Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2297-2301
Interaction Between Terminal Complement Proteins C5b-7 and Anionic
Phospholipids
By
Clive Liu,
Patricia Marshall,
Ian Schreibman,
Ann Vu,
Weiming Gai, and
Michael Whitlow
From the Division of Dermatology, Manhattan Veteran's Administration
Medical Center, New York; and Ronald O. Perelman Department of
Dermatology, New York University School of Medicine, New York,
NY.
 |
ABSTRACT |
We have recently shown that C5b-6 binds to the erythrocyte membrane
via an ionic interaction with sialic acid before the addition of C7 and
subsequent membrane insertion. In this study we assessed the role of
anionic lipids in the binding of the terminal complement proteins to
the membrane and the efficiency of subsequent hemolysis. Human
erythrocytes were modified by insertion of dipalmitoyl
phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine (DPPS),
dipalmitoyl phosphatidylethanolamine (DPPE), or dipalmitoyl
phosphatidic acid (DPPA). Lipid incorporation and the hemolytic assays
were done in the presence of 100 µmol/L sodium orthovanadate to
prevent enzymatic redistribution of lipid. We found that the neutral
lipids, DPPC and DPPE, did not affect C5b-7 uptake or hemolysis by
C5b-9. In contrast, the two acidic phospholipids, DPPS and DPPA, caused
a dose-dependent increase in both lysis and C5b-7 uptake. We conclude
that the presence of anionic lipids on the exterior face of the
membrane increases C5b-7 uptake and subsequent hemolysis. It is known
that sickle cell erythrocytes have increased exposure of
phosphatidylserine on their external face and are abnormally sensitive
to lysis by C5b-9. The data presented here provide a plausible
mechanism for this increased sensitivity.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE MEMBRANE ATTACK complex of the
complement system consists of five proteins: C5, C6, C7, C8, and
C9.1-4 Upon activation, these proteins expose hydrophobic
residues, insert into membranes, and form transmembrane channels. The
process of channel formation can be divided into two phases: the
initial interaction between the cell membrane, C5b-6, and C7 to form a
stably inserted C5b-7 complex; and the subsequent addition of C8 and
multiple C9 molecules to form a transmembrane channel.1-4
It is well established that the terminal complement proteins interact
with membrane lipids via hydrophobic interactions.2,5,6 Ionic interactions between C5b-6 and the cell membrane are also important.7,8 We recently showed that C5b-6 binds to sialic acid moieties on glycophorin, and that this binding increases the
membrane uptake of C5b-6 and subsequent hemolysis by
C5b-9.8
Silversmith and Nelsestuen7 used light scattering to show
that the interaction of C5b-6 and C5b-7 with phospholipid vesicles is
dependent on the lipid headgroup composition of the vesicles. C5b-6
bound to vesicles made of phosphatidic acid or phosphatidylglycerol but
did not bind to vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), or phosphatidylinositol.7
The major lipids of the erythrocyte membrane are sphingomyelin (SM),
PC, phosphatidyl ethanolamine (PE), and PS.9 Under normal
circumstances, the distribution of erythrocyte membrane lipids is
asymmetrical; SM and PC are localized to the external lipid monolayer,
while PS and PE are localized to the interior leaflet.9
This nonequilibrium condition is maintained in part by the slow rate of
"flip-flop" of lipids between the inner and outer monolayer, and
in part by a lipid "flippase" that transports the
amino-phospholipids PE and PS from the outer monolayer to the inner
monolayer.10,11 The actual structure of this lipid flippase
is not known, but it is inhibited by vanadate.9,11
One pathological condition in which the normal distribution of
phospholipids is disrupted is sickle cell anemia. Membranes of
deoxygenated sickle erythrocytes have significant quantities of PS and
PE in the outer monolayer and a compensatory increase in PC in the
inner monolayer.12 Sickle red cells that have undergone repeated cycles of oxygenation/deoxygenation assume an irreversibly sickled shape, and in contrast to the reversibly sickled cell, these
cells have abnormal phospholipid asymmetry even under fully oxygenated
conditions.12
It is well known that sickle cell erythrocytes are capable of
activating complement via the alternative pathway due to the exposure
of PS and PE on the outer monolayer.13,14 Test and Woolworth15 showed that erythrocytes from sickle cell
anemia patients also showed a marked increase in susceptibility to
lysis by the terminal complement proteins, C5b-9. The mechanism of this increased sensitivity to complement was due to increased binding of
C5b-7 to the surface of the sickled erythrocytes.15
Based on our studies showing that sialic acid caused increased binding
of C5b-7 to erythrocytes, we would predict that the presence of anionic
lipids on the exterior monolayer of the erythrocytes may also increase
lysis by increasing C5b-7 uptake.
To test this theory directly we incorporated exogenous synthetic lipids
with different headgroups into normal human erythrocytes. We find that
presence of anionic lipid in the outer monolayer of the erythrocytes
increases both deposition of C5b-7 and subsequent lysis with the
addition of C8 and C9, thus strengthening the hypothesis that ionic
interactions between C5b-6 and the membrane are important in modulating
membrane damage by the terminal complement proteins.
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MATERIALS AND METHODS |
Buffers and solutions.
All buffers were prepared with glass-distilled water and ultrafiltered
before use. Phosphate-buffered saline (PBS): 150 mmol/L NaCl, 5 mmol/L
sodium phosphate, pH 7.4; DGVB2+:
veronal-buffered saline containing 71 mmol/L NaCl, 139 mmol/L dextrose,
2.5 mmol/L sodium veronal (pH 7.4), 0.1% gelatin, 0.15 mmol/L
CaCl2, and 1 mmol/L MgCl2; GVB-EDTA:
veronal-buffered saline containing 142 mmol/L NaCl, 2.5 mmol/L sodium
veronal (pH 7.4), 0.1% gelatin, and 10 mmol/L EDTA.
Reagents.
Human erythrocytes were collected by venipuncture from volunteers.
Blood was initially collected in buffered EDTA solution, washed, and
stored in DGVB+2. C5b-6 was purchased from Advanced
Research Technologies (San Diego, CA). C7, C8, and C9 were purchased
from Quidel (San Diego, CA) or Advanced Research Technologies.
Dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine
(DPPS), dipalmitoyl phosphatidyl ethanolamine (DPPE), and dipalmitoyl
phosphatidic acid (DPPA) were obtained from Avanti (Alabaster, AL). The
fluorescent derivatives, nitrobenzoxadiazole (NBD)-DPPC,
NBD-DPPS, NBD-DPPE, and NBD-DPPA were obtained from Avanti. The
structure of these synthetic derivatives was similar in that the
R1 fatty acid was 16:0 and the R2 fatty acid
was 6:0 with the NBD group at the end of the acyl chain. 14C-DPPC was obtained from New England Nuclear (Boston, MA).
Radiolabeling.
C7 was iodinated using IodoGen (Pierce Chemical Co, Rockford, IL) and
125I (New England Nuclear). The specific activity was
between 0.3 and 3.2 × 107 cpm/µg.
Hemolytic assays.
Hemolytic assays were performed as previously described.16
Briefly, one volume of erythrocytes (1.5 × 108/mL)
was incubated with one volume of C5b-6 (3.33 µg/mL) or buffer at
30°C for 30 minutes. One volume of C7 (2 µg/mL) was then added and the mixture incubated at 30°C for an additional 15 minutes. Cells were then washed and incubated with guinea pig serum in the
presence of 10 mm EDTA at 37°C for 60 minutes. Lysis was determined spectrophotometrically. Percent lysis was calculated as (EXP 
BK)/(CL BK),
where EXP is the OD412 of the sample containing C5b-9, BK is the OD412 of erythrocytes treated with
C5b-7 only, and CL is the OD412 of 100% lysis.
Lipid incorporation.
We incorporated specific lipids into the external leaflet of the
erythrocyte membrane by incubating human erythrocytes with phospholipid
vesicles. Lipid uptake was monitored by incorporation of
fluorescent-labeled phospholipid. An individual dipalmitoyl phospholipid (DPPA, DPPS, DPPE, or DPPC) was mixed with its fluorescent derivative (NBD-DPPA, NBD-DPPS, NBD-DPPE, or NBD-DPPC). Experiments were also performed with 14C-DPPC and unlabeled DPPC. Molar
ratios of unlabeled lipid to fluorescent probe were 3:1. In the case of
14C-DPPC, the molar ratio of unlabeled to labeled lipid was
8:1. Phospholipid/probe mixtures were dissolved in chloroform and dried under nitrogen. Vesicles were then prepared by addition of PBS and
sonication for 10 minutes at room temperature with a probe sonicator.
Contaminating large multilamellar vesicles were removed from the
suspension by centrifugation at 2,000g for 15 minutes. Erythrocytes (final concentration 4.5 × 107/mL) were
incubated with 15 mL of phospholipid vesicles for 30 minutes at
37°C. The lipid concentration was varied between 0 and 10 µg/mL.
When cells were incubated with higher concentrations of lipid, the
cells became excessively fragile, leading to increases in background
lysis. In addition, when higher concentrations of lipid vesicles were
incubated with cells, the lipid tended to bind to the cells as vesicles
rather than incorporating into the membrane. Lipid incorporation and
the hemolytic assays were done in the presence of 100 µmol/L sodium
orthovanadate. This concentration has been shown to inhibit the human
erythrocyte "flippase," thus preventing enzymatic redistribution
of lipid.9-11
A standard curve was generated by removing cells with a known
concentration of lipid, lysing the cells with 1% sodium dodecyl sulfate (SDS) and reading the fluorescence in a Perkin-Elmer
(Norwalk, CT) Fluorimeter (excitation 
, 470 nm;
emission , 525 nm). Lipid incorporation was estimated reading the
fluorescence of 107 lipid-enriched cells in the presence of
1% SDS and comparison with the standard curve.
To determine whether adherent vesicles were present in the treated
erythrocyte preparations, fluorescence of treated erythrocytes was
determined in the presence and absence of 2% Triton X-100. Because of
he self-quenching properties of the labeled phospholipids, the
phospholipid vesicles show about a 10-fold increase in fluorescence after lysis. Lipid that has inserted into the erythrocyte membrane is
dequenched by dilution with membrane lipids, and its fluorescence is
not increased by detergent solubilization. The erythrocytes used for
complement activation studies did not show increased fluorescence after
lysis, indicating minimal contamination with intact
vesicles.17
Incorporation of 14C DPPC was done in a similar fashion
except that the molar ratio of 14C DPPC to cold DPPC was
1:8. 14C was counted in a liquid scintillation counter
(Beckman, Fullerton, CA) with quench correction.
Uptake of 125I-C7.
Phospholipid-enriched erythrocytes were incubated with C5b-6 and
125I-C7 to assess C5b-7 uptake. A total of 0.1 mL of 1.5 × 108 erythrocytes were incubated with C5b-6 at
30°C for 30 minutes in GVB-EDTA. 125I-C7 was then added
and the mixture incubated at 30°C for an additional 15 minutes.
Cells were then pelleted at 750g for 3 minutes in a Eppendorf
tabletop centrifuge (Brinkman, Old Westbury, NY). Erythrocytes were
washed once with GVB-EDTA and pelleted at 750g. This procedure
pellets only whole cells and not erythrocyte membranes that have been
lysed; thus, we measured the C7 counts that were bound to cells, not
ghost membranes. Cells were then lysed with 5 mmol/L sodium phosphate
(pH 7.5) and membranes pelleted at 16,000g, washed once with
PBS, and membranes pelleted at 16,000g and counted for
125I. Background binding was estimated by incubating the
cells in the presence of 125I-C7, but in the absence of
C5b-6. Background counts were generally about 10% of the counts in the
presence of C5b-6 and were subtracted from the total counts per minute
to give specific counts per minute.
| |
RESULTS |
PS increases C5b-7 uptake and lysis by C5b-9.
We wished to determine the effect of lipids with different head groups
on uptake of C5b-7 and eventual lysis by C5b-9. Based on our earlier
data, we would predict that the presence of anionic lipids such as DPPS
and DPPA would increase both C5b-7 uptake and lysis.
Figure 1 shows that incorporation of DPPS
significantly increases both the uptake of C5b-7- and C5b-9-mediated
lysis in a dose-dependent fashion. Under normal conditions, the human
erythrocyte contains 446 µmol/L of PS per liter of cells, however
virtually none of the PS is exposed on the outer
monolayer.18
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| Fig 1.
PS increases C5b-7 uptake and lysis by C5b-9. Various
concentrations of PS were incorporated into erythrocytes in the
presence of vanadate. The cells were washed and used in a hemolytic
assay to determine percent lysis ( ). The same cells were used to
measure uptake of C5b-7 using 125I-C7 ( ). Lysis before
the addition of C8 and C9 varied between 0% for the buffer control and
14% for the highest concentration of DPPS.
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Phosphatidic acid increases C5b-7 uptake and lysis by C5b-9.
Figure 2 shows that DPPA also increased
C5b-7 uptake and subsequent lysis by C5b-9. Phosphatidic acid is a
minor component of the erythrocyte membrane (less than 80 µmol/L
phosphatidic acid per liter of cells), thus the added PA represents a
significant increase in the PA content of the
erythrocyte.18
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| Fig 2.
Phosphatidic acid increases C5b-7 uptake and lysis by
C5b-9. Various concentrations of phosphatidic acid were incorporated
into erythrocytes in the presence of vanadate. The cells were washed
and used in a hemolytic assay to determine percent lysis (). The
same cells were used to measure uptake of C5b-7 using
125I-C7 (). Lysis before the addition of C8 and C9
varied between 0% for the buffer control and 18% for the highest
concentration of DPPA.
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PC has no effect on C5b-7 uptake and lysis by C5b-9.
The two major neutral phospholipids of the erythrocyte are PC and PE.
PC is normally the predominant phospholipid on the exterior face of the
erythrocyte membrane. Figure 3 shows that
addition of exogenous PC to the erythrocytes has no effect on either
C5b-7 uptake or lysis by C5b-9. In normal erythrocytes, PC content is 944 µmol/L cells.18
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| Fig 3.
PC has no effect on C5b-7 uptake and lysis by C5b-9.
Various concentrations of PC were incorporated into erythrocytes in the
presence of vanadate. The cells were washed and used in a hemolytic
assay to determine percent lysis (). The same cells were used to
measure uptake of C5b-7 using 125I-C7 (). Lysis before
the addition of C8 and C9 varied between 0% for the buffer control and
12% for the highest concentration of DPPC.
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PE has no effect on C5b-7 uptake and lysis by C5b-9.
PE is the other major neutral phospholipid of the erythrocyte membrane.
Under normal circumstances, the vast majority of the PE is localized to
the interior leaflet of the lipid bilayer. In
Fig 4, we incorporated increasing
quantities of PE into the erythrocyte, and as can be seen, there is no
increased lysis or increased uptake of C5b-7. Erythrocyte membranes
contain 1040 µmol of PE per liter of cells; however, only 20% of the
PE is present on the outer leaflet of unmodified erythrocytes (208 µmol/L). Thus the added PE is a significant percentage of the exposed
PE.18
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| Fig 4.
PE has no effect on C5b-7 uptake and lysis by C5b-9.
Various concentrations of PE were incorporated into erythrocytes in the
presence of vanadate. The cells were washed and used in a hemolytic
assay to determine percent lysis (). The same cells were used to
measure uptake of C5b-7 using 125I-C7 (). Lysis before
the addition of C8 and C9 varied between 0% for the buffer control and
8% for the highest concentration of DPPE.
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|
Using 14C PC as a marker for total PC gives results
identical with the fluorescent probe.
We wished to verify our results using a nonfluorescent probe to be
sure our results were not affected by the presence of the fluorescent
NBD moiety, so we used 14C PC. The results are shown in
Fig 5. The use of the radioactive probe
gave results that were identical to the fluorescent probe: addition of
DPPC causes no increase in either C5b-7 uptake or lysis by C5b-9.
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| Fig 5.
14C PC has no effect on C5b-7 uptake and
lysis by C5b-9. DPPC was incorporated into the erythrocytes as in Fig
3; however, we used 14C-DPPC as a marker of DPPC
incorporation. The cells were washed and used in a hemolytic assay to
determine percent lysis (). The same cells were used to measure
uptake of C5b-7 using 125I-C7 (). Lysis before the
addition of C8 and C9 varied between 0% for the buffer control and 5%
for the highest concentration of DPPC.
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| |
DISCUSSION |
The results of this study show that the presence of anionic lipids on
the external face of the erythrocyte membrane results in increased
hemolysis by the terminal complement proteins. The increased lysis is
due to increased deposition of C5b-7, as the C5b-7 uptake parallels the
amount of increased lysis. Addition of two neutral phospholipids, DPPC
and DPPE, showed no increase in either hemolysis or in C5b-7 uptake. In
comparing the slopes of both lysis and C5b-7 uptake, DPPS is somewhat
more efficient than DPPA in C5b-7 binding and subsequent hemolysis.
This indicates that negative charge density alone is not responsible
for the increased uptake of C5b-7. This result is similar to what we
found in the case of C5b-6 binding to sialic acid. The negative charge of the sialic acid was essential for the interaction; however, other
structural modifications also affected the ability of gangliosides to
interact with C5b-6.8 In similar fashion, the serine moiety may serve to increase the affinity of C5b-6 for the membrane.
These data provide further evidence for the importance of ionic
interactions between C5b-6 and anions on the cell membrane. Our model
of this interaction is shown in Fig 6. When
C5 is cleaved by the classical or alternative pathway C5 convertase,
C5a is released into the fluid phase and C5b remains bound to the
convertase.19 C5b then binds to C6 to form C5b-6. This
complex then dissociates from the C5 convertase and binds to the
anionic lipid headgroups via ionic forces. When C7 binds to C5b-6, the
ionic bond between C5b-6 and the membrane is disrupted, C5b-7 exposes
hydrophobic residues and the C5b-7 complex inserts into the membrane.
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| Fig 6.
Model for interaction between membrane anions, C5b-6 and
C7. (A) Binding of C5b-6 to anionic lipids via the grouped positive
charges of the conserved TS1 domain in C6. (B) The effect of C7
addition. Binding of C7 via anionic residues disrupts the
membrane-C5b-6 interaction and leads to exposure of hydrophobic
residues, resulting in the dissociation of the C5b-7 complex from the
anionic molecule and insertion into the lipid bilayer. TS represents
the highly conserved TS1 domain in C6. H represents the hydrophobic
domains of C5b, C6, or C7.
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|
The amino acid sequence or sequences of C5b-6 that interacts with
membrane anions is not known. The terminal complement proteins share
several structural domains that are found in other proteins, including
the thrombospondin type 1 motif (TS1).20 C6 has three and
C7 has two TS1 domains. This sequence of amino acids binds to
negatively charged molecules such as heparin, heparan sulfate proteoglycan, sulfatides, and cholesterol sulfate and contain grouped
positive charges making them likely candidates to bind to anions.
Previous studies have investigated the role of lipid structure in
complement lysis. Shin and coworkers showed that increasing the
fluidity of the membrane increased the efficiency to lysis by
C5b-9.21-23 These studies focused on altering the
hydrophobic domain of the membrane in distinction to the current
studies that examine the hydrophilic domain of the lipid molecule.
Obviously both are important in controlling susceptibility of membranes to damage by C5b-9.
One pathological condition in which there is exposure of PE and PS on
the outer monolayer of erythrocytes is sickle cell disease. The
mechanism of this flip-flop is unclear and may be due to recently identified calcium-dependent phospholipid "scramblase," as it is
known that sickle cell erythrocytes do have elevated levels of
calcium.24
Test and Woolworth found that sickle erythrocytes are abnormally
sensitive to lysis by C5b-9 and that this abnormal sensitivity was due
to increased deposition of C5b-7. Our current data supply a likely
mechanism for the increased sensitivity of sickle erythrocytes to
hemolysis by C5b-9.
There are some interesting similarities in the ways the terminal
complement proteins and the alternative pathway activation proteins
interact with membrane lipids. Exposure of PS and PE on the external
membrane leaflet leads to increased activation of the alternative
complement pathway.13 Thus, the exposure of PS on the
external face of the sickle erythrocyte makes these cells more
susceptible to complement lysis by at least two mechanisms, increased
activation of the alternative pathway and increased channel formation.
| |
FOOTNOTES |
Submitted July 23, 1998; accepted November 20, 1998.
Supported by a Veteran's Administration Merit Review and a fellowship
from the Charles Culpeper Foundation.
Address reprint request to Michael Whitlow, MD, PhD,
Dermatology Service, Veteran's Administration Medical Center, 423 E
23rd St, New York, NY 10010-5050.
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.
| |
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TOP
ABSTRACT
INTRODUCTION
