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The Distribution of Erythrocyte Phospholipids in Hereditary Spherocytosis
Demonstrates a Minimal Role for Erythrocyte Spectrin on Phospholipid
Diffusion and Asymmetry
By Frans A. Kuypers, Bertram H. Lubin, Maggie Yee, Peter Agre, Philippe F. Devaux, and Danielle Geldwerth
In the human erythrocyte membrane phosphatidylcholine
and sphingomyelin reside mainly in the outer leaflet,
whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine,are mainly found in the inner
leaflet. Maintenance of phospholipid asymmetry has been
assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin.
To investigate whether spectrin contributesto maintaining
the phospholipid transbilayer distribution and kinetics of
redistribution, w e studied erythrocytes from hereditary
spherocytosis patients whose spectrin levels ranged from
34%to 82%of normal. The phospholipid composition and
the accessibility of membrane phospholipids to hydrolysis
by phospholipases were in the normal range. Spin-labeled
phosphatidylserine and phosphatidylethanolamine ana-
logues that had been introducedinto the outer leaflet were
rapidly transported at 37°C to the inner leaflet, whereas
the redistributionof spin-labeled phosphatidylcholine was
slower. The kinetics of transbilayer movement of these spinlabeled phospholipidin all samples was in the normal range
and was not affected by the level of spectrin. Although these
erythrocyte membranes contained as little as 34%of the
normal level of spectrin and were characterized by several
physical abnormalities, the composition, distribution, and
transbilayer kinetics of the phospholipids were found to be
normal. W e therefore conclude that spectrin plays, at best,
only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.
0 1993 by The American Society of Hematology.
T
spectrin but retained their ability to redistribute spin-labeled
phospholipids introduced in the outer monolayer.” The distribution of the endogenous phospholipids was not studied
in these fragments. No data are reported on the distribution
of phospholipids in intact erythrocytesthat contain significant
lower levels of spectrin. Previous analyses of erythrocyte
membranes from a group of individuals with spherocytosis
demonstrated a close correlation between reduced erythrocyte
spectrin content, reduced relative membrane spectrin density,
and changes in several physical properties of the membranes.
Shear modulus, bending stiffness, and viscosity were all reduced.” In addition, the mechanical stability of the membranes was reduced’’ and the lateral redistribution of band
3 protein and glycophorins was increased as measured by
fluorescence recovery after photob1ea~hing.l~
Because the
changes in these parameters indicate that the structural integrity of the membrane is altered in spherocytic RBCs, they
also suggest that alterations in the membrane phospholipid
distribution in these cells may occur.
To investigate the relationship between erythrocyte spectrin
content and phospholipid distribution, erythrocytes from the
same group of spherocytic individuals (ie, with defined re-
HE PHOSPHOLIPIDS of the erythrocyte membrane are
distributed asymmetrically between the inner and outer
leaflet of the lipid bilayer. Whereas the choline-containing
phospholipids, phosphatidylcholine (PC) and sphingomyelin
(SM), are localized mainly in the outer monolayer, the aminophospholipids, phosphatidylethanolamine (PE) and phosphatidylserine (PS), are mainly or exclusively found in the
inner monolayer.’ The architecture of the membrane phospholipids is very dynamic, and the asymmetric distribution
reflects the differential equilibrium of the movement of phospholipid molecular species across the bilayer. The rates of
transbilayer movement are dependent on phospholipid classes
as well as molecular species within each class. The aminophospholipids are actively transported by an adenosine triphosphate (ATP)-dependent translocation system, the aminophospholipid translocase, or “flipa~e.”’-~Although this
protein transports both PE and PS, it exhibits a high affinity
for the latter. The half-times of transbilayer movement of
spin-labeled PS and PE introduced into the outer monolayer
are on the order of a few minutes and half an hour, respectively, at 37°C. In addition, all phospholipids are transported
by an ATP-independent system at rates that are significantly
slower, and that vary strongly with the acyl chains of the
phospholipid molecular
Spin-labeled PC is transported by this ATP-independent process from the outer to
inner monolayer with a half-time on the order of a few hours.
Several proteins embedded in the lipid bilayer interact with
the underlying membrane skeleton, a highly organized network composed primarily of spectrin and actin’ and it has
been suggested that spectrin, protein 4.1, and other components of the membrane skeleton interact directly with the
lipid bilayer. This interaction, which has been demonstrated
in model systems,’ could contribute to the asymmetric distribution of phospholipids in the erythrocyte? Although
published studies indicate that phospholipids, in particular
PS, interact with membrane skeleton proteins, neither the
nature of this interaction nor its contribution to the asymmetric distribution of phospholipids in the intact erythrocyte,
is well understood. Membrane fragments released from red
blood cells (RBCs) after heating to 50°C contained little
Blood, Vol81, N o 4 (February 15). 1993: pp 1051-1057
From the Children ‘s Hospital Oakland Research Institute, Oakland,
CA; the Johns Hopkins Medical School, Baltimore, MD;and the
Institut de Biologie Physico-Chimique, Paris, France.
Submitted June 22, 1992; accepted October 9, 1992.
Supported by National Institutes of Health Grants No. HL36255,
DK32094, HL20985, HL21061, HL27059, and HL33991; by the
Centre National de la Recherche Scientifique (URA 526); by the Institut National de la Santb et de la Recherche Mbdicale (No. 900104);
and by the Universtite‘Paris VU. P.A. is an Established Investigator
of the American Heart Association.
Address reprint requests to Frans A . Kuypers, PhD, Children’s
Hospital Oakland Research Institute, 747 52nd St, Oakland, CA
94609.
The publication costs of this article were defayed 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.
0 1993 by The American Society of Hematology.
0006-4971/93/8104-0023$3.00/0
1051
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1052
KUYPERS ET AL
duced spectrin levels) were evaluated for the distribution of
endogenous phospholipids as well as the redistribution kinetics of spin-labeled phospholipids introduced into the outer
monolayer. Our results indicate that the reduction in spectrin
levels has no effect on phospholipid distribution or kinetics
i n hereditary spherocytosis and support the conclusion that
the contribution of spectrin t o the maintenance of phospholipid asymmetry, if it exists, is only a minor one.
Table 2. Relative Abundance of the Major Phospholipid Classes
in Membranes of Control Cells and Erythrocytes
From Eight HS Patients
Subject
MATERIALS AND METHODS
Erythrocytes. Human erythrocyte suspensions were prepared
from fresh human venous blood collected in heparinized tubes after
informed consent was obtained from individuals diagnosed with hereditary spherocytosis (HS) or from laboratory volunteers. Erythrocytes were pelleted by centrifugation; washed twice with 0.9% NaC1,
and once with incubation buffer; and finally diluted in incubation
buffer to the appropriate hematocrit level. The buffer used for the
phospholipase experiments contained 50 mmol/L NaH2P04, 4.95
mmol/L KCI, 1.25 mmol/L KH2P04, 0.25 mmol/L MgCI2, 0.5
mmol/L CaCI2, 120 mmol/L NaC1, pH 7.4.The buffer for the spin
labeling and incubation studies was composed of 10 mmol/L HEPES,
pH 7.4,containing 144 mmol/L NaC1, I mmol/L MgC12,0.5 mmol/
L EGTA, 10 mmol/L glucose, 5 mmol/L KCI.
Lipid analysis. Erythrocyte membrane lipids were extracted according to the method of Rose and Oklander.14Phospholipid classes
were separated by two-dimensional thin layer chromatography according to the method of Roelofsen and Zwaal,15and lipid phosphorus
was determined according to the method of Rouser et a1.I6
Phospholipase treatment of erythrocytes. The hydrolysis of
membrane phospholipids by phospholipase A2 from bee venom
(Sigma, St Louis, MO) and sphingomyelinaseC from Staphylococcus
aureus (Sigma) was determined as described by Roelofsen and
Zwaa1.l' After the incubation with the combination of phospholipases,
erythrocytes were pelleted by centrifugation, and the relative amount
of hemoglobin in the supernatant was used to calculate the percentage
of cells that hemolysed during phospholipase treatment. Based on
the assumption that all phospholipids in a cell become available for
phospholipase hydrolysis as hemoglobin is lost, the following mathematical correction was made to determine the fraction of phospholipid in the outer monolayer. PLO,, = 100* (A - H)/(100 - H),
in which PLO,,* = percentage of phospholipid on the outside, A =
percentage of phospholipid hydrolyzed, and H = percentage of hemolysis.
Table 1. Characterization of Erythrocytes in HS Patients
and Controls
Subject
A Recessive HS
B Recessive HS
C Recessive HS
D Recessive HS
E DominantHS
F DominantHS
G DominantHS
H Heterozygote
N Normal
Spectrin
(% of normal)
34
45
52
59
76-82
76-82
76-82
100
100
PC
PE
28.5 f 2.7
28.0f 0.8
28.6f 1.2
27.4f 0.6
31.2f 0.7
28.0f 1.2
31 .O f 0.2
27.8 k 0.5
30.0k 1.8
31.2f 2.8
30.5f 0.4
30.3f 1.8
29.7f 0.8
29.1 f 0.7
32.0f 0.8
29.8f 0.6
29.5 f 1.5
29.0f 0.7
PS
13.1f 1.8
13.0f 0.3
13.4f 1.2
13.4f0.2
11.8f 0.7
14.8f 2.5
11.8*0.5
13.62 0.7
12.4k 1.3
SM
27.1 f 2.0
28.3 k 0.4
27.5 f 0.3
29.8? 0.1
27.5 ? 0.1
25.3 t 0.8
27.5f 0.6
28.9 f 0.1
29.1 k 2.3
The average of three determinations f SD is given for samples A
through H. The average of six determinations ? SD is given for control
cells (N).
Determination of the trans membrane redistribution of spin-labeled
lipids. The spin-labeled phospholipids used contain a short nitroxide-labeled fatty acyl group in the sn-2 position. The partial solubility
of these probe molecules facilitates their incorporation into membranes and enables the measurement of lipid translocation by the
determination of the probe fraction in the outer monolayer that can
be extracted by bovine serum albumin (BSA)." Before being labeled,
erythrocytes (30%hematocrit) were incubated for 5 minutes in buffer
at 37°C with 5 mmol/L (final concentration) di-isopropyl fluorophosphate to minimize the hydrolysis of the spin-labeled phospholipids." I-palmitoyl-2-(4-doxyl-pentanoyl)-phosphatidy~choline
(spPC), I-palmitoyl-2-(4-doxyl-pentanoyl)-phosphatidy~ethanolamine (spPE), and I-palmitoyl-2-(4-doxyl-pentanoyl)-phosphatidylserine (spPS) were synthesized as recently described." The spinlabeled phospholipids were dried from a chloroform solution and
resuspended in buffer." Labeling was performed at 30% hematocrit
with label at a final concentration of approximately 10 pmol/L. Determination of the amount of spin label in the outer monolayer of
the erythrocyte was performed by a back-exchange technique using
BSA as described." Erythrocytes were separated from the spin-labeled-BSA supernatant by a I-minute spin at 7,600gin an Eppendorf
(Madison, WI) centrifuge. Quantitation of the spin-label of the supernatants was performed on a Varian EPR spectrometer (Varian,
San Fernando, CA).
ATP measurement. Intracellular ATP concentration was measured in a 30% cell suspension in the buffer used for the spinlabel
studies. ATP was extracted from the cells in 0.5% trichloroacetic
acid, 2 mmol/L EDTA, and measured with the Pharmacia LKB luciferin-luciferase assay (Pharmacia, Piscataway, NJ).
Spectnn/Bd
0.54
0.73
0.74
0.68
0.90
0.90
0.90
1
1
The genetic background of the patients is given. The spectrin levels
were determined by radioimmunoassay, and the relative spectrin to band
3 (63)ratio, by sodium dodecyl sulfate-polyacrylamide electrophoresis
in membranes of erythrocytes from individuals with HS (A through H)
and control cells (N).
RESULTS
Characterization of erythrocytes.
T h e individuals with
spherocytosis included in this study were deficient in spectrin
with levels that ranged from 34% to 82% of n ~ r m a l " * ~ ~ . * ~
(Table 1). Smaller reductions in ankyrin, band 4.1, a n d other
minor proteins were noted. Details are given in previous
studies of these patients.11,19,20
The designation of the samples
(A through H) from the patients a n d control (N) was used
to identify these samples in the tables and figures.
phospho'ipComposition and organization Of
The Kdative amounts o f t h e major Phospholipid C ~ a s s e S
of these erythrocyte membrane samples are given in Table
2. No significant differences were found between the phosidS.
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PHOSPHOUPlDS IN HEREDITARY SPHEROCYTOSIS
1053
Fig 1. The distributionof endogenous PC
PE 0,and PS
C)between the inner and outer
monolayers for control erythrocytes (N) and erythrocytesfrom
eight individuals with HS (A
through H).
m,
pholipid composition of erythrocytes from normal controls
(sample N)and those of erythrocytes from HS patients with
various amounts of spectrin (samples A through H).
A combination of phospholipase A2 from bee venom and
sphingomyelinase C from S airrciis was used to determine
thedistribution ofthe phospholipid in the bilayer. The erythrocytes from normal controls (sample N) and those containing
reduced amounts ofspectrin (samplesA through H) exhibited
only small differences in the accessibility of PC and PE to
phospholipase A2 hydrolysis (Fig l). No PS degradation was
observed in any of the samples, indicating that PS is exclusively located in the inner monolayer of the membrane. A p
proximately 70%0to 78% of PC and 18% to 26% of PE in the
membrane were degraded by phospholipase A2, indicating
that endogenous PC is mainly located in the outer monolayer,
whereas most of the PE is located in the inner monolayer.
Sphingomyelinase C treatment indicated that approximately
80% of SM is located in the outer monolayer. These results,
similar to previous reports on normal erythrocytes,*' indicate
that spectrin levels as low as 34% of normal have no effect
on the asymmetric organization of endogenous phospholipids
in the erythrocyte.
A correction on the results describing the phospholipid
asymmetry as measured with phospholipases was needed as
the fragility of the membrane of the spectrindeficient erythrocytes led to substantial hemolysis of some samples during
phospholipase treatment. The normal control samples (N)
as well as the heterozygous control sample (H)exhibited less
than 2% hemolysis after treatment with both enzymes. The
spectrin-deficient samples, with the exception of A, showed
I % to 4.5% hemolysis after treatment with phospholipase A2.
This level of hemolysis was similar after sphingomyelinase
treatment for samples C through E. However, the samples
containing the lowest amount ofspectrin (A and B)exhibited
substantial hemolysis during combined phospholipase treatment (25% and 12%, respectively), whereas sample A also
exhibited 20% hemolysisduringtreatment with phospholipase
A2 alone. On the assumption that all phospholipids in the
bilayer of the hemolyzed cell fraction are available to phospholipase degradation. a correction for hemolysis, as indicated
in the method section, was made on the data shown in
Fig I.
Transhilayer kinelies qf spin-labeled phospholipid analogires. A typical result of the redistribution of spin-labeled
Fii 2. Typicalkineticsof the inward tm
*
of spin-labeled
PS (o),PE 0, a, pc ( ) in mmbranes of=asl,.
The curyes
shown are fined to the experimental data by least quare, exponential nonlinearregression.
I
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1054
KUYPERS ET AL
phospholipids across the membrane bilayer of normal cells
is shown in Fig 2. The lines connecting the experimental data
points are derived from a least-square, exponential nonlinear
regression fit. The half-times of equilibration across the bilayer, as calculated from these fits, are approximately 2, 40,
and 120 minutes for PS, PE, and PC, respectively. The plateaus, as calculated from these fits, that were reached for the
distribution across the bilayer for PE (79%) and PC (28%)
closely resembled the asymmetric distribution found for these
phospholipids based on their availability to phospholipase
degradation as depicted in Fig 1. The redistribution kinetics
of spin-labeled PS indicates that approximately 10%of the
spPS introduced into the membrane resides in the outer
monolayer at equilibrium and is extractable by BSA. In contrast, no endogenous PS could be hydrolyzed by phospholipases (Fig 1). These data on rate and equilibrium distribution
of spin-labeled phospholipids are in agreement with published
results," and the difference between the equilibrium distribution of spin-labeled PS and the availability of endogenous
PS to phospholipases was noted previ~usly.~
The data on the kinetics of spin-labeled phospholipid redistribution of erythrocytesin association with varying degrees
of spectrin deficiency are depicted in Fig 3. The redistribution
of spPS was measured in all samples (Fig 3A), whereas the
redistribution of spPC was determined in a limited number
100
I
I
I
I
of samples because of the total sample size available from
various individuals (Fig 3C). For each class of phospholipids
the intersample differences that were detected were minor.
No correlation was found between the level of spectrin and
the redistribution of these spin-labeled probes.
The activity of the aminophospholipid translocase can be
affected by ATP d e p l e t i ~ n We
. ~ therefore tested the intracellular ATP concentration of the samples used for the redistribution studies with the spin-labeled probes. The ATP
level was similar in all HS samples (average value, 0.99 mmol/
L k 0.15 mmol/L) and did not differ significantly from the
ATP levels determined in the normal control cells (1.03
mmol/L k 0.08 mmol/L). No correlation was found between
the ATP levels and the kinetics of transbilayer movement.
DISCUSSION
The asymmetric distribution of phospholipids in membranes was postulated 20 years ago by Bret~cherz'.'~and has
been demonstrated in a wide variety of cells by a variety of
techniques. All techniques allow distinction between both
leaflets and between head groups of the different membrane
phospholipid molecular species and include measurements
of accessibility of phospholipids to chemical reagents, phospholipid transfer proteins, and phospholipases as well as
measurements of redistribution of phospholipid probe mol-
I
I
100
g
I
I
I
200
300
400
80
0
'0
.-
60
60
I
-n
c
40
n
:
20
o +
0
I
I
100
200
A
U
Time
I
300
(minutes)
0
I
400
500
0
6
100
500
Time (minutes)
6 0 {
-
0 4
0
C
I
100
I
I
200
300
Time (minutes)
I
I
400
500
Fig 3. The kinetics of the inward translocation of three spinlabeled phospholipid classes. (A) Spin-labeled PS in erythrocytes
from two normal individuals (+, A) and eight HS patients: A (0).
6
(0).C (0).
D (XI. E (+I, F (A),G (+) and H (m). (6)
Spin-labeled PE in
erythrocytesfrom two normal individuals (+, A) and seven HS patients: A (0)B
.(U), D (X), E (+), F (A), G (+) and H .).( (C) Spinlabeled PC in erythrocytes from two normal individuals (+, A) and
four HS patients: D (X), E (+), G (+) and H (D).
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PHOSPHOLIPIDS IN HEREDITARY SPHEROCYTOSIS
e ~ u l e s . ~It. became
~ ~ , ~ ~apparent that this asymmetric distribution is the equilibrium in a dynamic movement of phospholipids across the erythrocyte membrane bilayer. In
contrast to pure lipid systems, which exhibit very slow transbilayer movement,26the erythrocyte lipids show a wide range
of transbilayer movement, with half-times on the order of a
few minutes (for PS species) to more than 20 hours for certain
PC specie^.^,^.'^ In a normal erythrocyte, PC and sphingomyelin reside at equilibrium mainly in the outer leaflet,
whereas the aminophospholipids PE and PS are primarily
found in the inner leaflet.
Three separate factors have been implicated in this transbilayer movement and asymmetric distribution: (1) an ATPindependent, or “passive” transbilayer movement of all endogenous phospholipid molecular species in which integral
membrane proteins have been implicated to play a role27;( 2 )
an ATP dependent, or “active” aminophospholipid transl ~ c a t i o n , ~ -a~process
. ~ ’ that facilitates the transbilayer movement of PE and PS, with a high affinity for PS; (3) the interaction of phospholipids (in particular, PS) with membrane
skeleton protein^.^.'^ This role of the membrane skeleton in
phospholipid distribution was supported by results that
showed the interaction of spectrin with PS on monolayers
and vesicle^.^^^^^^^ In addition to spectrin, other membrane
skeleton proteins such as protein 4.1 were found to interact
with p h o s p h ~ l i p i d . In
~ ~other
? ~ ~ cells, proteins such as inculin,33al~ha-actinin,~~
and p r ~ f i l i ninteracted
~~
with phospholipids, and modification of the membrane skeleton in platelets
had a profound effect on the phospholipid di~tribution,~~
although conditions that led to a loss of PS asymmetry in platelets did not coincide with a loss of interaction of PS with the
platelet cyt~skeleton.~~
Relatively few studies have assessed the importance of the
phospholipid-membrane skeletal interaction in intact erythrocyte membranes. Membrane fragments released from RBCs
as a result of heat treatment contain significantly lower
amounts of spectrin but largely retain their ability to redistribute spin-labeled phospholipids introduced in the outer
monolayer.” No data are available on the distribution of
endogenous phospholipids in these fragments. Similar treatment of erythrocytes at 50°C led to a loss of normal spectrin
structure in the remnant cells, whereas the phospholipid distribution was not affected as indicated by phospholipase
treatment and fluorescamine or merocyanine 540 labeling.38
In addition, a study that used photolabeling of spectrin with
a photoactivable PE analogue did not support a model in
which spectrin is involved in maintaining the asymmetric
phospholipid distribution.”
Sickled erythrocytes have been used as a model in which
to study the effect the disruption of the normal interactions
in the membrane has on phospholipid distribution as a result
of the extreme shape changes under low oxygen tensions.
Deoxygenated sickle erythrocytes exhibit an altered asymmetric distribution of endogenous phospholipid^^^ and a
higher transbilayer movement of PC.4’ Under these conditions, the activity of the aminophospholipid translocase is
also decreased as measured by the redistribution of spin-labeled aminophospholipids, and spin-labeled PC also exhibits
a faster transbilayer movement.42This organizational altered
1055
bilayer was interpreted to be located in the spectrin devoid
spicules formed during the sickling proce~s.~~.‘”’
The combination of ATP depletion45and shape change4’ was suggested
to be responsible for the loss of asymmetry. Other factors
implicated in membrane abnormalities, such as oxidative
in particular to the aminophospholipid translocase,
could also play a role in altering the phospholipid distribution
in sickle cells.
In this study we postulated that using erythrocytes that
contain significantlylower amounts of spectrin would provide
a more direct approach to delineate this effect of the membrane skeleton protein on phospholipid distribution. Hereditary spherocytosis, a congenital hemolytic
results
in abnormally fragile erythrocytes that are more spherical
than discoid. The membranes of these cells are reported to
have a partial reduction in their total p h o ~ p h o l i p i d , ~a~ , ~ ’
condition that seems to be corrected by splenectomy. All
spherocytic samples in our study were obtained from splenectomized individuals (A through G). No significant differences in phospholipid composition were found between these
samples and those from nonsplenectomized individuals (H
and N). Hence, our data suggest that the loss of phospholipid
as a result of splenic conditioning is a random process that
is not selective for specific phospholipid classes. Furthermore,
because no significant differences in the phospholipid distribution were found, our data suggest that splenectomy does
not affect phospholipid distribution in erythrocytes. However,
these conclusions are only indicative because we could not
compare erythrocytes before and after splenectomy.
Previous analyses of erythrocyte membranes from this set
of spherocytic individuals demonstrated a close correlation
among erythrocyte spectrin content, relative membrane
spectrin density, and several physical properties of the membrane, including a reduction in its shear modulus, bending
stiffness, and viscosity,’I a lower mechanical stability,12and
an increased lateral redistribution of band 3 protein and glyc~phorins.‘~
Because of the reduction in the ratio of surface
area to volume of spherocytes as compared with discocytes,
the spherocytes are more sensitive to hypotonic lysis. A correlation was noted between the sensitivity towards hypotonic
lysis and the quantity of spectrins2 as well as deformability,
as measured by ektacytometry.” This instability of the membrane could be the basis for the increased hemolysis, which
is correlated with the degree of spectrin deficiency, that was
found during phospholipase treatment. Although these parameters indicate that the membrane organization is altered,
the hydrolysis of endogenous phospholipids with phospholipase was not correlated with the level of spectrin. These
data indicate that the increased ratio of lipid bilayer relative
to membrane skeleton does not affect endogenous phospholipid asymmetry. The substantial level of hemolysis after
phospholipase treatment in the samples with low levels of
spectrin necessitated a correction of the data based on the
assumption that all phospholipids were available to phospholipase hydrolysis as soon as hemoglobin was lost from
the cell. The validity of this correction and the conclusion
that phospholipid asymmetry is retained was confirmed by
the measurement of the redistribution of the spin-labeled
phospholipids across the bilayer. Also, in this set of experi-
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KUYPERS ET AL
1056
ments the equilibrium reached was similar for the erythrocytes
of the heterozygous individual (H), those with spectrin levels
as low as 34% of normal (A through G), and those of the
normal control (N). In addition, the rate of redistribution of
each class of spin-labeled phospholipids was virtually identical
in these samples, indicating that the activity of the aminophospholipid translocase is not affected in these erythrocytes.
In conclusion, the composition, distribution, and transbilayer kinetics of phospholipids in patients with spectrindeficient hereditary spherocytosis were normal although the
membranes contained as little as 34% of the normal level of
spectrin, and previous studies of these patients had established
that the disorder alters several physical properties of these
membranes. Our data show that any aminophospholipid
molecule that moves to the outer monolayer is rapidly transported back to the inner monolayer as long as the aminophospholipid translocase is active even if the membrane is
destabilized. Although the interaction of phospholipids with
the membrane skeleton could restrict the outward movement,
thereby stabilizing the membrane and reducing the ATP
needed to maintain the asymmetric equilibrium, the loss of
almost 70%of the spectrin does not seem to affect membrane
phospholipid organization. Our data do not exclude a role
for spectrin in the stabilization of the phospholipid bilayer
but indicate that this protein plays at best only a minor role
in the maintenance of erythrocyte membrane phospholipid
organization as long as the aminophospholipid translocase
is active. The inactivation of the amino phospholipid translocase, whether by the absence of ATP or some other disturbance such as oxidation, would ultimately lead to loss of
phospholipid asymmetry, driven by a concentration gradient.
ACKNOWLEDGMENT
We thank Paulette HervC and Pierre Fellmann for the gift of the
spin-labeled phospholipids.
REFERENCES
1. Verkleij A, Zwaal R, Roelofsen B, Comfurius P, Kastelijn D,
van Deenen L: The asymmetric distribution of phospholipids in the
human red cell membrane: A combined study using phospholipases
and freeze-etch electron microscopy. Biochim Biophys Acta 323:178,
1973
2. Connor J, Schroit AJ: Determination of lipid asymmetry in
human red cells by resonance energy transfer. Biochemistry 26:5099,
1987
3. Daleke DL, Huestis WH: Incorporation and translocation of
aminophospholipids in human erythrocytes. Biochemistry 24:5406,
1985
4. Devaux P F Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30: 1163, 1991
5. Middelkoop E, Lubin BH, Op den Kamp JAF, Roelofsen B:
Flip-flop rates of individual molecular species of phosphatidylcholine
in the human red cell membrane. Biochim Biophys Acta 855:421,
1986
6 . van Meer G, Op den Kamp J: Transbilayer movement of phosphatidylcholine species in intact human erythrocytes. J Cell Biochem
19:193, 1982
7. Gardner K, Vann Bennett G: Recently identified erythrocyte
membrane skeletal proteins and interactions, in Brinkhous K, Stass
S (eds): Hematology. New York, NY,Marcel Dekker, 1989, p 1
8. Mombers C, de Gier J, Demel RA, van Deenen LLM: Spectrinphospholipid interaction, a monolayer study. Biochim Biophys Acta
603:52, 1980
9. Haest CWM, Plasa G , Kamp D, Deuticke B: Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte.
Biochim Biophys Acta 436:353, 1978
IO. Calvez J-Y, Zachowski A, Hermann A, Morrot G, Devaux
P F Asymmetric distribution of phospholipids in spectrin-poor
erythrocyte vesicles. Biochemistry 27:566, 1988
I 1 . Waugh RE, Agre P: Reductions of erythrocyte membrane viscoelastic coefficients reflect spectrin deficiencies in hereditary spherocytosis. J Clin Invest 8 1:133, 1988
12. Chassis J, Agre P, Mohandas N: Decreased membrane mechanical stability. J Clin Invest 82:617, 1988
13. Golan D, Palek J, Age P Red cell membrane spectrin content
regulates band 3 and glycophorin lateral diffusion. Blood 74:24, 1990
14. Rose HG, Oklander M: Improved procedure for the extraction
of lipids from human erythrocytes. J Lipid Res 6:428, 1965
15. Roelofsen B, Zwaal RFA: The use of phospholipases in the
determination of asymmetric phospholipid distribution in membranes. Methods Membrane Biol 7:147, 1976
16. Rouser G, Fleisher S, Yamamoto A: Two dimensional thin
layer chromatographic separation of polar lipids and determination
of phospholipids by phosphorus analysis of spots. Lipids 5:494, 1970
17. Morrot G, HervC P, Zachowski A, Fellmann P, Devaux PF:
Aminophospholipid translocase of human erythrocytes: Phospholipid
substrate specificity and effect of cholesterol. Biochemistry 28:3456,
1989
18. Brown A: ATP and ATPase determinations in red blood cells,
in Ellory J, Young J (eds): Red Cell Membrane. A Methodological
Approach. London, UK, Academic, 1982, p 223
19. Agre P, Omnger E, Bennet V: Deficient red-cell spectrin in
severe, recessively inherited spherocytosis. N Engl J Med 306: 1 155,
1982
20. Agre P, Casella JF, Zinkhin WH, McMillan C , Bennett V:
Partial deficiency of erythrocyte spectrin in hereditary spherocytosis.
Nature 3 14:380, 1985
21. Roelofsen B: Phospholipases as tools to study the localization
of phospholipidsin biological membranes: A critical review. J Toxicol
1:87, 1982
22. Bretscher M: Asymmetrical lipid bilayer structure for biological
membranes. Nature 236: 1 1, I972
23. Bretscher M: Phosphatidylethanolamine: Differential labeling
in intact cells and cell ghosts of human erythrocytes by a membrane
impermeable reagent. J Mol Biol 7 I :523, 1972
24. Op den Kamp J: Lipid asymmetry in membranes. Ann Rev
Biochem 48:47, 1979
25. Etemadi AH: Membrane asymmetry. A survey and critical
appraisal of the methodology. 11. Methods for assessing the unequal
distribution of lipids. Biochim Biophys Acta 604:423, 1980
26. de Kruijff B, Wirtz K Induction of a relatively fast transbilayer
movement of phosphatidylcholine in vesicles. A 13CNMR study.
Biochim Biophys Acta 468:3 18, 1977
27. van Deenen L: Topology and dynamics of phospholipids in
membranes. FEBS Lett 123:3, 1981
28. Haest C, Plasa G, Kamp D, Deuticke B: Protein-lipid interactions in the erythrocyte membrane: Relevance for structural properties, in Lassen U, Ussing H, Wieth J (eds): Membrane Transport
in Erythrocytes. Copenhagen, Denmark, Munksgaard, 1980, p 108
29. Maksymiw R, Sui S, Gaub H, Sackmann E: Electrostatic coupling of spectrin dimers to phosphatidylserine containing lipid lamellae. Biochemistry 26:2983, 1987
30. Cohen AM, Lui SC, Lawler J, Derick L, Palek J: Identification
of the protein 4.1 binding site to phosphatidylserine vesicles. Biochemistry 27:614, 1988
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PHOSPHOLIPIDS IN HEREDITARY SPHEROCYTOSIS
3 1. Eder P, Soong C, Tao M: Phosphorylation reduces the affinity
of protein 4.1 for spectrin. Biochemistry 25:1764, 1986
32. Rybicki AC, Heath R, Lubin B, Schwartz RS: Human erythrocyte protein 4.1 is a phosphatidylserine binding protein. J Clin
Invest 81:255, 1988
33. N i d i V. Dimitrov D, Brunner J, Burger M: Interaction of
the cytoskeletal component vinculin with bilayer structures analyzed
with a photoactivable phospholipid. J Biol Chem 261:6912, 1986
34. Burn P, Rotman A, Meyer R, Burger M: Diacylglycerol in
large mactininlactin complexes and the cytoskeleton of activated
platelets. Nature 3 14:469, 1985
35. Lasing I, Linberg U: Specific interaction between phosphatidylinositol 4,Sbiphosphate and profilactin. Nature 3 14:472, 1985
36. Verhallen P, Bevers E, Comfurius P, Zwaal R: Fluoride dependent calcium-induced platelet procoagulant activity shows that
calpain is involved in increased phospholipid transbilayer movement.
Biochim Biophys Acta 942:150, 1988
37. Comfurius P, Bevers E, Zwaal R: Interaction between phosphatidylserine and the isolated cytoskeleton of human blood platelets.
Biochim Biophys Acta 983:212, 1989
38. Gudi S, Kumar A, Bhakuni V, Gokhale S, Gupta C: Membrane
skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes. Biochim
Biophys Acta 1023:63, 1990
39. Pradhan D, Williamson P, Schlegel R: Bilayer/cytoskeleton
interactions in lipid symmetric erythrocytes assessed by a photoactivable phospholipid analogue. Biochemistry 30:7754, 1991
40. Lubin B, Chiu D, Bastacky J, Roelofsen B, van Deenen L:
Abnormalities in membrane phospholipid organization in sickled
erythrocytes. J Clin Invest 67:1643, 1981
41. Franck P, Chiu D, Op den Kamp J, Lubin B, van Deenen L,
Roelofsen B: Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process. J Biol Chem
258:8436, 1983
42. Blumenfeld N, Zachowski A, Galacteros F, Beuzard Y, Devaux
P: Transmembrane mobility of phospholipids in sickle erythrocytes:
1057
Effect of deoxygenation on diffusion and asymmetry. Blood 77349,
1991
43. Franck P, Bevers E, Lubin B, Comfurius P, Chiu D, Op den
Kamp J, Zwaal R, van Deenen LLM, Roelofsen B: Uncoupling of
the membrane skeleton from the lipid bilayer. The cause of accelerated
phospholipid flip-flop leading to an enhanced procoagulant activity
of sickled cells. J Clin Invest 75:183, 1985
44. Liu S, Derick L, Zhai S, Palek J: Uncoupling of the spectrinbased skeleton from the lipid bilayer in sickled red cells. Science 252:
574, 1991
45. Middelkoop E, Lubin BH, Bevers EM, Op den Kamp JAF,
Comfurius P, Chiu DTY, Zwaal RFA, van Deenen LLM, Roelofsen
B: Studies on sickled erythrocytes provide evidence that the asymmetric distribution of phosphatidylserine in the red cell membrane
is maintained by both ATP-dependent translocation and interaction
with membrane skeletal proteins. Biochim Biophys Acta 937:28 1,
1988
46. Hebbel R P Beyond hemoglobin polymerization: The red
blood cell membrane and sickle disease pathophysiology. Blood 77:
214, 1991
47. Dacie J: The life span of the red blood cell and circumstances
of its premature death, in Wintrobe M (eds): Blood, Pure and Eloquent. New York, NY, McGraw Hill, 1980, p 21 1
48. Lux S: Disorders of the red cell membrane skeleton: Hereditary
spherocytosis and hereditary elliptocytosis, in Stanburry J, Wyngaarden J, Frederickson D, Goldstein J, Brown M (eds): The Metabolic
Basis of Inherited Disease. New York, NY, McGraw Hill, 1983,
p 1573
49. Becker P, Lux S Hereditary spherocytosisand related disorders.
Clin Haematol 14:15, 1985
50. Cooper R, Jandl J: The role of membrane lipids in the survival
of red cells in hereditary spherocytosis. J Clin Invest 48:736, 1969
5 1. Reed CF, Swisher SN: Erythrocyte lipid loss in hereditary
spherocytosis. J Clin Invest 45:777, 1966
52. Agre P, Asimos A, Casella J, McMillan C: Inheritance pattern
and clinical response to splenectomy as a reflection of erythrocyte
spectrin deficiency in hereditary spherocytosis. N Engl J Med 3 15:
1579, 1986
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1993 81: 1051-1057
The distribution of erythrocyte phospholipids in hereditary
spherocytosis demonstrates a minimal role for erythrocyte spectrin
on phospholipid diffusion and asymmetry
FA Kuypers, BH Lubin, M Yee, P Agre, PF Devaux and D Geldwerth
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