Vol.
167, No. 3, 1990 March 30, 1990
BIOCHEMICAL
ISOLATION
OF
(DESIALYLATED) FROM
BLOOD
OF
Vladimir
of
February
blood
3rd
(1,2).
PATIENTS:
NATIVE
Igor A. Sobenin,
USSR Cardiology
Cardiology,
reported
intracellular
lipid
neuraminidase
induced
Abbreviati agglutinin;
%S,
of
G. Tonevitsky,
N.Smirnov
Str.
15A,
Moscow
Research
Russia
121552,
8, 1990
that
with
It was demonstrated
uptake
Alexander
and Vladimir
low density
coronary
intimal that
heart
cells this
derived
level.
Furthermore
desialylated
LDL (3,4).
: LDL, low density phosphate buffered
OOO6-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
LDL of
lipid
intracellular
lipoprotein disease from
isolated
(LDL)
induces
(CHD) grossly
normal
from
lipid
human aorta
sialic acid (3,4); a negative correlation being and the LDL ability to elevate the
LDL has
as compared with LDL from healthy subjects established between the sialic acid content
greater
LIPOPROTEIN
CHROMATOGRAPHY
N. Orekhov,
in cultured
accumulation
DENSITY
Cherepkovskaya
patients
of
LIPOPROTEIN
Experimental
We have recently the
LOW
AFFINITY
V. Tertov,
Center,
Received
MODIFIED
FROM
Alexander
Institute
ATHEROGENIC
ATHEROSCLEROTIC
SEPARATION BY
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1122-1127
a lower
healthy
accumulation Proceeding
content
of
subjects which from
treated may be due
these
with to
observations,
lipoprotein. RCA saline; BSA,' bovi@ker%c&%%ir?mmuniS
1122
a
Vol. 167,
3,
No.
we have
BIOCHEMICALANDBIOPHYSICALRESEARCH
1990
concluded
that
desialylation
LDL modifications
atherogenic
bond
(5,6).
Sialic
one of
represents
possible
the
of LDL in vivo.
The molecule of apolipoprotein polysaccharide chain (high-mannose
glycosidic
COMMUNICATIONS
B (apo and
sialated
acid is a terminal
contains
B)
two types
biantennary)
linked
of by N-
saccharide of the biantennary
The gangliosides of LDL also contain a terminal sialic acid (7). type (5,6). in N-linked chains sialic acid is preceded by galactose residue, desialylation results in exposure of galactose, suggesting that LDL can bind
Since
to galactose-specific lectins such as the Ricinus communis agglutinin D-galactose and low RCA120 exhibits high affinity for terminal (RCA12rJ. affinity for other saccharides of the LDL molecule (8). In the present study we have
attempted
Sepharose-linked
to isolate RCAl20
human aortic
in cultured
desialylated
LDL by affinity
and to investigate
intimal
LDL effect
chromatography on lipid
on
accumulation
cells.
MATERIALSAND METHODS
LDL (d=1.019-1.063 mg/ml) was isolated by ultracentrifugation Lindgren ( and desialylated by treatment with neuraminldase pf;Twhere ?i ). Sialic acid content was determined according
with a he ane-isopropanol mixture (3:l v/V) (14x). The otal cholesterol &tent Wt< Boehringer Mannheim Kits etermined by the method of Siedel et al. (15 1 usin Ma nheim F.R.G.). Cell protein was measured by t ll e method of Lowry et al.
the
Lipids
according to as described to Svennerholm
method
were extracted of Hara and
Radin
167.
Significance
Program
package
of differences (17).
was evaluated
using
a BMDPIV
statistical
RESULTS was not washed with PBS-NaCl or to RCA120- Se p harose PBS/Z% BSA. Fig. 1 shows LDL binding to the sorbent depending on the amount of Part
of LDL applied
1123
Vol.
167, No. 3, 1990
01
BIOCHEMICAL
1
Protem
aw.
LDL
applied
(mg)
applied
that
LDL
the
amount
(Fig. the
to
the
was eluted of
column.
0.78
with
2). All LDL was eluted molecule (sialic
that the
preparation
galactose
(Fig.
solution.
increased
acid,
2).
obtained
proportion (from
eluted only subjects,
the
of
the
It
should
but
not
other
D-glucose,
D-mannose,
This finding indicates that LDL binding to RCA120-Sepharose.
from
patients' derived
LDL capable
20 to 64X),
that
with galactose
by 25 mM galactose,
in the LDL preparation
average
12.50
concentration
can be seen
sites for Fig. 3 shows the elution profile by a linear isolated from healthy subjects and CHD patients.
LDL
3.13
Sugar
50.00
(mM)
of
and these
dependence
is
of RCA120-Sepharose.
LDL in the eluate
LDL
acetylglucosamine) serve as specific
It
mg protein/ml
0.25-1.5
Bound
0
to RCA -Sepharose dePendin on the amount o,f LDL amount'# LDL isolated rom poo ed blood was applied to Sepharose and processed as described in Materials and as measured after washing with 50 mM galactose.
htmb~~t”~~u,“d’t~r~~~~~~
linear within
02
z
LDL binding The indicated
lipoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
blood from
binding
the
saccharides L-fucose,
galactose
of N-
residues
share of bound LDL exceeds subjects. In CHD patients,
RCA120-Sepharose
LDL preparations
by high galactose concentrations (30-50 proportion of bound LDL was ~15%. 1124
mentioned
concentration
galactose gradient for LDL It can be seen that in the
healthy to
be
contained mM) (Fig.
was
48+7%,
on
LDL which were 3).
In healthy
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
30
Fraction kk&edE#k?& blkodf?? applied to a 5-ml column linear galactose gradient aliquots.
It can be 2-fold lower healthy subjects. and
unbound
TABLE
seen
LDL,
1.
Table 1 of sialic Patients’ LDL from
content
a
1.5-fold
Effects of cultured
LDL
cells
number
-Sepharose b l'near alactose and ZHD patienk Rhc,!l?fly subjects Bound LDL was with RCA Sepharok' 8&l cholest’erol was measured and
that
LDL
acid bound
higher
isolated as compared of sialic
intracellular
on
normal
human
pg 1 mg p$otein Healthy
Total
Bound Unbound
log 92
subjects
:E 26:9 CHD patients
Total
Bound Unbound
'308 62
14.7 I&%
Desialylated
in
vitro
blood
LDL had
acid
as
had
obtained a 3-fold
compared
cholesterol aortic intima
Cholesterol accumulation % over contrbl
WC co tent
e1ut.e with a in 0.5-ml
patients' with
RCA ,2D-Sepharose
to
content
subfractions from grossly
from
rad'ent. LDL ?$~).were
content
a
from lower
with
in
the
Vol.
167, No. 3, 1990
total
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS
The sialic
LDL fraction.
obtained
healthy
from
preparation
acid content
subjects
The
total
the
preparation
Z-fold
higher
than
that
in
sialic
acid
content
in
the
was
of CHD patients.
in
of LDL the LDL unbound
lipoprotein
fraction of healthy subjects was similar to that in the total LDL fraction while in bound LDL it was Z-fold lower. It can be concluded from these observations that LDL isolated both from patients’ and healthy subjects' blood and capable of binding to RCA 12D-Sepharose are desialylated lipoproteins. To confirm
subjects 3-fold
hypothesis,
this
were desialylated
fall
LDL bound
in
sialic
the
LDL isolated
from the blood of
by neuraminidase treatment acid content.
About
healthy
which brought
90% of -in vitro
about
a
desialylated
to RCAlzO-Sepharose.
Incubation of human aortic intimal cells with the total preparation of LDL blood had no effect on the intracellular isolated from healthy subjects' cholesterol level (Table 1). By contrast, the bound LDL fraction induced
cholesterol with
accumulation.
neuraminidase
intracellular
or
Incubation
the
bound
with both
total
LDL subfraction
preparation
The total
of LDL isolated
from
patients’
increase in intracellular cholesterol content effect (4-fold increase in intracellular cholesterol bound
LDL subfraction.
intracellular Table from
treated
increase
in
The unbound
blood produced
(Table
1).
level)
LDL subfraction
a 2.5-
A more
potent
was observed
induced
with
in
no increase
cholesterol. 1 shows
pooled
results
to a 3-fold
cholesterol.
fold
the
LDL preparations
led
the
healthy
results
of experiments
subjects'
(n=lO)
with
LDL preparations
and CHD patients’
isolated
(n=lO) blood. Similar
with LDL preparations isolated from 2 pools of healthy subjects' and 3 pools of patients’ blood. Analysis of these data revealed a negative correlation between the sialic acid content of both unbound and bound were
obtained
fractions and their ability accumulation (r=-0.85, n=Zl, ~~0.05).
LDL
to
induce
intracellular
cholesterol
DISCUSSION finding
The major can
be separated
RCAIZD-Sepharose.
from
of this study is that a subfraction total LDL preparation by affinity
this subfraction ricin and can be eluted with galactose, but suggests that LDL bears a terminal galactose. The fact
that
of desialylated
LDL
chromatography
binds to the
agglutinin
on of
not with other saccharides, As is known, galactose is a
constituent of the biantennary polysaccharide chain which generally contains a terminal sialic acid. The appearance of lectin-accessible galactose indicates the
loss of
sialic
acid.
Direct
determination 1126
of the sialic
acid content
Vol.
167, No. 3.1990
it findings indicate Desialylated
showed
that
BlOCHEMlCALANDBlOPHYSlCALRESEARCH
was markedly that
the
in the
reduced
bound
LDL separated
contain
subfractions from
the
bound
LDL preparation
COMMUNICATIONS
LDL subfraction.
desialylated isolated
These LDL. from
CHD
subfraction, induced cholesterol accumulation in cultured human aortic intimal cells. The atherogenic properties of the whole LDL pool may be conditioned by the subfraction of desialylated LDL. The desialylated LDL isolated from healthy subjects’ blood also induced intracellular lipid accumulation but to a lesser extent than desialylated LDL from patients’ blood which may be attributed to a lower degree of desialylation judging by the fact that this LDL is eluted by a lower concentrations of galactose. On the other hand, the total LDL preparations isolated from healthy subjects' blood, unlike LDL from patients' blood, induced no increase in intracellular cholesterol. This may be due to a lower degree of desialylation of this LDL and a lower content of desialylated LDL in the total LDL pool. We hope that the method described will be useful for biochemical and physico-chemical studies on desialylated lipoproteins. Besides, it might be helpful in elucidating the mechanisms of their formation and interaction with cells as well as pathogenic and diagnostic significance of desialylated LDL presence in the blood of atherosclerotic patients. patients'
blood,
unlike
the
unbound
REFERENCES 1.
2. 3. 4. 5. 6. 7.
Vauhkonen Biochem.
Ianiguchi,.l
C Viitala, 152 2;43-50
uiocnem. uiophys Fluecklger, R. n---l
Ishikawa
J., Y
Parkkinen,
J.,
Tsunemitsu, M., 273 197-265. (19851 Monogr. Atheroscler.,
and Rauvala, and Fukuzaki, v.
13,
pp.
J. H. (1989) Arch.
H. (1985)
Eur.
53-62,
Karger,
3: 1::
‘,’ kic%ci.
Biophys. _
Acta
266, 543-
15: 12: (1983) Clin. t , J75;1080. 16. Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. &:l’y ‘Ckt - '193 265-275 17. Dixon W.3 and Brown M B. (1977 Biomedical Computer Programs. PSeries’, pp.‘i85-198, Uniders’ity of Ca1 ifornia Press, Berkeley.
1127
167, No. 3, 1990 March 30, 1990
BIOCHEMICAL
ISOLATION
OF
(DESIALYLATED) FROM
BLOOD
OF
Vladimir
of
February
blood
3rd
(1,2).
PATIENTS:
NATIVE
Igor A. Sobenin,
USSR Cardiology
Cardiology,
reported
intracellular
lipid
neuraminidase
induced
Abbreviati agglutinin;
%S,
of
G. Tonevitsky,
N.Smirnov
Str.
15A,
Moscow
Research
Russia
121552,
8, 1990
that
with
It was demonstrated
uptake
Alexander
and Vladimir
low density
coronary
intimal that
heart
cells this
derived
level.
Furthermore
desialylated
LDL (3,4).
: LDL, low density phosphate buffered
OOO6-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
LDL of
lipid
intracellular
lipoprotein disease from
isolated
(LDL)
induces
(CHD) grossly
normal
from
lipid
human aorta
sialic acid (3,4); a negative correlation being and the LDL ability to elevate the
LDL has
as compared with LDL from healthy subjects established between the sialic acid content
greater
LIPOPROTEIN
CHROMATOGRAPHY
N. Orekhov,
in cultured
accumulation
DENSITY
Cherepkovskaya
patients
of
LIPOPROTEIN
Experimental
We have recently the
LOW
AFFINITY
V. Tertov,
Center,
Received
MODIFIED
FROM
Alexander
Institute
ATHEROGENIC
ATHEROSCLEROTIC
SEPARATION BY
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1122-1127
a lower
healthy
accumulation Proceeding
content
of
subjects which from
treated may be due
these
with to
observations,
lipoprotein. RCA saline; BSA,' bovi@ker%c&%%ir?mmuniS
1122
a
Vol. 167,
3,
No.
we have
BIOCHEMICALANDBIOPHYSICALRESEARCH
1990
concluded
that
desialylation
LDL modifications
atherogenic
bond
(5,6).
Sialic
one of
represents
possible
the
of LDL in vivo.
The molecule of apolipoprotein polysaccharide chain (high-mannose
glycosidic
COMMUNICATIONS
B (apo and
sialated
acid is a terminal
contains
B)
two types
biantennary)
linked
of by N-
saccharide of the biantennary
The gangliosides of LDL also contain a terminal sialic acid (7). type (5,6). in N-linked chains sialic acid is preceded by galactose residue, desialylation results in exposure of galactose, suggesting that LDL can bind
Since
to galactose-specific lectins such as the Ricinus communis agglutinin D-galactose and low RCA120 exhibits high affinity for terminal (RCA12rJ. affinity for other saccharides of the LDL molecule (8). In the present study we have
attempted
Sepharose-linked
to isolate RCAl20
human aortic
in cultured
desialylated
LDL by affinity
and to investigate
intimal
LDL effect
chromatography on lipid
on
accumulation
cells.
MATERIALSAND METHODS
LDL (d=1.019-1.063 mg/ml) was isolated by ultracentrifugation Lindgren ( and desialylated by treatment with neuraminldase pf;Twhere ?i ). Sialic acid content was determined according
with a he ane-isopropanol mixture (3:l v/V) (14x). The otal cholesterol &tent Wt< Boehringer Mannheim Kits etermined by the method of Siedel et al. (15 1 usin Ma nheim F.R.G.). Cell protein was measured by t ll e method of Lowry et al.
the
Lipids
according to as described to Svennerholm
method
were extracted of Hara and
Radin
167.
Significance
Program
package
of differences (17).
was evaluated
using
a BMDPIV
statistical
RESULTS was not washed with PBS-NaCl or to RCA120- Se p harose PBS/Z% BSA. Fig. 1 shows LDL binding to the sorbent depending on the amount of Part
of LDL applied
1123
Vol.
167, No. 3, 1990
01
BIOCHEMICAL
1
Protem
aw.
LDL
applied
(mg)
applied
that
LDL
the
amount
(Fig. the
to
the
was eluted of
column.
0.78
with
2). All LDL was eluted molecule (sialic
that the
preparation
galactose
(Fig.
solution.
increased
acid,
2).
obtained
proportion (from
eluted only subjects,
the
of
the
It
should
but
not
other
D-glucose,
D-mannose,
This finding indicates that LDL binding to RCA120-Sepharose.
from
patients' derived
LDL capable
20 to 64X),
that
with galactose
by 25 mM galactose,
in the LDL preparation
average
12.50
concentration
can be seen
sites for Fig. 3 shows the elution profile by a linear isolated from healthy subjects and CHD patients.
LDL
3.13
Sugar
50.00
(mM)
of
and these
dependence
is
of RCA120-Sepharose.
LDL in the eluate
LDL
acetylglucosamine) serve as specific
It
mg protein/ml
0.25-1.5
Bound
0
to RCA -Sepharose dePendin on the amount o,f LDL amount'# LDL isolated rom poo ed blood was applied to Sepharose and processed as described in Materials and as measured after washing with 50 mM galactose.
htmb~~t”~~u,“d’t~r~~~~~~
linear within
02
z
LDL binding The indicated
lipoprotein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
blood from
binding
the
saccharides L-fucose,
galactose
of N-
residues
share of bound LDL exceeds subjects. In CHD patients,
RCA120-Sepharose
LDL preparations
by high galactose concentrations (30-50 proportion of bound LDL was ~15%. 1124
mentioned
concentration
galactose gradient for LDL It can be seen that in the
healthy to
be
contained mM) (Fig.
was
48+7%,
on
LDL which were 3).
In healthy
Vol.
167,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
30
Fraction kk&edE#k?& blkodf?? applied to a 5-ml column linear galactose gradient aliquots.
It can be 2-fold lower healthy subjects. and
unbound
TABLE
seen
LDL,
1.
Table 1 of sialic Patients’ LDL from
content
a
1.5-fold
Effects of cultured
LDL
cells
number
-Sepharose b l'near alactose and ZHD patienk Rhc,!l?fly subjects Bound LDL was with RCA Sepharok' 8&l cholest’erol was measured and
that
LDL
acid bound
higher
isolated as compared of sialic
intracellular
on
normal
human
pg 1 mg p$otein Healthy
Total
Bound Unbound
log 92
subjects
:E 26:9 CHD patients
Total
Bound Unbound
'308 62
14.7 I&%
Desialylated
in
vitro
blood
LDL had
acid
as
had
obtained a 3-fold
compared
cholesterol aortic intima
Cholesterol accumulation % over contrbl
WC co tent
e1ut.e with a in 0.5-ml
patients' with
RCA ,2D-Sepharose
to
content
subfractions from grossly
from
rad'ent. LDL ?$~).were
content
a
from lower
with
in
the
Vol.
167, No. 3, 1990
total
BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS
The sialic
LDL fraction.
obtained
healthy
from
preparation
acid content
subjects
The
total
the
preparation
Z-fold
higher
than
that
in
sialic
acid
content
in
the
was
of CHD patients.
in
of LDL the LDL unbound
lipoprotein
fraction of healthy subjects was similar to that in the total LDL fraction while in bound LDL it was Z-fold lower. It can be concluded from these observations that LDL isolated both from patients’ and healthy subjects' blood and capable of binding to RCA 12D-Sepharose are desialylated lipoproteins. To confirm
subjects 3-fold
hypothesis,
this
were desialylated
fall
LDL bound
in
sialic
the
LDL isolated
from the blood of
by neuraminidase treatment acid content.
About
healthy
which brought
90% of -in vitro
about
a
desialylated
to RCAlzO-Sepharose.
Incubation of human aortic intimal cells with the total preparation of LDL blood had no effect on the intracellular isolated from healthy subjects' cholesterol level (Table 1). By contrast, the bound LDL fraction induced
cholesterol with
accumulation.
neuraminidase
intracellular
or
Incubation
the
bound
with both
total
LDL subfraction
preparation
The total
of LDL isolated
from
patients’
increase in intracellular cholesterol content effect (4-fold increase in intracellular cholesterol bound
LDL subfraction.
intracellular Table from
treated
increase
in
The unbound
blood produced
(Table
1).
level)
LDL subfraction
a 2.5-
A more
potent
was observed
induced
with
in
no increase
cholesterol. 1 shows
pooled
results
to a 3-fold
cholesterol.
fold
the
LDL preparations
led
the
healthy
results
of experiments
subjects'
(n=lO)
with
LDL preparations
and CHD patients’
isolated
(n=lO) blood. Similar
with LDL preparations isolated from 2 pools of healthy subjects' and 3 pools of patients’ blood. Analysis of these data revealed a negative correlation between the sialic acid content of both unbound and bound were
obtained
fractions and their ability accumulation (r=-0.85, n=Zl, ~~0.05).
LDL
to
induce
intracellular
cholesterol
DISCUSSION finding
The major can
be separated
RCAIZD-Sepharose.
from
of this study is that a subfraction total LDL preparation by affinity
this subfraction ricin and can be eluted with galactose, but suggests that LDL bears a terminal galactose. The fact
that
of desialylated
LDL
chromatography
binds to the
agglutinin
on of
not with other saccharides, As is known, galactose is a
constituent of the biantennary polysaccharide chain which generally contains a terminal sialic acid. The appearance of lectin-accessible galactose indicates the
loss of
sialic
acid.
Direct
determination 1126
of the sialic
acid content
Vol.
167, No. 3.1990
it findings indicate Desialylated
showed
that
BlOCHEMlCALANDBlOPHYSlCALRESEARCH
was markedly that
the
in the
reduced
bound
LDL separated
contain
subfractions from
the
bound
LDL preparation
COMMUNICATIONS
LDL subfraction.
desialylated isolated
These LDL. from
CHD
subfraction, induced cholesterol accumulation in cultured human aortic intimal cells. The atherogenic properties of the whole LDL pool may be conditioned by the subfraction of desialylated LDL. The desialylated LDL isolated from healthy subjects’ blood also induced intracellular lipid accumulation but to a lesser extent than desialylated LDL from patients’ blood which may be attributed to a lower degree of desialylation judging by the fact that this LDL is eluted by a lower concentrations of galactose. On the other hand, the total LDL preparations isolated from healthy subjects' blood, unlike LDL from patients' blood, induced no increase in intracellular cholesterol. This may be due to a lower degree of desialylation of this LDL and a lower content of desialylated LDL in the total LDL pool. We hope that the method described will be useful for biochemical and physico-chemical studies on desialylated lipoproteins. Besides, it might be helpful in elucidating the mechanisms of their formation and interaction with cells as well as pathogenic and diagnostic significance of desialylated LDL presence in the blood of atherosclerotic patients. patients'
blood,
unlike
the
unbound
REFERENCES 1.
2. 3. 4. 5. 6. 7.
Vauhkonen Biochem.
Ianiguchi,.l
C Viitala, 152 2;43-50
uiocnem. uiophys Fluecklger, R. n---l
Ishikawa
J., Y
Parkkinen,
J.,
Tsunemitsu, M., 273 197-265. (19851 Monogr. Atheroscler.,
and Rauvala, and Fukuzaki, v.
13,
pp.
J. H. (1989) Arch.
H. (1985)
Eur.
53-62,
Karger,
3: 1::
‘,’ kic%ci.
Biophys. _
Acta
266, 543-
15: 12: (1983) Clin. t , J75;1080. 16. Rosenbrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. &:l’y ‘Ckt - '193 265-275 17. Dixon W.3 and Brown M B. (1977 Biomedical Computer Programs. PSeries’, pp.‘i85-198, Uniders’ity of Ca1 ifornia Press, Berkeley.
1127
