physiologic event.
J Lab Clin Med
108:315,
1986