Differences in erythrocyte membrane proteins and glycoproteins in sickle cell disease

Vol.

74,

No.

1, 1977

BIOCHEMICAL

DIFFERENCES AND

IN

GLYCOPROTEINS

November

BIOPHYSICAL

ERYTHROCYTE IN

Michael G. Massachusetts Cambridge,

Received

AND

RESEARCH

MEMBRANE

PROTEINS

SICKLE

CELL

Vernon of

M. Ingram Technology 02139 USA

Riggs

and Institute Massachusetts

COMMUNICATIONS

DISEASE

4,1976

Summary: Erythrocyte membrane proteins obtained from individuals with sickle cell anemia show an SDS polyacrylamide gel pattern that differs in five regions from the normal pattern. These membranes when compared with membranes from normal individuals also show a marked decrease in sialic acid content which correlates with a marked reduction of the periodic acid-Schiff staining of the three major glycoprotein components. The observed membrane protein and glycoprotein changes are a characteristic of all the red cells in sickle cell anemia and do not correlate with the proportion of irreversibly sickled cells. When bizarre on

shapes

ing

but

a permanent

that

renders of

molecular (4)

the

throcyte

the

redissolves irreversibly

remains

in

the

deoxygenated

of

the

internal

and

most

irreversibly

sickled

sickled the

membranes

are

similar.

the

membrane

cell

membranes

patterns

and

normal

However, protein

we and

the

cells

is

rigid

(1). have

been

(AA) have glycoprotein

for

looked

conflicteven-

changes

Durocher and

and

sickle for

patterns

to

the

in

the

Conrad

cell and

a

Apparent-

approach

look

cells.

Up-

cycle

Another to

assume

assume

sickle-unsickle

(2,3).

such

they hemoglobin.

There

the

(ISC) of

from

of

membrane.

during

protein

among

become

sickled

fragmentation

irreversibly of

anemia

crystallization

the

tons

cell

remain

cell

composition

reported

al terat

many

cell the

sickle

hemoglobin

alteration

reports

problem

by

the

shape,

tually

in

caused

reoxygenation

normal ly

erythrocytes

(SS)

ery-

have

found

of

SS ery-

throcytes.

METHODS Cells and membranes were prepared as in reference 5, except that O.OlM Tris, 0.15M NaCI, pH 7.4 was used for washing cells, and O.OlM Tris pH 7.4 was used for preparing membranes. Protein determination was performed by the Lowry procedure (6). SDS-polyacrylamide gel electrophoresis of 25pg dissolved membranes was performed as in reference5,except 4.5% acrylamide, 0.17% bisacrylamide; 0.4% SDS and 80mM dithiothreitol in sample buffer; Quantitation of staining intensity 0.2% SDS in electrophoresis buffer. (Coomassie blue) of bands was with a Gilford spectrophotometer Model 6040/ 2410-S attached to an Autolab digital integrator Model 6300. For glyco-

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

191

Vol.

74,

30-

No.

1, 1977

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

40

2 A

0

AA . . . . . ss

-IS

06-

-7 i

E

AA . . . . ss

E

- 1.0 0 2 a

a

0 04x

-05

Figure 1: Proteins and erythrocyte membranes. stained with Coomassie 16% irreversibly sickled different since the gels (B) Glycoprotein gels. protein (total protein conditions and scanned mean and range of five

glycoproteins of normal and sickle cell anemia (A) Protein gels loaded with 25 pg total protein, blue; bands numbered as in (7). This SS sample had cells. The full scale absorbance of the gels is were run and destained on separate occasions. All gels were loaded with equal amounts of membrane minus hemoglobin), handled together under the same at the same full scale absorbance. Bars indicate normal and seven sickle cell samples.

Table

Analysis Band

Pre-I 1 2 2.1 2.2 2.3 2.1

:::A 4.58

65 ii Globin

(a) (b) (c)

of Proteins

I

in Normal

AA(a)

sdb)

and Sickle

Cell Membranes

Significance(c) of Difference

Direction Difference

of (AA+sB)

0.8 14.7 12.4 3.9 2.0 1.3 29.1 4.5

ko.28 50.67 20.55 to.70 kO.20 20.20 20.90 20.17

0.7 13.6 11.3 3.7 I.8 1.0 27.1 4.7

k0.42 k1.41 kl.11 20.49 k0.20 kO.17 20.32 +1.15

co.01 co.01

6.8 4.6

ko.37 20.57 ko.36

4.3 8.5 3.0

to.26 k1.17


+

2.9 5.0 5.3 3.4 1.6

20.24 50.66 io.39 50.45

6.3 5.1 2.6 4.3

*1.07 ko.46 k0.81

co.01 co.01 co.01

+ +

0.6

f0.22

2.7

20.97

co.01

+

20

samples;

expressed

as

% of

total

protein

less

15

samples;

expressed

as

% of

total

protein

less

Mean +S.D., 15 donors, globin. Mean ?S.D., IO donors, globin. From t distribution.


kO.39

192

Vol. 74, No. 1, 1977

BIOCHEMICAL

protein globin, areas

quantitation gels stained with PAS cut from photocopies In vitro “sick1 ing” wholeblood at 37°C under Penicillin and streptomycin tively.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were loaded with 15Opg protein, not including reagent (5), quantitated by weighing the peak of gel scans. is an overnight incubation with gentle shaking of a humidified atmosphere of 5% CO2 in nitrogen, were present at 100 U/ml and 100 ug/ml respec-

RESULTS Our sickle

gel cell

from

in

tate.

high weight These

changes

same

SS blood

These

changes when

are

known

under

increased

further

(data

are

contain

The the

membranes

sense

they

in

the

molecular

be

due

to

the

PAS = periodic

energy

shown

are

in

Table

persons

might

adsorb

weight lower

high

levels

of

(11).

Such

1 are with

sialic

acid-Schiff’s

also

sickle more

range

of acid

of

plasma 4.5

content

of

reagent.

193

when

among

the

molecular

aliquots

of

the

procedure. are

among

(9).

those

Sickle

(lo),

is

where-

events.

conditions It

cells

especially favor

possible

the

that

the

effects. with

disease proteins

bands

lower

calcium

compatible cell

in

calcium

(3).

induced

patient, are

the

is

precipi-

decreases

proteins

presence

calcium

to

“sickling”

the

cells

globin

degradative

vitro

membrane

depletion

patient

shown)

signifi-

components;

an occluded

several not

are

as

begin

or

A (Fig.

several

expected,

the

an --in

of

sickled

observe

from

that

in

irreversibly we

data

lysed abnormally

of

to

amounts

all

obtained

As

from

increases one

AA cells

which

the

suggesting

relative

3 overlapping

probably

with

hemoglobin

2 or

While

subjected

only

small,

varies

constant.

are

have

donors

patterns

fairly

(8), changes

from

with

8 appears.

band

I),

conditions

changes

into

(Table

sample

of

and

are

proteins,

in

generation

splits

who

weight

are

to

and

these

remain

classes

seen

of

samples

molecular

changes

proteins

compared

white,

preparations

magnitude

normal

or

the

7 increase

SS membrane

The

changes

black all

6 and

membrane

several

3 decreases

4.5A,

present

show

While

Band

bands

erythrocyte

individuals, 1).

cant.

of

anemia

normal

lA,Table

as

patterns

to

are or

8.

sickle

the

hypothesis

“sticky”

intracellular

Such

stickiness

cell

membranes

that

in

the proteins

might which

we

BIOCHEMICAL

Vol. 74, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

Analysis of Glycoproteins

II

in Normal and Sickle Ce2.1Membranes

/da)

Glycoprotein(c)

592

db)

460

+21

% Change AA + ss

+26

-22.3

p<
+ 2.7

-19.0

p co.01

31

*

1.5

25

Neutral Carbohydrate(e)

55

f

5.5

55 + 6.9

N-acetyl glucosamine(f)

92

A

8

88

69

+

5

64 i 7

Sial

ic

Acid(d)

N-acetyl galactosamine

(f)

Significance of Difference

*12

Mean +S.D., 6 donors. Counts of ISCs in oxygenated Mean +S.D., 7 donors. 16 to 61%. Arbitrary units, determined as in Methods. ug/mg membrane protein, determined by the method of hydrolysis in 0.1 N H2SOh at 80”~ for 1 hour. ug/mg membrane protein, determined by the method of n moles/mg membrane protein determined on the amino (Durrum D 500) after hydrolysis in 4 N HCI at 100°C

(a) (b) (c) (d)

report

Rehfeld

below.

bumin

to

e cells.

sick1

polypeptides

1 to

observed. thesis

that

ed

cells

red

under

4.5,

6,

our

increase

in

in

of

serum

or

7,

8 and

globin.

described

by

vitro

an --in we

feel

with

sickling certain

NaF In

Thus our

Table incubation

of

the

4.5

in

which

indicate

merely

due

Tris-saline case

quantitative

I for in

the

occurrence

cell absence of

194

the

of

to

as

was

an

protein

fact

hypowashsickled

buffer

in of

the bands

polypeptide are

serum

of

binding,

increase in

al-

in

artificially

membranes of

is

the

albumin

Ca++

(14).

amounts

normal,

were

after

human

relative

changes

sickle

(13)

against

ImM

there

Warren

from

Dubois et al. acid analyser for 6 hours.

the

than

anemia

varied

binding

then

smaller

cell

every the

case,

be

is

sickle

20mM

increased

the to

band

plasma.

in

were

experiments in

N1 with

describe

appear

four

persons

(12)

this

would

,

position

though

4.2

from

al. If

However

5% CO2

absence

et

smears

com-

increased

proteins.

changes

Alin

the

gel

Vol.

74,

No.

1, 1977

patterns

from

these

BIOCHEMICAL

sickle

cell

membranes,

BIOPHYSICAL

RESEARCH

we

cannot

yet

be

the

three

major

lower

staining

COMMUNICATIONS

sure

of

the

cause

of

changes. The

PAS

quantitative

I,

II

three

and

examination

III

-

showed

glycoproteins

the

PAS

in

a given

are

in

reactivities sample;

of

significantly

seven

of

SS samples

all

the

three

relative

(Fig.

16,

glycoproteins

human

of

Table

the

-

intensities

are

mobilities

glycoproteins

II).

lower

of

all

Interestingly, by

the

same

glycoproteins

in

amount

the

gel

unchanged. Erythrocyte

sialic

acid,

these

classes

PAS

membranes

sial

ic

of

all

three

highly

II

performed

ability.

In

acid

(Table

no

We conclude in

servation

that

acid

but

weakly,

in

susceptible

whether

acid

plot

removal

particular

of

for

preparations

from in of

made.

that

two

conditions

Table

IL

an

were

of of from

frequencies unusual

195

r = 0.89,

~~0.01).

no

statistically content. which of

to

the of

amount

negatively,

and

percent

irreversibly

sialic

acid

total

residues

neutral

are

sickling.

donors.

acid

of

irreversibly

and

white

ob-

treated

correlate

irreversible

is

the

content

among sialic

are

neuraminidase

These

the

content

intensity

light

proportion

content

thirds

under

group

in

glycoprotein acid

a lower

material

proportion

the

of

AA samples

and

reactive

especially

content, sialic

showing

PAS

reduction

staining

SS and

of

N-acetylgalactosamine

acid,

were

of

blood

the

each

is

the

carbohydrate

Counts

SS samples

the

origin,

and

sialic

16).

the

through

PAS staining

(5,

sialic

AA samples

responsible

is

and

all

neutral

of

membrane

suggests to

in

of

(15):

for

1B there

content

almost

portion

decreased

The cells

The

of

the

with

carbohydrate. sickled

the

carbohydrates

nature Fig.

N-acetylglucosamine

liberated

cells

of

acid

total

of

Assays

the

SS samples

line in

in that

show

determine

glycoproteins

change

gels

classes

carbohydrate.

Sialic

SS membranes

erythrocytes

sickled

the

I I).

changes

decreased

neutral

(regression

shows

three

to

PAS-positive

significant

sialic

and

were

correlated

Table

contain

hexosamines

staining

of

be

AND

black

To

determine

individuals

distribution,

might samples

not

Vol.

74, No.

from

four

globin

white type

acid

content In

of

sialic

acid,

to

fractionate from

tensity

of

of

percentage

is ISCs

in

in

it

factor

the in

AA cells

The

was

position

on

SS serum

removes

such

heparinized

37°C.

at

that

at

(18)

Bertles levels and

with blue

decrease as

loss

content

which

the protein

for

a higher

also

contain-

in

a whole

of

the

of

SS cell

as AA cells

decreased

to

in

red

only

and

65%

plasma,

(lg),

terminal

washed

and

the

staining

Table

II.

The

incubation 3 percent,

the

some

it

sickle and

cell not

of

AA control

respectively.

test

of

staining

the

incubation after

also the

was incubation that

the

were

in-

glycoprotein

the 84%

washed

performed.

of

hand

to

some

idea,

heterologous other

known

AA plasma

intensity

either

acid

that this

were

intensity

We conclude

196

To in

sialic are

possible

incubations

in

the

is

SS cells

On

before

of which

residues.

and

which of

cell

homologous

after

glycoproteins 71

the

least

glycoproteins

The

isolated

determined

heterologous

population

the

ISC

except

acid

cell

of

Coomassie

fraction

process.

and

correlated

fractions,

sialic

membrane

SS plasma

were

fell

red

ISCs

acid

not

sialic

disease

of

the

bottom

the

assume

the

of

plasma

the

to

surface

logous

and

reasonable

overnight

proteins

trol,

is

membranes

bands

the

external

in

cubated

that

the

Sialic

the

particular.

a terminal

be on

of

the

in

containing

were

PAS-

and

technique

the

of seen

level

Furthermore,

We feel

a characteristic

the

and

among

as

levels

with

population.

5%) in

vs.

the

fractions

slightly

changes

(16%

(45%).

ISCs

cell

Hemo-

glycoprotein

connected

obtaining

fraction. no

globin

most

Since is

showed

of

red

compared.

differences

ultracentrifugation

only

the

any

between

COMMUNICATIONS

electrophoresis in

were

be

sample, the

gel

PAS positive

a connection

of

in

nor lower

the

were

differences

therefore

varied

ISCs

pattern

anemia

45%

PAS stain

percentage

the

for

SS blood

5 to

acid,

must

we employed an

varying

staining

look

no

RESEARCH

donors

acrylamide

observed

SS cells to

pH 8.9

sialic

The

BIOPHYSICAL

AA unrelated

We found

or

of

order

on

(17).

pattern.

AND

black

determined

glycoprotein

protein

the

five

previously

positive

ed

AA and

was

described

gel

BIOCHEMICAL

1, 1977

the

glyco-

or

homo-

PAS

staining

of

the

in

homologous

loss

AA con-

of

sial-

in-

BIOCHEMICAL

Vol. 74, No. 1, 1977

ic

acid

from

caused

by

SS membranes

a plasma

is

an

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

intrinsic

property

of

the

cells

and

not

factor.

DISCUSSION The as

is

mechanism

the

impact

havior

and

sialic

acid

ing acid

and

ISCs

fate

of

retain

acid

sialic

is

acid It

(16,

look

for

have

on

has

been

suggested

Since

the

the

membranes

may

provide

of

further

SS cell

could 20).

characteristic

sickle

from

erythrocytes

cells

must

their

of

older

membrane we

sialic

SS erythrocyte.

these

not,

of

loss

the

content

or to

of

its

loss

which

destroying

from

. sickled

of

all

the

is

subsequent

be-

that

a means partial

a lower

of

loss

SS erythrocytes,

membrane

unclear,

recogniz-

of

sialic

irreversibly

defect

which

causes

shape.

We thank the following for help in obtaining SS and AA blood samples: Ors. Louis Sullivan, George Buchanan, Warren Point, Murray Eden, Joseph Votano and Allison Hagerich. Dr. Lisa Steiner and Mr. John Novosad performed the amino sugar analyses. Supported by the Boston Sickle Cell Center, USPHS - HL 15157.

REFERENCES 1.

Chien,

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Usami, Diggs,

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2.

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Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (1971) Biochemistry, 10, 2606-2617. Lowry, D.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1851) J. Biol. Chem, 183, 265-275. Steck, T.L. (1972) J. Mol. Biol, 66, 295-305. Fischer, S., Nagel, R.L., Bookchin, R.M., Roth, E.F., Jr., and TellezNagel, I. (1975) Biochem. Biophys. Acta, 375, 422-433. Triplett, R.B., Wingate, J.M., and Carraway, K.L. (1972) Biochem. Biophys. Res. Commun, 48, 1014-1020. Skelton, T.D., Swofford, H.S., Kolpin, C.E., and Jacob, Eaton, J.W., H.S. (1873) Nature, 246, 105-106. Weed, R.I., LaCelIe, P.L., and Merrill, E.W. (1969) J. Clin. Invest, 48, m-809. Rehfeld, S.J., Eatough, D.J., and Hansen, L.D. (1875) Biochem. Biophys. e Res. Commun, 66, 586-591. Warren, L. (1959) J. Biol. Chem, 234, 1971-1975. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. (1856) Anal. Chem, 28, 350-356.

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Blood, R. Sot.

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225-305. Bertles, Marchesi, New York. Balduini,

J.F., V.T. C.,

and Milner, (1975) Cell Balduini,

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