Rigid - Semantic Scholar · Supported in part by Grants AM 26263. AM 16095, and AM 2157/from the National Institutes of Health. Presented in part at the American Society ofHematology

Blood. Vol. 63, No. 6 (June), 1984: pp. 1385-1392 1385

Rigid Membranes of Malayan Ovalocytes: A Likely Genetic Barrier

Against Malaria

By N. Mohandas, L. E. Lie-Injo, M. Friedman, and J. W. Mak

A high frequency of nonhemolytic hereditary ovalocytosis

in Malayan aborigines is thought to result from reduced

susceptibility of affected individuals to malaria. Indeed.

Kidson et al. recently showed that ovalocytes from Melane-

sians in Papua New Guinea are resistant to infection in

culture by the malarial parasite Plasmodium falciparum. In

order to determine if protection against parasitic invasion

in these ovalocytes might be the result of some altered

membrane material property in these unusual cells. we

measured their membrane and cellular deformability char-

acteristics using an ektacytometer. Ovalocytic red cells

were found to be much less deformable in comparison to

normal discoid red cells. Similar measurements on isolated

membrane preparations revealed a marked reduction in

ovalocytic membrane deformability. To produce equal

deformation of ovalocytic and normal membranes. ovalo-

cytes required an 8-10-fold increase in applied shear

T HE GEOGRAPHIC COINCIDENCE of sickle

cell mutation and malaria first drew attention to

the possibility that the sickle cell gene might confer

resistance to this disease,’ but direct experimental

support for this hypothesis awaited the development of

an in vitro culture system for the malarial parasite.2 In

addition to elucidating the cellular mechanism of

sickle cell resistance,3 the culture system has also

helped to study a number ofother red cell variants that

appear to offer protection against malarial infection.�8

These include red cells containing hemoglobins C, E,

and F, thalassemic red cells, cells deficient in glucose-

6-phosphate dehydrogenase (G6PD), and En (a - ) red

cells. The mechanisms of protection against parasite

infection of these variants are different. For example,

in En (a-) red cells, the absence of glycophorin A

decreases the attachment of the parasite to the cell

membrane, thus inhibiting the first step in parasite

invasion of the red cell.7 On the other hand, in thalas-

semic cells, hemoglobin F-containing cells, and G6PD-

deficient red cells, the parasite appears to enter the cell

normally but is subsequently destroyed during replica-

tion, probably by the increased oxidant damage gener-

ated in these red cell variants.6

Based on an epidemiologic study of Malayan ab-

origines from an area where malaria was endemic,

Baer et al.9 put forward the hypothesis that ovalocyto-

sis, which occurs with high frequency (-30%) in this

population, might represent yet another red cell van-

ant genetically selected by its associated protection

against malaria. Lie-Inj&#{176} and Amato and Booth�

found that hereditary ovalocytosis is widespread in the

Southeast Asian region, occurring in several different

ethnic groups, with over 20% of the population of

stress. indicating that their membrane was capable of

deforming under sufficient stress. To test the possibility

that this increased membrane rigidity might confer resis-

tance to parasitic invasion. we performed an in vitro

invasion assay using Plasmodium falciparum merozoites

and Malayan ovalocytes of varying deformability from

seven different donors. The level of infection of the ovalo-

cytes ranged from 1 % to 35% of that in control cells. and

the extent of inhibition appeared to be closely related to

the reduction in membrane deformability. Moreover. we

were able to induce similar resistance to parasitic invasion

in nonovalocytic normal red cells by increasing their mem-

brane rigidity with graded exposure to a protein crosslink-

ing agent. Our findings suggest that resistance to parasite

invasion of Malayan ovalocytes is the result of a genetic

mutation that causes increased membrane rigidity.

Papua New Guinea exhibiting this phenotype in some

areas. The hereditary ovalocytosis in these regions,

sometimes also referred to as hereditary elliptocytosis,

is not associated with clinical symptoms on anemia.’2

Kidson et al.’3 recently found that these ovalocytic red

cells from Melanesians in Papua New Guinea were

indeed resistant to infection in culture by the malarial

parasite, Plasmodiumfalciparum, and suggested that

membrane skeletal changes might be responsible for

this resistance. More recently, Hadley et al.’4 found

that these ovalocytes were resistant to invasion not only

by Plasmodium falciparum but also by Plasmodium

knowlesi. Based on these findings and the knowledge

that these parasites recognize different surface recep-

tons,5 the authors suggested that the resistance of these

ovalocytes to malarial parasite infection must involve a

feature common to the invasion pathways of both

parasites, rather than a specific receptor defect.

In order to define the mechanism of protection

against parasite invasion in these ovalocytes, we stud-

From the Departments of Laboratory Medicine and Epidemiol-

ogy and International Health. Cancer Research Institute. Univer-

sity ofCalifornia. San Francisco, CA. and the Institutefor Medical

Research, Kuala Lumpur, Malaysia.

Supported in part by Grants AM 26263. AM 16095, and AM

2157/from the National Institutes of Health.

Presented in part at the American Society ofHematology Annual

Meeting, Washington. D.C.. December /982 fBlood 60(Suppl 1):

38a, 1982/.

Submitted September 30, 1 983,’ accepted January 6, 1984.

Address reprint requests to Dr. Narla Mohandas, Cancer

Research Institute, M-I 282, University of California. San Francis-

co, CA 94143.

© 1 984 by Grune & Stratton, Inc.

0006-4971/84/6306--OO/9$03.OO/O

For personal use only.on October 23, 2017. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

1386 MOHANDAS ET AL.

ied membrane and cellular deformability properties of

these cells. We have been able to show that the

resistance to invasion is directly related to the

increased membrane rigidity of the ovalocytes. More-

over, we were able to induce similar resistance to

parasite invasion in normal red cells by increasing their

membrane rigidity. Our results suggest that resistance

to parasite invasion of ovalocytes in Malayan ab-

onigines is the result of a genetic mutation that results

in increased membrane rigidity.

MATERIALS AND METHODS

Blood was obtained from seven Malayan aborigines who had

previously been identified, on the basis of red cell morphology, as

having hereditary ovalocytosis, and from four normal Malayan

subjects. Blood drawn into acid citrate dextrose was shipped on ice

from Kuala Lumpur to San Francisco. The delay from the time

blood was drawn to the time of analysis was less than 36 hr.

Resealed ghosts for membrane deformability and stability mea-

surements were prepared by a procedure adapted from Johnson.’5

The cells were first washed 3 times in 140 mM NaCl, 5 mM

Tris-HC1, pH 7.4 (“resealing buffer”). They were then lysed in 40

vol of ice-cold hypotonic medium, consisting of 7 mM NaCI, 5 mM

Tris-HCI, pH 7.4 (“lysing buffer”). After hemolysis was complete,

the hemolysate was centrifuged at 1 5,000 rpm for 10 mm in a Sorvall

RC-5 centrifuge, and the supernatant was removed. Ghosts were

resuspended in 10 vol of “resealing buffer,” after which they were

incubated at 37#{176}Cfor I hr to promote resealing. A subsequent

centrifugation at I 5,000 rpm for 5 mm produced a concentrated

ghost suspension for the membrane deformability and stability

measurements.

Normal red cells with increased membrane rigidity were prepared

by treating cells with very low concentrations of glutaraldehyde. For

treatment, the red cells were washed 3 times in buffered saline,

containing potassium and glucose (BSKG: 134 mM NaCl, S mM

KC1, 8.6 mM Na2HPO4, 1.4 mM NAH2PO4, and 11 mM glucoseadjusted to 290-295 mosmole/kg and pH 7.4), and resuspended to a5% hematocrit. A 0.8% stock solution of glutaraldehyde (Poly-

sciences, Inc., Warrington, PA) in BSKG was then added to the red

cell suspension to give the final desired concentration in the range of

0.008%-0.032%. The cell suspension was incubated for 5 mm atroom temperature, and the red cells washed twice in BSKG and

resuspended in BSKG to a final hematocrit of 40%. Treatment at

these low concentrations of glutaraldehyde resulted in increased

membrane rigidity in the absence of detectable hemoglobin cross-

linking or consequent increases in cytoplasmic viscosity. Before

addition to P. falciparum cultures, these cells were washed 3 times

with tissue culture medium.

The deformability of intact red cells and resealed erythrocyte

ghosts was measured in an ektacytometer.”’8 This device imposes a

well defined laminar shear stress field on the cells, while simulta-

neously monitoring the extent of cell deformation by laser diffrac-

tometry. A “deformability index” (DI) is obtained, which is equiva-

lent to the ellipticity of the deforming cells. In the standard mode of

operation, DI is recorded continuously as a function of shear rate.

For measurement of intact red cell deformability, 10 �il of a 40% red

cell suspension was thoroughly mixed with 3 ml of polyvinyl pyrroli-

done (PVP, mol wt 360,000, 4 g/dl w/v, 32.6 cp at 20#{176}C290

mosmole/kg, pH 7.4). This suspension produced a maximum shear

stress of 170 dynes/sq cm at 100 rpm. Numerical values of the

maximum deformability index reached (defined as DI,,,�,), were used

to compare the deformability of different samples. For measurementof the deformability of resealed membranes,’9 30 �l of packed

resealed ghosts (approximately 250 x 106) were suspended in 3 ml

Stractan (25 cp viscosity 290 mosmole/kg, pH 7.4). The ektacytom-

eter was also used to measure whole cell deformability of red cells asa continuous function of the suspending medium osmolality at a

constant applied shear stress of 1 70 dynes/sq cm (osmotic gradient

ektacytometry). For these studies, the DI of red cells was continu-

ously recorded as the suspending medium osmolality was linearly

increased from 50 to 500 mosmole/kg. As previously shown, the

curve showing the variation of DI with suspending medium osmolal-

ity can be analyzed to provide information about initial cell surface

area, surface area-to-volume ratio, and cell water content.2#{176}

Membrane stability was measured by a membrane fragmentationassay using the ektacytometer.2’ Resealed ghosts were suspended in

a dextran solution of97 cp viscosity (dextran mol wt 40,000, 35 g/dl

w/v, 290 mosmole/kg, pH 7.4). Rotation of the ektacytometer

chamber at a speed of 1 10 rpm generated a shear stress of 575

dynes/sq cm. Continuous application of this stress resulted in

progessive fragmentation of the intact membranes into small unde-formable spherical fragments. This process was detected as a

decrease in the DI, which was monitored as a function of time. The

time required for the DI to fall to half of its maximum value was

taken as a measure of the susceptibility of ghosts to shear-induced

fragmentation and, hence, of membrane stability.

To confirm the information about the influence of cell watercontent, and hence internal viscosity, of whole cell deformability

derived from osmotic gradient deformability profiles, we analyzed

the red cell density distribution on discontinuous Stractan density

gradients.2223 As red cell density is largely determined by cell

hemoglobin concentration, the distribution of cells along density

gradients gives a measure of heterogeneity of hemoglobin concentra-

tion and hence internal viscosity in a given cell population.

Invasion of red cells by Plasmodium falciparum merozoites was

measured by culturing late-stage parasites (schizonts) with unin-

fected cells.3’6’24 Red blood cells to be tested were washed twice in

RPM! 1640 (GIBCO Laboratories, Grand Island, NY) and resus-

pended at a hematocrit of 1% in complete medium (RPM! !640, 25

mM HEPES, 40 �g/ml gentamicin, 10% human AB serum).

Schizonts of P. falciparum, collected by gelatin sedimentation, were

added to produce a concentration of 2 schizonts/ 100 red cells.

Duplicate aliquots of 500 z1 of this cell suspension were incubated

overnight in 16-mm culture wells at 37#{176}Cunder a gas phase of 5%

02, 3% CO2. and 92% N2. Invasion of red cells by parasites was

evaluated either by direct microscopic examination or by quantitat-

ing the incorporation of 3H-hypoxanthine by the newly invaded

parasites. For evaluation by direct microscopic examination, the red

cells from 24-hr cultures were spread on slides, fixed, and stained in

Giemsa. The fraction of red cells containing ring forms was deter-mined by counting sufficient individual cells to give less than 10%

standard error (usually 2,000 red cells). For evaluation of parasite

invasion by hypoxanthine incorporation, 5 jzl of 20 �Ci/ml 3H-

hypoxanthine (New England Nuclear, Boston, MA) was added to

25-hr cultures and the cultures incubated for an additional 18 hr.

The cells were resuspended, collected on GF/c filters (Whatman,

Clifton, NJ), washed in 10% trichloroacetic acid (TCA), 5% TCA,and ethanol. The tritiated counts associated with the precipitated

nucleic acid on the filters was determined using a Packard Tri-Carb,

model 3375. The viability of young parasites was confirmed by

microscopy.

RESULTS

The whole cell deformability profiles of discoid red

cells from four normal Malayan subjects and ovalo-

cytes from the seven aboriginal subjects are shown in

Fig. 1 . The ovalocytic red cells had markedly reduced




co 200

SHEAR STRESS (dynes/cm2)

100 140 180 220 260 300 340 380 420 460

OSMOLALITY (mosmol/kg)

RIGID OVALOCYTES PROTECT AGAINST MALARIA 1387

Ui

z

>.

I-.-j

Ui

Fig. 1 . Deformability index versus shear stress for discoid redcells from four normal Malayan subjects (dashed lines) and ovalo-cytes from seven Malayan aborigines (solid lines). The shaded arearepresents the variation seen in red cells from 20 normal controlsubjects. There is a marked reduction in the deformability index at

all values of applied shear stress for ovalocytic red cells.

deformability indices (DI) values at all levels of

applied shear stress. Deformable normal discoid red

cells become ellipsoidal and orient in the direction of

flow at all levels of applied shear stress in the ektacy-

tometer, generating positive deformability index val-

ues. In contrast, the more rigid ovalocytes appear to

orient in a direction perpendicular to flow without

undergoing deformation at low levels of applied stress,

producing a negative value for deformability index.

Only when higher levels of shear stress are applied do

the ovalocytes begin to deform and start orienting in

the direction of flow, giving positive values for de-

formability index. The marked reductions in the DI

values at all levels of applied stress, as well as in the

maximum value of the deformability index attained by

the ovalocytes compared to normals, indicate that

these red cells have significantly reduced whole cell

deformability.

The observed reductions in whole cell deformability

of ovalocytosis could result from increased internal

viscosity, decreased surface area, or increased mem-

brane rigidity of these cells. These three cellular

factors can be distinguished by osmotic gradient ekta-

cytometry in which the deformability is measured as a

continuous function of suspending medium osmolali-

ty.2#{176}We obtained the osmotic gradient deformability

profiles of normal and ovalocytic red cells, and these

are shown in Fig. 2. The osmotic gradient deformabil-

ity profiles of red cells from the four control Malayan

subjects were nearly normal, with minimal changes

consistent with the 36-hr shipping time. However, the

deformability profiles of the seven ovalocytic samples

were markedly abnormal. The salient features that

distinguished these abnormal profiles were:

( I ) The osmolality at which the deformability index

reached a minimum value in the hypotonic region was

shifted to a lower value, indicating that the ovalocytes

had a more favorable surface area-to-volume ratio and

hence increased osmotic resistance. This indicates that

neither reduced surface area nor reduced surface area-

to-volume ratio could play a role in their reduced whole

cell deformability.

(2) The reduction in the maximally attained value

for DI and the asymmetric nature of the deformabil-

ity profile after the maximum value of the deformabil-

ity index was attained were characteristic features

of red cell membranes with increased elastic shear

modulus,2#{176} and strongly suggested that increased

membrane rigidity may be significantly limiting whole

cell deformability.

(3) The accelerated loss of deformability in the

hypertonic region suggested that increased internal

viscosity may also contribute to the reduced whole cell

deformabililty of ovalocytes.

To obtain more direct evidence with regard to the

possible role of increased internal viscosity, we deter-

mined the density distribution of red cells from two

ovalocytic and two normal samples on discontinuous

Stractan density gradients. The results are shown in

Fig. 3. The density distribution of ovalocytes was very

similar to that of normal red cells. Populations of

ovalocytic red cells with elevated density were not

detected, indicating that subpopulations of red cells

with increased hemoglobin concentration, and hence

increased internal viscosity, are not responsible for

reduced whole cell deformability of ovalocytes.

Ui

z

2-I--i

0

0

Ui0

Fig. 2. Osmotic deformability profiles of discoid red cells fromnormal Malayan subjects (dashed lines) and ovalocytes from sevenMalayan aborigines (solid lines). The shaded area represents thevariations for 20 normal controls. The reduction in the maximally

attained deformability index (the maximum height of the curve). aswell as the asymmetric nature of the curve for ovalocytes.suggests increased membrane rigidity. The shift to the left of thedeformability minimum in the hypotonic range for ovalocytes is theresult of increased surface area-to-volume ratio of these cells.




- -- - - - - - -

24re

-0.2

200 400 600 800 000

SHEAR RATE (sec�’)

Fig. 4. Deformability index versus shear rate for discoid redcells (dashed lines) and ovalocytes (solid lines) in suspendingmedia with viscosities of 24 and 1 00 cp. Note the use of shear raterather than shear stress in the abscissa (shear stress = shearrate x viscosity). The disproportionately higher increase in thedeformability index value of ovalocytes with increasing suspendingmedium viscosity (hence increased applied shear stress) and theability of ovalocytes to reach near-normal maximal deformabilityindex values implies that reduced whole cell deformability of thesecells is limited by increased membrane rigidity.


Fig. 3. Analysis of the density distribution of Malayan ovalo-

cytes (A and B) and normal red cells (C and D) on discontinuousStractan density gradients. The density increased from 1 .070 to1 .124 in 12 equal increments. Note the minimal differences in redcell density distribution between ovalocytes and normal red cells.The absence of dehydrated ovalocytes in the high density regionsof the gradient indicates that subpopulations of cells withincreased cell hemoglobin concentration. and hence increasedinternal viscosity. are not present in the whole blood samples ofMalayan ovalocytes.

Taken together, these results imply that neither

reduced surface area nor increased internal viscosity

can account for the observed reductions in deform-

ability of ovalocytes and suggest that increased

membrane rigidity is the dominant cause of reduced

deformability.

If, as suggested, the deformability characteristics of

ovalocytes are indeed dominated by membrane rigidity

and not by surface area limitation or increased cyto-

plasmic viscosity, then it should be possible to obtain

increased cell deformation by increasing the applied

shear stress sufficient to overwhelm the resistance of

the rigid membrane. Figure 4 shows the effects of

increasing applied shear stress on deformability of

normal and ovalocytic red cells. The abscissa in this

figure is the applied shear rate. The value for applied

shear stress is obtained by multiplying the shear rate

by suspending medium viscosity. It can be seen that by

increasing the applied shear stress by increasing the

suspending medium viscosity, the deformability indeA

of ovalocytes can be almost normalized. However, to

obtain deformation equivalent to normals, the ovalo-

cytic red cells required the application of 8-10-fold

greater shear stress, again emphasizing that reduced

deformability was not limited by surface area in these

cells and confirming membrane rigidity as the basis of

deformability defect.

Direct evidence for increased membrane rigidity of

ovalocytes was also obtained by measuring the de-

formability characteristics of membranes as opposed

to whole cells. Figure 5A shows the deformability

index versus shear rate for resealed ovalocytic mem-

branes in two different suspending media, with viscosi-

ties of 25 and 100 cp. Again, ovalocytic membranes

had reduced deformability compared to control mem-

branes at all values of applied shear stress. When the

deformability data for the resealed membranes were

replotted as deformability index versus logarithm of

shear stress, a linear relationship was seen between the

two variables (Fig. SB). The lines for the ovalocytic

membranes were parallel to those of normal mem-

branes, but were displaced to higher values of applied

shear stress. This implied that to obtain equivalent

membrane deformation, the ovalocytic membranes

required higher values of applied shear stress com-

pared to normal membranes. The magnitude of the

displacement is a direct measure of increased mem-

brane rigidity. For the seven ovalocytic membrane

samples examined, the increased rigidity ranged from

sevenfold to eighteenfold. These observations indicated

that ovalocytic membranes have markedly increased

rigidity. Interestingly, although the ovalocytic red cell

membrane rigidity was significantly elevated, the

mechanical stability of these membranes, as deter-

mined by membrane fragmentation assay in the ekta-

cytometer, was similar to that of normal membranes.

Thus it appeared that although the ovalocytic mem-

branes offer increased resistance to deformation, their

xUi0z

2-I--J

.(

2Ui0




UiCz

>-I--I

4

UiC

UiCz2-

-I

04

0

UiC

A

SHEAR RATE (sec�’)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

xw02

2-

-J

4

2

Fig. 6. Deformability index versus shear stress for normal redcells treated with increasing concentrations of glutaraldehyde.There is a gradual reduction in the slope of the curves withincreasing glutaraldehyde concentration. reflecting the increasingshear rigidity of the red cell membrane.

SHEAR STRESS (dynes/cnt2)


/�/�

� ��9�2�’ II. ;�

�;;�P’ /J.’,//�,

i/�, B111111 1 � � hull � I

5 0 50 00 200300

SHEAR STRESS ( dynes/cm2)

Fig. 5. (A) Deformability index versus shear rate for resealedmembranes prepared from normal discoid red cells (dashed lines)and from ovalocytes (solid lines) in Stractan solutions with viscosi-ties of 24 and 100 cp. (B) Deformability index versus log shearstress for resealed membranes from normal discoid red cells(dashed lines) and from ovalocytes (solid lines). In comparison tonormal membranes. 7-1 8-fold higher values of applied shearstress must be applied to ovalocyte membranes to obtain equiva-lent deformability.

susceptibility to undergo membrane fragmentation at

elevated levels of applied fluid shear stress was the

same as that of normal membranes. This is in contrast

to other forms of hereditary elliptocytosis in which

membrane rigidity was almost normal but membrane

stability was appreciably decreased.2’

The consequences of increased membrane rigidity of

ovalocytes on the ability of malarial parasites to infect

these red cells was studied using the in vitro culture

assay. The results of these experiments are shown in

Table 1 . It can be seen that in general a reduction in

deformability paralleled a reduction in parasitic

invasion, except in one case (subject MS), in which

invasion was lower than would be expected for the

measured decrease in deformability. The decreased

Table 1 . Relationship Between Deformability and Plasmodium

falciparum Infection of Malayan Ovalocytes

3H-Hypoxanthine Incorporated

by 24-hr Culture

cpm Percent Deformabulity

(Mean ± SEM) Normal (% Normal(

Malayan normals

(n = 4) 2,409 ± 499 100 100

Malayan ovalocytes

AC. 847 35 40

MS. 178 7 33

TB. 452 19 23

ND. 320 13 22

S.S. 223 9 10

C.N. 33 1 6

K.A. 100 4 5

parasitic infectivity of ovalocytes was also confirmed

by direct microscopic examination of the red cells after

culture.

In order to further define the relationship between

membrane rigidity and invasion, we studied the rela-

tionship between these two parameters in modified

normal red cells. Red cells with progessively increasing

membrane rigidity were prepared by treating washed,

normal cells with micromolar concentrations of glu-

taraldehyde. The deformability characteristics of such

treated cells are shown in Fig. 6, where it can be seen

that such glutaraldehyde treatment resulted in a

graded loss of red cell deformability. Measurement of

the deformability of resealed membranes prepared

from these treated cells established that the loss in

whole cell deformability was due to increased mem-

brane rigidity.

In order to compare the invasion susceptibility of

these treated rigid normal cells with the native ovalo-

cytes, we determined the ability of malarial parasites





to infect them in vitro. The results are shown in Table

2. It can be seen that as the normal cells become

progressively less deformable, their susceptibility to

infection also progessively decreased. Thus it appeared

that membrane rigidity also influences the ability of

parasites to infect normal red cells.

DISCUSSION

We have found a strong correlation between

increased membrane rigidity and decreased malarial

parasite invasion in both hereditary Malayan ovalocy-

tosis and in vitro-modified normal red cells. This

suggests that membrane rigidity per se is a sufficient

cause for the resistance of ovalocytes to parasitic

invasion, and further implies that red cell membrane

rigidity may have been a genetic trait under positive

selection in malarial areas. At least two different

mechanisms could contribute to the resistance of rigid

membranes to parasite entry: (1 ) the indeformable

membrane may prevent close juxtaposition of cell and

parasite membranes, which is necessary for multiva-

lent receptor interactions, and (2) the indeformable

membrane may limit the process of membrane invagi-

nation and block an attached parasite from entering

the cell cytoplasm.

Additional properties of ovalocytes in vitro suggest

that both of these factors may contribute to reduced

invasion. One of these is a decrease in the expression of

several blood group antigens.25 We have observed that

antigen expression in normal cells can be similarly

decreased by glutaraldehyde treatment (unpublished

observations). The assay for expression is based on

antibody-induced aggregation, and as we can assume

that the glutaraldehyde-treated cells do not lack anti-

gen, lack of expression may be caused by the rigid

membrane inhibiting efficient cell-cell contact. In

some cases, lack of expression may also be caused by

decreased steric accessibility of antibody to antigen. In

either case, however, it is clear that cell-cell interac-

tion, which is dependent on receptor-ligand binding, is

suppressed in a nondeformable cell.

Malayan ovalocytes and glutaraldehyde-treated

normal cells are also both deficient in their ability to

Table 2.

falcipa

Relationship Between Deformability and Plasmodium

rum Infection of Glutaraldehyde-Fixed Normal Cells

Deformability Parasite Invasion

(% Normal) 1% Normal(

100 100

86 71

57 29

24 19

� 5 7

Young parasites had normal morphology. There was no evidence of

glutaraldehyde toxicity.

undergo drug-induced discocyte-stomatocyte transfor-

mation and membrane endocytosis (unpublished

observations). These results could be explained as a

consequence of the rigid membrane preventing the

appreciable curvature changes and skeletal protein

rearrangement demanded by the formation of endo-

vesicles.26 Hence, the influences of increased mem-

brane rigidity on cell-cell interactions and on the

membrane invagination process may both contribute

to reduced parasitic invasion of ovalocytes.

Parasite invasion of the red cell is a complex process

involving multiple steps.24’27 The invading parasite first

attaches to the red cell membrane, weakly and reversi-

bly. After attaining a specific orientation on the mem-

brane, with its apical end in contact with the mem-

brane, the parasite forms an intimate junction with the

red cell membrane that mediates dramatic changes in

red cell membrane protein structure and topology. The

parasite is then enclosed in a localized invagination of

the membrane and is subsequently internalized by

endocytosis. Protection of the host against malaria

could be achieved by blocking any of these stages in

parasite entry and replication. For example, the pro-

tection against malaria seen in Duffy-negative cells

against P. knowlesi and in En (a - ) cells against P.

I alciparum appears to result from the absence of

specific recognition factors for these parasites on the

cell membranes.5 However, in both these instances, the

red cells are susceptible to invasion by the other type of

malarial parasite, as their membranes possess the

factors recognized by that parasite. This is in contrast

to Malayan ovalocytes, which are resistant to invasion

by both P. knowlesi and P. falciparum.’4 Our data

indicate that this general protection against both forms

of parasites could be the result of the rigid membranes

of these cells, which may limit both the attachment of

the parasites to the membrane and also their subse-

quent internalization.

A number of cellular features appear to distinguish

Malayan ovalocytes from other forms of congenital

elliptocytes so far examined. First, Malayan ovalocytes

undergo thermally induced fragmentation when

heated to 52#{176}C,whereas normal red cells undergo

similar morphological changes between 49#{176}and 50#{176}C

and other forms of hereditary elliptocytes between 48#{176}

and 49#{176}C.’3’28Second, the membrane stability of Ma-

layan ovalocyte membranes is normal, whereas that

of the other forms of elliptocytes is markedly re-

duced.21’29’3#{176}Finally, the increase in membrane rigidity

seen for Malayan ovalocytes is far greater than that

observed in other forms of elliptocytes or red cells from

other hemolytic anemias, including even irreversibly

sickled cells. An altered membrane skeleton, resulting

from abnormal skeletal protein interactions due to a





mutant protein, might be expected to confer this

increased rigidity, as the elasticity of the red cell

membrane is primarily regulated by membrane skele-

tal proteins and their interactions.3’ However, sodium

dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) of the ovalocyte membrane prepara-

tions showed no gross changes in the protein composi-

tion of these membranes. It is tempting to suggest that

a mutant spectrin, which might not be detected by

SDS-PAGE analysis, but which had an increased

ability to crosslink, could confer increased rigidity.

Indirect evidence to support such a hypothesis can be

derived from thermal fragmentation studies. It has

previously been shown that the difference between

normal red cell fragmentation, which occurs between

49#{176}and 50#{176}C,and that of hereditary pyropoikilocytes

(HPP), which occurs at 46#{176}C,is the result of the

normal and the mutant HPP spectrin undergoing

thermal transition at these different temperatures.32

Similarly, the fragmentation of Malayan ovalocytes at

52#{176}Ccould result from a spectrin variant with stronger

than normal intermolecular associations.

Our findings suggest that the resistance of parasite

invasion of Malayan ovalocytes is the result of a

genetic mutation that causes increased membrane

rigidity. The molecular and biochemical basis for the

increased membrane rigidity has yet to be defined. As

more knowledge of the biochemical abnormalities

involved in Malayan ovalocytes is acquired, it may well

provide insights into the role of skeletal proteins in

modulating parasite entry into red cells and also

insights into the role of skeletal proteins and their

interactions in the regulation of red cell membrane

material properties.

ACKNOWLEDGMENT

We would like to thank Drs. Stephen B. Shohet and Joel Anne

Chasis for critical reading of the manuscript. We would also like to

thank James Harris for his assistance in the preparation of the

manuscript.

REFERENCES

1. Allison AC: Protection afforded by sickle-cell trait against

subtertian malarial infection. Br Med J 1:290, 1954

2. Trager W, Jensen JB: Human malaria parasites in continuous

culture. Science 193:673, 1976

3. Friedman MJ: Erythrocyte mechanism of sickle cell resistance

to malaria. Proc Natl Aced Sci USA 75:1994, 19784. Pasvol G, Weatherall Di, Wilson RJM: Effects of faetal

haemoglobin on susceptibility of red cells to Plasmodium falcipa-

rum. Nature 270:171, 1977

5. Miller LH, Haynes JD, McAuliffe FM, Shiroshi T, Durocher

JR. McGinniss MH: Evidence for differences in erythrocyte surface

receptors for the malarial parasites, Plasmodium falciparum and

Plasmodium knowlesi. J Exp Med 146:227, 1977

6. Friedman MJ: Oxidant damage mediates variant red cell

resistance to malaria. Nature 280:245, 1979

7. Pasvol G, Wainscoat .15, Weatherall Di: Erythrocytes defi-

cient in glycophorin resist invasion by the malarial parasite Plas-

modiumfalciparum. Nature 297:64, 1982

8. Pasvol G, Wilson RiM: The interaction of malaria parasites

with red blood cells. Br Med Bull 38:133, 1982

9. Baer A, Lie-Injo LE, Welch QB, Lewis AN: Genetic factors

and malaria in the Temuan. Am J Hum Genet 28:179, 1976

10. Lie-Injo LE: Hereditary ovalocytosis and hemoglobin E-

Ovalocytosis in Malayan aborigines. Nature 208:1329, 1965

1 1 . Amato D, Booth PB: Hereditary ovalocytosis in Melanesians.

Papua New Guinea Med J 20:26, 1977

12. Lie-!njo LE, Fix A, Bolton JM, Gilman RH: Hemoglobin

E-Hereditary elliptocytosis in Malayan aborigines. Acta Haematol

47:210, 1972

13. Kidson C, Lamont G, Saul A, Nurse GT: Ovalocytic erythro-

cytes from Melanesians are resistant to invasion by malaria parasite

in culture. Proc NatI Acad Sci USA 78:5829, 1981

14. Hadley T, Saul A, Lamont G, Hudson DE: Resistance ofMelanesian elliptocytes (ovalocytes) to invasion by Plasmodium

knowlesi and Plasmodium falciparum malaria parasites in vitro. J

Clin Invest 71:780, 198315. Johnson RM: The kinetics of resealing washed erythrocyte

ghosts. J Mem Biol 22:231, 1975

16. Bessis M, Mohandas N: A diffractometric method for the

measurement ofcellular deformability. Blood Cells 1:307, 1975

17. Groner W, Mohandas N, Bessis M: New optical technique for

measuring erythrocyte deformability with the ektacytometer. Clin

Chem 26:1435, 1980

18. Mohandas N, Clark MR, Jacobs MS. Shohet SB: Analysis of

factors regulating erythrocyte deformability. J Clin Invest 66:563,

I 980

19. Heath BP, Mohandas N, Wyatt JL, Shohet SB: Deformabil-

ity of isolated red blood cell membranes. Biochim Biophys Acta

691:211, 1982

20. Clark MR. Mohandas N, Shohet SB: Osmotic gradient

ektacytometry: Comprehensive characterization of red cell volume

and surface maintenance. Blood 61:899, 1983

21. Mohandas N, Clark MR. Heath BP, Rossi M, Wolfe L, Lux

SE, Shohet SB: A technique to detect reduced mechanical stability

of red cell membranes: Relevance to elliptocytic disorders. Blood

59:768, 1982

22. Corash LM, Piomelli 5, Chen HC, Seaman C, Gross E:Separation of erythrocytes according to age on a simplified density

gradient. J Lab Clin Med 84:147, 1974

23. Clark MR. Mohandas N, Embury SH, Lubin B: A simple

laboratory alternative to irreversibly sickled cell (ISC) counts. Blood

60:659, 1982

24. Friedman Mi, Blankenberg T, Sensabaugh G, Tenforde T:

Recognition and invasion of human erythrocytes by malarial para-

sites. Contribution of sialoglycoproteins to attachment and host

specificity. J Cell Biol (in press)

25. Booth PB, Serjeantson 5, Woodfield DG, Amato D: Selective

depression of blood group antigens associated with hereditary ovalo-

cytosis among Melanesians. Vox Sang 32:99, 1977

26. Haest CWM, Fischer TM, Plasa G, Deuticke B: Stabilization

of erythrocyte shape by a chemical increase in membrane shear

stiffness. Blood Cells 6:539, 1980

27. Wilson Ri: How the malarial parasite enters the red blood

cell. Nature 295:368, 1982





28. Tomaselli MB, John KM, Lux SE: Elliptical erythrocyte

membrane skeletons and heat-sensitive spectrin in hereditary ellipto-

cytosis. Proc NatI Acad Sci USA 78:191 1, 1981

29. Tchernia G, Mohandas N, Shohet SB: Deficiency of skeletal

membrane protein band 4. 1 in homozygous hereditary elliptocytosis.J Clin Invest 68:454, 1981

30. Liu SC, Palek J, Prchal J: Defective spectrin dimer-dimer

association in hereditary elliptocytosis. Proc Natl Aced Sci USA79:2072, 1982

31 . Evans EA, Hochmuth RM: A solid-liquid composite model of

the red cell membrane. J Memb Biol 30:351, 1977

32. Chang K, Williamon JR. Zarkowsky HS: Altered circular

dichroism of spectrin in hereditary pyropoikilocytosis. J Clin Invest

64:326, 1979




1984 63: 1385-1392

N Mohandas, LE Lie-Injo, M Friedman and JW Mak malariaRigid membranes of Malayan ovalocytes: a likely genetic barrier against

http://www.bloodjournal.org/content/63/6/1385.full.htmlUpdated information and services can be found at:

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of


http://www.bloodjournal.org/content/63/6/1385.full.html

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





Documents

Rigid - Semantic Scholar · Supported in part by Grants AM 26263. AM 16095, and AM 2157/from the National Institutes of Health. Presented in part at the American Society ofHematology