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.
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Blood, R. Sot.
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225-305. Bertles, Marchesi, New York. Balduini,
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