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Phosphatidylserine Transport in RhNullErythrocytes
By Robert E. Smith and David L. Daleke
Phosphatidylserine transport in normal and Rh,,,, red blood
cells was determined by measuring characteristic morphologic changes induced by synthetic phospholipids. Treating
normal A+ cells with commercial anti-A antisera, antiRh,(D) antisera, or with saturating concentrations of purified Rh,(D) antibodies had no effect on phosphatidylserine
transport. Normal B- cells treated with purified anti-B
antibodies transported phosphatidylserine a t rates equal to
those of cells not treated with antibody. Rh,,,, cells,
deficient in t h e protein bearing t h e Rh,(D) antigen, incorporated dimyristoylphosphatidylcholineand dimyristoylphosphatidylserine at rates and to extents similar t o normal
cells. Furthermore, incorporated phosphatidylserine, but
RHNUbL
SYNDROME is a rare blood phenotype characterized by the lack of all erythrocyte Rh determinants,
including the polypeptide bearing the Rh,(D) antigen.’.’
These erythrocytes display secondary defects such as altered
cation regulation, increased osmotic fragility, and a stomatocytic m o r p h o l ~ g y . ~Membrane
.~
phospholipid organization is
altered in these cells. In normal erythrocytes the choline
phospholipids (sphingomyelin [SM] and phosphatidylcholine [PC]) are located primarily in the cell outer monolayer,
while the aminophospholipids (phosphatidylethanolamine
[PE] and phosphatidylserine [PSI) are localized in the inner
m ~ n o l a y e r . ~In. ~Rh,,,, cells, an increased amount of P E is
accessible to hydrolysis by exogenous phospholipases, and all
of the PC can be exchanged with exogenous PC from
synthetic membranes,’ suggesting a loss of membrane asymmetry and an increase in passive phospholipid flip-flop.
Recent evidence indicates that the asymmetric distribution
of phospholipids in erythrocytes is maintained, in part, by an
aminophospholipid-specific, adenosine triphosphate (ATP)dependent, and sulfhydryl reagent-sensitive protein that
transports PS and PE from the membrane outer leaflet to the
inner leaflet.”” Active transport of phosphatidylserine has
been measured in a number of cell types using a variety of
spin-, radio-, or fluorescent analogs of pho~phatidylserine?”~
Another method relies on the characteristic effects of incorporated phospholipids on cell shape.”.” Short, saturated acyl
chain phospholipids transfer spontaneously from sonicated
vesicles into cell^.'^^'^*'^ These lipids induce shape changes
indicative of their bilayer location, and can be used to
measure lipid incorporation and transport.”
Although aminophospholipid transport activity has been
well-characterized, the protein or proteins responsible have
not been identified. Recently, a 31- to 32-Kd protein that is
labeled by a phosphatidylserine photoaffinity probel7*”and
comigrates with a protein that reacts with membrane permeant sulfhydryl reagents” has been proposed as a candidate
for the PS tran~porter.’~.’~
The Rh polypeptide has several
unusual characteristics that are similar to those of the
putative PS transporter. It has a molecular weight of 28 to 33
Kd21.22and possesses 3 to 4 sulfhydryl g r o ~ p s *that
~ * ~are
~
necessary for antigenicity,” and may be palmitoylated.26
Other features of the Rh polypeptide imply it is buried in the
membrane and may play a role in maintaining membrane
integrity. The Rh protein is not glycosylated, requires phosBlood, Vol 7 6 , No 5 (September 1). 1990: pp 1021-1027
not phosphatidylcholine. w a s rapidly transported across
t h e membrane bilayer. Energy depletion or treatment with
sulfhydryl reagents inhibited phosphatidylserine transport
equally in normal and Rh,,,, cells. These results indicate
that, although Rh,,,, cells have numerous membrane defects, they a r e capable of adenosine triphosphate-dependent transport of exogenously added dimyristoylphosphatidylserine. Normal phosphatidylserine transport in t h e
presence of anti-Rh,(D) antibodies or in cells deficient in
t h e Rh,(D) polypeptide indicates that this protein is not t h e
aminophospholipid transporter.
0 1990 by The American Society of Hematology.
pholipid for antigenicity, and retains antigenicity after trypsin
or pronase treatrr~ent.’~*’~
Phosphatidylserine transport activity is similarly resistant to exogenous protease^.^^**^ The Rh
protein is associated with the c y t o ~ k e l e t o n ~and
~ . ~its
’ absence
in Rh,,,, cells results in several membrane a b n o r m a l i t i e ~ , ~ . ~ . ~
implying that it serves an important structural role. Although Rh antigens are present only on human erythrocytes,
the Rh protein may be ubiq~itous.~’
The apparent similarity
of the physical characteristics of the Rh protein and the
putative phosphatidylserine transporter has lead to the suggestion that the proteins are related and may be identica1.20,26
These reports prompted the present investigation to (1)
determine if the defects in membrane structure of Rh,,,, cells
include alterations in aminophospholipid transport, and (2)
to determine if the Rh protein is required for phosphatidylserine transport.
MATERIALS AND METHODS
Dimyristoylphosphatidylserine(DMPS), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylcholine(DOPC) were
purchased from Avanti Polar Lipids (Pelham, AL). Anti-A, anti-B,
and anti-Rh,(D) antisera were obtained from American Dade
(Miami, FL). Protein A-Sepharose CL-4B and PD-10 (Sepharose
G-25M) columns were purchased from Pharmacia (Piscataway,
NJ). Carrier free NaIZ5Iwas obtained from New England Nuclear
(Boston, MA), and Iodo-gen reagent was obtained from Pierce
(Rockford, IL). Pronase, phospholipase A, (bee venom and Naja
naja), sphingomyelinase C (Staphylococcus aureus), N-ethylmaleimide (NEM), and the luciferin-luciferase ATP assay kit were
purchased from Sigma (St Louis, MO). All other chemicals were
reagent grade.
Cells. Human erythrocyteswere obtained from adult volunteers
From the Department of Chemistry, Indiana University, Bloomington.
Submitted January 3, 1990; accepted May 10, 1990.
Supported by a Grant-in-Aid from the Indiana AJiliate of the
American Heart Association.
Address reprint requests to David L. Daleke. PhD, Department of
Chemistry, Indiana University. Blmmington. IN 47405.
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.
0 1990 by The American Society of Hematology.
0006-4971/90/7605-0006$3.00/0
1021
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1022
by venipuncture and collected into EDTA or citrate anticoagulant.
Rh,,,, cells (donor H.H.) were obtained from Dr C. Kruppe (St
Mary’s Hospital, Long Beach, CA). Erythrocytes were pelleted by
centrifugation (3,00Og, 5 minutes), washed three times with 4 vol of
138 mmol/L NaCI, 5 mmol/L KC1, 6.1 mmol/L Na,HPO,, 1.4
mmol/L NaH,PO,, pH 7.4 (PBS) and stored at 4OC. Cells were used
within 24 hours of collection. Before use, intracellular ATP concentrations were determined by the luciferin-luciferase method according to the manufacturer’s (Sigma) instructions. Glutathione concentration was measured by the method of B e ~ t l e r . ~ ~
Phospholipase treatments. Transmembrane phospholipid asymmetry in normal (B-) and Rh,,,, red blood cells (RBCs) was
measured by phospholipase dige~ti0n.I~
Briefly, 0.2 mL cells were
suspended in 0.8 mL 10 mmol/L HEPES, 150 mmol/L KCI, pH 7.4
(HBS) containing 0.5 mmol/L CaCI,. Bee venom phospholipase A,
(20 IU) was added and, after a 1-hour incubation at 37OC,
sphingomyelinase C (0.1 IU) was added. After an additional 1-hour
incubation, 0.1 mL HBS containing 10 mmol/L EDTA was added
and the cells were collected by centrifugation. The cells were washed
twice with HBS containing 1 mmol/L EDTA. In separate experiments, cells were treated with a combination of bee venom phospholipase A, (20 IU/mL), Naja naja phospholipase A, (20 IU/mL),
and sphingomyelinaseC (1 IU/mL), conditions similar to those used
previously to measure phospholipid asymmetry in Rh,,,, cell^.^"^
Hemolysis was measured using Drabkin’s reagent.33
Phospholipid analysis. Cells (0.2 mL) were lysed in 10 mmol/L
Tris, 2 mmol/L EDTA, pH 7.4, collected by centrifugation (16,OOOg
for 10 minutes), and washed twice with Tris/EDTA buffer. Ghost
lipid was extracted with 3.6 mL chloroform:methanol(l:2), followed
by the addition of 1.2 mL chloroform and 1.2 mL water. The organic
phase was collected and the solvent was evaporated with a nitrogen
stream. After reconstitution in 4: 1 chloroform:methanol, lipids were
separated by two-dimensional thin layer chromatography on silica
gel HL plates (Analtech) using chloroform:methanol:water:30%
ammonium hydroxide (90:505.5:5.5) in the first dimension and
ch1oroform:methanol:formicacid (55:25:5) in the second dimension.
Lipid spots were visualized with iodine vapor and amine-containing
lipids were detected by reaction with fluorescamine reagent (0.05%
wt/vol in acetone). Lipid spots were scraped from the plate and
analyzed for ph~sphate.~’
Antibody purification. For some experiments, blood group specific antibodies were purified from the corresponding commercial
antiserum. Briefly, 10 mL of antiserum was loaded onto a 2 mL
Protein A-Sepharose column equilibrated with 0.1 mol/L phosphate
(pH 8.0). The column was washed with 10 mL of 0.1 mol/L
phosphate (pH KO), and bound antibody was eluted with 0.1 mol/L
citrate (pH 3.0). Fractions were neutralized with 1 mol/L Tris-HCI
(pH 9.0) and analyzed for protein content by absorbance at 280 nm.
Neutralized buffer was exchanged for PBS (pH 7.4) with Sephadex
G-25. Protein purity was determined by gradient (5% to 15%)
polyacrylamide gel electrophoresis (PAGE) and protein concentration was determined by the method of Lowry et al.36Each antibody
was purified twice as described above, freeze-dried, and stored at
- 7OOC. This procedure resulted in antibody preparations that were
greater than 95% pure as indicated by PAGE under both reducing
and nonreducing conditions.
Antibody iodination and binding measurements. In experiments
measuring antibody binding to cells, anti-Rh,(D) antibody (1
mg/mL) was further purified by adsorption with washed B- erythrocytes at 15% HCT. After centrifugation, the antibody-containing
supernatant (0.5 mg) was radioiodinated with 1.43 mCi Na’251in 0.2
mL PBS + 30 pmol/L NaI, using 50 p g Iodo-gen. Iodinated
antibody was diluted to 2.5 mL and separated from free Na’*’I with
a PD-IO column. Specific activity of ‘ZSI-anti-Rh,(D) was 1.5
mCi/mg. Before use, unlabeled anti-Rh,(D) antibody was diluted
SMITH AND DALEKE
with labeled antibody to a final concentration of 6 mg/mL and a
specific activity of 1.8 pCi/mg. Anti-Rh,(D) binding to A + cells was
measured by incubating 0.5 to 500 pg/mL of antibody with cells at
5% hematocrit (HCT) for 30 minutes at 37OC in PBS. Cells were
washed twice with PBS, and radioactivity associated with the cell
pellet was measured with a Beckman Biogamma counter. The
number of antibody binding sites was calculated according to
Scatchard.”
Antibody treatment of erythrocytes. Cells (20% HCT) were
treated with whole or purified antisera for 30 minutes at 37OC. After
centrifugation (16,000gfor 3 minutes) cells were washed three times
with PBS.
Inhibition ofaminophospholipid translocation. Sulfhydryl group
modification was accomplished by treating cells (10% HCT) with 1
mmol/L NEM for 20 minutes. ATP depletion was accomplished by
incubating cells in PBS containing penicillin (5 IU/mL) and
streptomycin (5 pg/mL) for 24 hours at 37OC and was confirmed by
the luciferin/luciferase assay. After these treatments, cells were
pelleted by centrifugation (16,OOOg for 3 minutes), washed three
times, and treated with vesicles as described below.
Cell-vesicle incubations. Sonicated unilamellar vesicles of phospholipid were prepared as described.’’.’’ DMPS or DMPC vesicles
were incubated with cells at 50% hematocrit, 37OC, for the specified
time intervals.
Pronase treatment. In some experiments, cells pretreated with
antibodies and phospholipid were treated with pronase (1 pg/mL)
for 1 hour at 25°C to remove bound antibody. Pretreatment with
pronase (or neuraminidase, trypsin, or ~hymotrypsin~~)
has no effect
on PS transport.20s29
After pronase treatment, cells were washed two
times by centrifugation and reincubated in PBS for 1 hour at 37OC.
Extraction of DMPS or DMPC. Sonicated vesicles of DOPC
can function as a passive receptor for short chain phospholipids
located in the erythrocyte outer monolayer. The extent of back
extraction depends on the relative surface areas of the cells and
acceptor vesicles in a given suspension;maximal back-extraction can
be achieved with a high vesicle-to-cell surface area ratio.Is To
accomplish back-extraction of DMPS or DMPC, cells treated as
described above were incubated with sonicated DOPC vesicles (20
mmol/L) at 5% HCT for 7 hours at 37OC.
Cell morphology. Erythrocyte suspensions were prepared for
morphologic analysis by fixing 5 pL aliquots of 5% to 50% HCT
suspensions in 50 pL of 1% glutaraldehyde in 0.15 mol/L NaCl.
Samples were analyzed by light microscopy. Echinocytes were
assigned scores of -t1 to + 5 (increasing value denoting more severe
crenation), discocytes were scored 0, and stomatocytes were given
scores of -1 to -4 (decreasing value denoting more severe
invaginati~n).’~,’’
The average score of a field of 100 cells is defined
as the morphological index (MI).
RESULTS
Effect of Rh,(D) antibodies on PS transport. The effect
of anti-Rh,(D), anti-A and anti-B antibodies on phosphatidylserine transport in B- and A + erythrocytes was measured
using a cell morphology
Normal discoid erythrocytes became echinocytic (positive MI) when treated with
DMPC (Table l ) , indicating that this lipid accumulated in
the cell outer m ~ n o l a y e r . ’ ~ ~I n’ ’contrast, DMPS induced a
discocyte-to-stomatocyte shape change (negative MI, Table
1) consistent with the accumulation of this lipid in the
erythrocyte inner
Anti-A antisera (diluted
1:lO) had no significant effect on erythrocyte shape, while
anti-Rh,(D) antisera induced a slight stomatocytic morphology. Purified anti-Rh,(D) antibody at higher concentrations
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PS TRANSPORT IN RH,,,,
1023
ERYTHROCYTES
Table 1. Effect of Blood Group Antisera on RBC
Phospholipid Transport
Treatment
No Antibody
Buffer
Buffer, pronase
DMPC
DMPC, pronase
DMPS
DMPS, pronase
-0.1
-0.2
2.7
2.8
-1.8
-2.1
Anti-A (1:lO)
AntiRh,lD)
0.2
-0.6
0.1
-0.5
3.1
2.9
- 1.3
-2.0
2.9
2.6
-1.5
-2.5
M I of A+ erythrocytes treated with buffer (PES),anti-A ( 1 : 10 dilution),
or anti-Rh,(D) antisera for 30 minutes at 37°C. washed, and treated
either with buffer, DMPC ( 1 mmol/L), or DMPS ( 1 mmol/L) for 2 hours at
37°C. After two additional washes an aliquot of each sample was treated
with pronase ( 1 Mg/mL) for 1 hour at 25°C. washed, and reincubated for
1 hour at 37°C.
did not show the same initial stomatocytosis, indicating that
a component of the commercial antisera was responsible for
this shape change. Treatment of cells with antisera had little
effect on shape changes induced by subsequent DMPC or
DMPS treatment, although anti-Rh,(D) antisera-treated
cells became slightly less stomatocytic when exposed to
DMPS (Table 1). However, purified anti-Rh,(D) antibody at
higher concentrations (up to 0.5 mg/mL) did not change the
extent of DMPS-induced shape change (see below). Finally,
treating cells with pronase (1 pg/mL) to remove bound
antibody did not significantly alter the morphology of cells
preincubated with buffer or DMPC. All cells treated with
DMPS became more invaginate after pronase treatment,
perhaps as a result of the additional incubation time (1 hour),
allowing more DMPS to accumulate in the inner monolayer.
The number of antigenic sites for anti-A and anti-Rh,(D)
antibodies is approximately 1 x lo6 sites/cell and 4 x IO4
sites/cell, re~pectively.~~.~'
Scatchard analysis of '251-antiRh,(D) antibody binding to the A+ cells used in this work
indicated approximately 6.5 x lo4sites/cell. To test antibody
effects on lipid-induced shape changes at high antibody-tocell ratios, anti-A or anti-Rh,(D) antibodies were purified by
Protein A chromatography and incubated with A+ erythrocytes. Purified anti-A or anti-Rh,(D) antibodies at concentrations as high as 0.12 fg/cell (3.5 x lo5 molecules/cell) and
2.5 fg/cell (7.6 x IO6 molecules/cell), respectively, had no
effect on normal cell morphology or on shape changes
induced by DMPC or DMPS (data not shown).
Immunologically complexing the Rh protein does not
necessarily refute the hypothesis that the Rh protein and the
putative 32-Kd transporter are equivalent, since antibody
binding may not alter protein function. Furthermore, recent
data demonstrate that polyclonal antibodies to erythrocyte
32-Kd proteins do not inhibit PS transport.20
PS transport in Rh,,,, cells. To directly investigate the
possible involvement of the Rh,(D) protein in aminophospholipid transport, Rh,,,, cells were tested for transport activity.
As shown in Fig Id, these cells were slightly more stomatocytic compared with B- cells (Fig la). The ATP content of
Rh,,,, cells was depressed (0.35 mmol/L), while the glutathione content was within the normal range (2.04 mmol/
L).34 B- cells had ATP and glutathione concentrations of
0.85 mmol/L and 2.04 mmol/L, respectively. When exposed
to DMPC for 2 hours, both Rh,,,, and B- erythrocytes
became echinocytic to a similar degree (Fig 1, b and e).
DMPS treatment for 2 hours induced a stomatocytic shape
change of similar extent in both types of cells (Fig 1, c and f).
The time course of shape change in response to DMPC and
DMPS treatment was similar for Rh,,,, and B- cells. As
shown in Fig 2, the rate and extent of shape change induced
by DMPC and DMPS was similar for both types of cells.
However, at most timepoints Rh,,,, cells exhibited a slightly
lower MI than B- cells, perhaps as a result of their slight,
initial stomatocytic morphology.
Transport of DMPS in Rh,,,, cells was dependent on ATP
and sensitive to sulfhydryl modification. Depleting ATP in
Rh,,,, and B- cells to 5 pmol/L and 50 pmol/L, respectively,
inhibited DMPS transport but not incorporation of the lipid
(data not shown). NEM (1 mmol/L) inhibited transport of
DMPS in Rh,,,, and B- cells (data not shown).
The transbilayer distribution of DMPC and DMPS incorporated into Rh,,,, and B- cells was determined by back
extraction with a high concentration of DOPC vesicles.
Under the conditions used (20 mmol/L DOPC vesicles and
5% HCT), most (98%) of the outer monolayer dimyristoylphospholipid should be removed from the cell." When Rh,,,,
and B- erythrocytes that had been treated with DMPC for 3
hours were treated with DOPC acceptor vesicles for 7 hours,
they returned to a discoid morphology, confirming outer
monolayer localization of DMPC (Fig 2). Cells exposed to
DMPS before DOPC treatment became slightly more stomatocytic, rather than returning to a discoid morphology,
indicating that this lipid was mostly localized in the cell inner
monolayer, inaccessible to extraction. The increase in stomatocytic morphology may have resulted from extraction of
residual DMPS from the cell outer monolayer.
Phospholipid asymmetry in normal and Rh,,,, cells. To
verify that the Rh,,,, cells used in this study have membrane
alterations similar to Rh,,,, cells used in earlier studies,
transmembrane phospholipid asymmetry was measured using purified phospholipases. As shown in Table 2, a combination of phospholipase A, (bee venom, 20 IU/mL) and
sphingomyelinase C (S aureus, 0.1 IU/mL) resulted in an
increased hydrolysis of PE and SM in Rh,,,, cells compared
with normal B- cells. Qualitatively similar results were
obtained with a combination of bee venom phospholipase A,
(20 IU/mL), Naja naja phospholipase A, (20 IU/mL), and
a higher concentration of sphingomyelinase C ( I IU/mL),
although hemolysis was greater under these conditions. The
extent of PE hydrolysis in normal cells is consistent with
earlier reports; however, less PE in the Rh,,,, cells used here
and less SM in both types of cells was accessible to
hydrolysis.8 These differences may reflect a diversity of the
Rh,,,, phenotype or differences in enzyme preparations and
conditions.
DISCUSSION
Although many physical properties of the aminophospholipid transporter have been characterized, its identity remains unknown. PS and PE transport in erythrocytes have
been shown to be inhibited by reagents that react with
s ~ l f h y d r y l ' ~ . "and
. ~ ~ arginine" residues, or reagents that
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SMITH AND DALEKE
1024
Fig 1. Light mkrogl.phs of nornul IB ) (a through c ) and Rh,
" d l L DMPC (b and e) or 1 " d l L DMPS (c and 1) for 2 hours.
n
Fig 2. T ~ ~ J W of
S B
O (0. 0)or Rh,
(0,
orythrocyre
shop. chongo during tr..tmMt with 1 " d l L DMPC (0.
.I or 1
" d l L DMPS (0.0)at 60% HCT and 37%. A f t a 3 hours the calls
wore eoltectd by centrifugation. washed. and treotod with 20
m d l L W P C &In at 6% HCT, 37% for 7 hours Idashod tinos).
Id through r) orythrocyrr k t o r o (a and d) and o f t r "
n
..
with 1
inhibit ATPases? Some of these properties have been exploited in attempts to identify the protein or proteins responsible for this activity. Sensitivity of the transporter to
sulfhydryl reagents (diamide or NEM) was one of the first
demonstrations of the proteic nature of the transporter.'o.i'
Subscquent studies confirmed the sensitivity of aminophospholipid transport to membrane-permeant sulfhydryl oxidizing and alkylating reagents. and revealed a 32-Kd protein in
erythrocytes that was particularly reactive with an '*'Ilabelcd sulfhydryl reagent.i9 This protein is also extensively
crosslinked with photoaffinity-labeled PS, but not with a
photoafinity-labeled PC,"." implying that the protein may
be the aminophospholipid transporter." Although coincident
PS binding and sulfhydryl reactivity of the 32-Kd protein
suggest that this protein might be the transporter. the data
remain inconclusive. Phenylazide photoaffinity probes uscd
in the aforementioned studies generate long-lived nitrenes on
irradiation that show a preference for reactivity with nucleophiles. such as free sulfhydryl groups.IRAlthough the headgroup specificity implies a specific lipid-protein interaction.
labeling with nitrene-generating photoafinity probes might
be unrelated to PS-transporter binding and may reflect
sulfhydryl group reactivity. Furthermore, a second report
using a short chain photoaffinity-labeled PS selectively
labeled a number of proteins in addition to a 27-Kd protein."
Although the protein bearing the Rh,(D) antigen possesses
characteristics similar to the putative 32-Kd transporter. the
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1025
PS TRANSPORT IN RH,,"^^ ERMHROCMES
Table 2. Digestion of Control (8-1 and Rh,,,, RBC Phospholipid by Phospholipases
% PhospholipidHydrolyzed
Enzyme Treatment
Control cells
Bee venom phospholipase A,
Bee venom phospholipase A,
sphingomyelinase C
A,
+
Rh,,,, cells
Bee venom phospholipase A,
Bee venom phospholipase A,
sphingomyelinase C
A,
+
+ sphingomyelinase C
PC
SM
PE
% Hemolysis
79.7
54.6
22.5
3.1
75.2
60.4
29.3
17.3
77.2
63.2
30.0
4.1
77.0
65.3
35.1
17.6
+ Naja naja phospholipase
+ sphingomyelinase C
+ Naja naja phospholipase
+
Naja naja phospholipase A, (20
Cells were treated with bee venom phospholipase A, (20 IU/mL) or with bee venom phospholipase A, (20 IU/mL)
IU/mL) for 1 hour at 37°C. Sphingomyelinase C (0.1 IU/mL) was added and, after an additional 1-hour incubation at 37°C. lipids were extracted and
analyzed for hydrolysis by thin-layer chromatography.
present work shows that normal cells treated with antiRh,(D) antibodies or Rh,,,, cells, missing the Rh polypeptide,
are capable of ATP-dependent PS transport, implying that
the Rh protein is not a PS transporter. Furthermore, the Rh
protein and the sulfhydryl-reactive 32-Kd protein are distinct; the '251-labeled sulfhydryl reagent previously used to
identify the 32-Kd protein" reacts with a 32-Kd protein in
both normal and Rh,,,, erythrocytes (D.L. Daleke, unpublished observations, April 1990). Recently, another protein
has been proposed as a PS transporter: Zachowski et aI3'
measured ATP-dependent PS transport in bovine adrenal
chromaffin granules and have proposed that the granule
ATPase I1 (molecular weight 115 Kd) is an aminophospholipid translocase. Positive identification of the aminophospholipid transporter awaits more definitive experiments.
Normal PS transport activity in Rh,,,, cells has several
important consequences for the function of the Rh protein.
The binding of the Rh protein to the cytoskeleton and
membrane alterations in Rh,,,, cells imply the protein has an
important structural role. However, the increased exchangeability of PC and phospholipase accessibility of P E in Rh,,,,
cells may result from a transient, rather than static, change
in P E distribution. The phospholipase assays used in these
studies' require long incubations (hours) during which reorientation of inner monolayer PE, or other membrane
perturbation^,^^ may occur. Inner monolayer P E may become accessible to externally applied phospholipases because
of rapid transmembrane flip-flop; however, this lipid may not
accumulate in the outer monolayer due to transport to the
inner monolayer by the aminophospholipid transporter.
Aminophospholipid transport in Rh,,,, cells has provided
additional information about the aminophospholipid transporter. Transport and stable accumulation of PS in the inner
monolayer of Rh,,,, cells suggests that the transporter is
capable of generating aminophospholipid asymmetry in the
presence of rapid lipid flip-flop. Similar membrane alterations are observed under conditions of altered cytoskeletal
structure, and have been used as evidence implicating cytoskeletal proteins in maintaining phospholipid asymmetry.
Studies with purified cytoskeletal proteins spectrin and band
4.1 have demonstrated a specific interaction of these proteins
with model membranes composed of pho~phatidylserine.4'-~~
Chemical44 or pathologic45 alterations in the structure of
cytoskeletal proteins in intact cells leads to a loss of P E
asymmetry. Furthermore, the association of the Rh protein
with the cytoskeleton suggests that the absence of R h protein
in Rh,,,, cells is directly responsible for cytoskeletal disruption and a loss of PE asymmetry. We propose that pathologic
conditions resulting in an increase in passive flip-flop should
not change the static distribution of phospholipids unless the
aminophospholipid transporter is damaged. Other data support this argument. Although altering cytoskeletal proteins
with chemical reagents apparently disrupts phospholipid
a~ymmetry,"~
studies reaching this conclusion measured
phospholipid asymmetry with phospholipases. Experiments
using a rapidly reacting chemical probe failed to confirm a
loss of a~ymmetry.~'
In addition, the PS-cytoskeletal protein
binding energy is too small to account for the maintenance of
PS asymmetry solely by binding of PS to cytoskeletal
proteins.46 Furthermore, Calvez et a147 have measured PS
transport in membrane preparations devoid of cytoskeletal
proteins and found that these preparations are capable of
transporting PS and generating transmembrane PS asymmetry. Cytoskeletal interactions may play a role in maintaining
static transmembrane PS asymmetry; however, the presence
of active aminophospholipid transport is sufficient for the
generation and maintenance of transmembrane PS asymmetry.
We have shown that the Rh protein is not the aminophospholipid transporter. The function of the cytoskeletal association of the Rh protein and the reasons for membrane
perturbations in its absence remain to be elucidated. Although the aminophospholipid transporter has not been
identified, the ability of Rh,,,, cells to maintain a transmembrane PS gradient indicates that the transporter can overcome membrane perturbations, such as increased flip-flop, to
generate and maintain an asymmetric membrane.
ACKNOWLEDGMENT
We are grateful for the generous cooperation of donor H.H. and
Dr C. Kruppe in these studies.
REFERENCES
1 . Vos GH, Vos D, Kirk RL, Sanger R: A sample of blood with no
detectable Rh antigens. Lancet 1:14, 1961
2. Sturgeon P: Hematological observations on the anemia associated with blood type Rh,,,,. Blood 36:310, 1970
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3. Ballas SK, Clark MR, Mohandas N, Colfer HF, Caswell MS,
Bergren MO, Perkins HA, Shohet SB: Red cell membrane and
cation deficiency in Rh null syndrome. Blood 63:1046, 1984
4. Bretscher M: Asymmetric lipid bilayer structure for biological
membranes. Nature 236:11, 1972
5. Verkleij AJ, Zwaal RFA, Roelofsen B, Comfurius P, Kastelijn
D, van Deenen LLM: The asymmetric distribution of phospholipids
in the human red blood cell membrane: A combined study using
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1990 76: 1021-1027
Phosphatidylserine transport in Rhnull erythrocytes
RE Smith and DL Daleke
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