From the
Lipid droplets resembling those seen in the extracellular space
of the arterial intima were generated in vitro when granule
proteases of rat serosal mast cells degraded the apolipoprotein B-100
(apoB-100) component of granule-bound low density lipoprotein (LDL),
and the particles fused on the granule surface (Paananen, K., and
Kovanen, P. T.(1994) J. Biol. Chem. 269, 2023-2031).
Moreover, the binding of the fused particles to the heparin
proteoglycan component of the granules was found to be strengthened. We
have now treated LDL particles with
The first visible change during atherogenesis is focal
accumulation of small extracellular lipid droplets in the extracellular
space of the arterial intima. Both chemical analysis
(1) and
size analysis
(2) of the droplets suggest that most of them are
derived from low density lipoprotein (LDL)
The
interaction of LDL with isolated proteoglycans has been studied in
detail. However, the ionic interaction of the LDL-derived extracellular
lipid droplets with the matrix components has not been studied so far.
Using an experimental model in which LDL particles are incubated with
the remnants of exocytosed rat mast cell granules, we could generate
lipid droplets with diameters in the same range as those of the
extracellular lipid droplets
(7) . The droplets were formed when
the two neutral proteases of the remnants, chymase and carboxypeptidase
A, degraded the apoB-100 of the heparin proteoglycan-bound LDL
particles. Moreover, the binding of these droplets to the heparin
proteoglycan component of the remnants was stronger than that of native
LDL
(7, 8) . As a possible mode of generation of lipid
droplets tightly bound to a proteoglycan matrix, these studies
suggested proteolytic modification of LDL.
In the current study, we
treated LDL particles with
The two lysine populations of
apoB-100 having different pK
The NMR
spectrum of the
The likely reasons for the observed increase in strength of
binding of proteolyzed LDL are more ionic interactions and/or stronger
ionic interactions between the residual apoB-100 and the proteoglycans.
Conformational changes during proteolysis and fusion could expose some
shielded proteoglycan binding regions (containing lysine and arginine
residues) in the remaining apoB-100 fragments. Indeed, despite the loss
of lysine residues from apoB-100 during proteolysis, the number of
active lysine residues did not decrease, reflecting either generation
of new active lysine residues during spatial reorganization of the
residual apoB-100 fragment and/or resistance of the basic regions of
apoB-100 to proteolytic cleavage (and subsequent release from the
particles). Similarly, since the number of active lysine residues/750
cholesteryl linoleate molecules increases from 26 to 42 during fusion,
the residual apoB-100 fragments must have undergone additional
conformational changes, with exposure of basic areas at the surface of
the enlarged particles. Moreover, when the residual apoB-100 fragments
of the fusing particles accumulate on the surface of the enlarging
particle, the total number of active lysine residues in the average
fused particle increases to 273. This 10-fold higher quantity of active
lysine residues, which reflects a greater number of potential
proteoglycan binding regions, per fused particle as compared with
native particles provides a plausible explanation for the significant
increase in the strength of binding upon proteolytic fusion of LDL.
A subendothelial proteoglycan-rich layer is present in arteries with
a well-defined thick intima, such as the coronary arteries and aorta.
The areas prone to atherosclerosis have been suggested to contain
proteoglycans with a unique structure that favors LDL
entrapment
(30, 31) . Another possible explanation for
the focal accumulation of LDL-derived cholesterol is modification of
LDL particles in the atherosclerosis-prone intimal areas with ensuing
strengthened binding to proteoglycans. One such modification might be
proteolytic fusion of LDL. As LDL enters the arterial intima, it
crosses the endothelial cell layer and becomes surrounded by smooth
muscle cells and three types of blood-borne cells: mast cells,
macrophages, and T cells. With regard to LDL fusion, of the proteolytic
enzymes secreted by these cell types, only mast cell chymase has been
studied so far
(8) . Although chymase and
After incubation of
100 mg of LDL in 8 ml of buffer A containing 5 mg of
The numbers of
active and normal lysine residues in native LDL, in proteolyzed but
unfused, and in proteolyzed and fused LDL particles are derived by
integration of the [
We are grateful to Dr. Nisse Kalkkinen for help in
amino acid analysis. The excellent technical assistance of Minna
Ahlstedt is gratefully acknowledged.
-chymotrypsin and examined the
strength with which the proteolytically modified LDL binds to human
aortic proteoglycans on an affinity column. We found that chymotryptic
degradation of the LDL particles triggered particle fusion. The higher
the degree of proteolytic degradation, the higher were the degree of
fusion and the strength of binding to the aortic proteoglycans.
Separation of the proteolyzed particles by size exclusion
chromatography into two fractions, unfused and fused particles, and
analysis of their binding strengths revealed that not only the fused
but also the unfused proteolyzed particles bound more tightly to the
proteoglycans than did the native LDL particles. To investigate the
mechanism underlying this increase in binding strength, we attached
[
C]dimethyl groups to the lysines and used NMR
spectroscopy to quantify the active lysine residues of apoB-100, which
are thought to be located in basic areas of apoB-100 and involved in
binding of LDL to proteoglycans. Analysis of the
C-labeled
particles showed that, despite loss of apoB-100 fragments from the
particles, the number of active lysine residues in the unfused
proteolyzed particles had not decreased. In the fused proteolyzed
particles, the number of active lysine residues was markedly increased.
Thus, proteolytic fusion appears to increase the number of basic
domains of apoB-100, which would explain the observed increase in the
strength of binding of the modified LDL particles to arterial
proteoglycans. Since the fused particles resemble the small lipid
droplets found in the atherosclerotic arterial intima, this LDL
modification offers a plausible mechanism for the focal accumulation of
lipid droplets in the extracellular proteoglycan matrix during
atherogenesis.
(
)
particles. At the light microscopic and electron
microscopic levels, the extracellular lipid droplets appear initially
in the subendothelial proteoglycan-rich layer
(3) , where they
are thought to be associated with matrix
proteoglycans
(4, 5) . Indeed, complexes of lipoproteins
containing apoB-100 and proteoglycans can be isolated from fatty
streaks and fibrous plaques of the human aorta
(6) .
-chymotrypsin, an enzyme with
specificity similar to that of mast cell chymase, and studied the
interaction of the proteolyzed particles with proteoglycans isolated
from human aortic intima. The electrostatic interactions which mediate
the binding of LDL particles to the negatively charged sulfate groups
of the proteoglycans are known to involve positively charged arginine
and lysine residues
(9, 10, 11, 12) . The
lysine residues of apoB-100 are of two forms having different
pK
values, 8.9 and 10.5
(13) . The
unusually low pK
of the former residues,
denoted as ``active lysines,'' is thought to depend on their
location in the basic areas, such as the proteoglycan binding areas, of
the protein. To gain information on the effects of proteolysis and of
fusion on the strength of binding of modified LDL particles to the
aortic proteoglycans, we investigated the populations of active lysine
residues in both unfused and fused proteolyzed particles.
Materials
-Chymotrypsin, bovine serum
albumin,
-aminocaproic acid, and soybean trypsin inhibitor were
from Sigma. Chondroitinase ABC and AC and the unsaturated
chondro-disaccharide kit for HPLC were from Seikagaku Kogyo.
[1,2-
H]Cholesteryl linoleate and
t-butoxycarbonyl-L-[
S]methionine
N-hydroxysuccinimidyl ester (the
S-labeling
reagent) were from Amersham. Phenylmethylsulfonyl fluoride was from
Boehringer Mannheim, Celite 545 (acid-washed) from Fluka, the Schrynel
nylon filter from Zürcher Beuteltuchfabrik AG, and
[
C]formaldehyde (99% isotope enrichment) as a
20% solution in water was from Isotec Inc. NaCNBH
from
Sigma was purified by recrystallization from dichloromethane prior to
use (14). Superose 6 HR 10/30 columns, HiTrap NHS-activated columns,
HiTrap Q columns, and Sephacryl 400-HR were from Pharmacia LKB
Biotechnology; Bio-Gel A-5m and Bio-Gel A-15m gel filtration media were
from Bio-Rad; the 5-µm NH
and S5 ODS (0.3
15
cm) columns were from Spherisorb. Cholesteryl ester transfer protein
was a kind gift from Drs. C. Ehnolm and M. Jauhiainen, National Public
Health Institute, Helsinki, Finland.
Preparation and Labeling of LDL
Human LDL (d = 1.019-1.050) was isolated from plasma of healthy
volunteers by sequential ultracentrifugation in the presence of 3
mM EDTA
(15, 16) .
[H]Cholesteryl linoleate was incorporated into
LDL essentially as described
(7) except that, instead of serum
we used cholesteryl ester transfer protein and LDL. For incorporation,
200 µCi of [
H]cholesteryl linoleate in
chloroform was first adsorbed onto 300 mg of the Celite. Then, 5 mg of
LDL and 500 µl of cholesteryl ester transfer protein (5-10
µmol of cholesteryl ester transferred/ml/h) in 2 ml of
Tris-chloride, pH 7.4, were added to the Celite, and the mixture was
incubated with gentle rotation in a Coulter mixer at 37 °C for
18-24 h. After incubation, the Celite was sedimented by
centrifugation. The supernatant containing LDL was chromatographed on a
Bio-Gel A-5 m column (1
40 cm) equilibrated in buffer A (150
mM NaCl, 1 mM EDTA, and 5 mM Tris chloride,
pH 7.4) with a flow rate of 7 ml/h. The specific radioactivities of the
H-LDL preparations ranged from 9,700 to 44,000 dpm/µg
of LDL.
S-LDL was prepared by labeling the protein
component of LDL by the Bolton-Hunter procedure
(17) with a
S-labeling reagent, as described previously
(7) . In
experiments, mixtures of
S-LDL and
H-LDL
(
S/
H) were used. For each experiment, labeled
LDL was diluted with unlabeled LDL to give the specific radioactivities
indicated in the figure legends. The concentration of LDL is expressed
in terms of its protein concentration.
Proteolytic Degradation of LDL
The standard
degradation assay was conducted at 37 °C in 100-400 µl of
buffer A containing 1.0 mg/ml of S-LDL/
H-LDL
and 0.1 mg/ml of
-chymotrypsin. After incubation for the indicated
times, degradation was stopped by adding trypsin inhibitor to give a
final concentration of 1 mg/ml. To determine the degree of proteolytic
degradation, 80 µl of ice-cold buffer A containing 5 mg/ml of
bovine serum albumin and 25 µl of 50% (w/v) trichloroacetic acid
were added to 20-µl aliquots of the incubation mixtures. After
incubation for 30 min at 0 °C, the mixtures were centrifuged for 10
min at 12,000
g, and
S radioactivities of
the supernatants were determined. The degree of apoB-100 degradation is
expressed as the amount of trichloroacetic acid-soluble radioactivity
produced. Blank values were obtained by incubating LDL in the absence
of proteolytic enzymes.
Analysis of Proteolyzed LDL Samples
After
incubation of LDL with the proteases, 100-µl aliquots of the
samples corresponding to 100 µg of undegraded LDL were run in
buffer A through two Superose 6 HR 10/30 columns connected in series.
Flow rate was 0.5 ml/min, and 500-µl fractions were collected. The
degree of fusion of the H-LDL was determined by measuring
the
H radioactivity of fractions 30-60 (the total
eluted radioactivity) and calculating the ratio of the radioactivity in
fractions 30-40 (void volume peak) to the total eluted
radioactivity. The degree of fusion is expressed in percent
((radioactivity in void volume peak/total eluted radioactivity)
100).
Preparation and Characterization of
Proteoglycans
Proteoglycans from intima-media of human aortas
obtained at necropsy within 24 h of accidental death were extracted
essentially as described by Hurt-Camejo et al.(18) .
Briefly, intima-media of the aortas was stripped, placed in a
preserving solution composed of 0.15 M NaCl, 0.5 mM
EDTA, 0.1 mM CuSO, and 5 mM Tris
chloride, pH 7.4, containing 0.2 mM phenylmethylsulfonyl
fluoride, 0.02% (w/v) NaN
, and 10 mM
-aminocaproic acid, and stored at -70 °C until use.
Proteoglycans were extracted from the intima-media at 4 °C for 24 h
with 15 volumes of 6 M urea, 1 M NaCl in the presence
of 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride,
0.02% (w/v) NaN
, and 10 mM
-aminocaproic
acid. Next, the mixture was filtered through a Schrynel 48-µm nylon
filter and centrifuged at 100,000
g for 60 min. The
supernatant was diluted with 6 M urea to give a final
concentration of 0.25 M NaCl and loaded on a Q HiTrap column
(5 ml) equilibrated with 6 M urea, 0.25 M NaCl, 10
mM CaCl
, and 50 mM acetate, pH 6.2, and
the protease inhibitors. The column was washed with the above buffer,
and the proteoglycans were eluted with a linear gradient of 0.25
M-1.0 M NaCl in the buffer (120 ml) at a flow
rate of 2 ml/min. The peaks at 280 nm were collected, dialyzed against
water, and lyophilized. Proteoglycan monomers were obtained by running
the proteoglycans through a 1
50-cm column of Sephacryl-400 HR
in 20 mM HEPES, 0.1 M Na
SO
,
25 mM EDTA, and 4 M guanidine-HCl, pH 7.0, at a flow
rate of 12 ml/h
(19) . The peaks at 280 nm were collected,
dialyzed against water, and lyophilized. The disaccharide composition
of the proteoglycans was analyzed by highperformance liquid
chromatography using a 5-µm NH
column after treatment
of the proteoglycans with chondroitinase ABC and AC
(20) . The
proteoglycan preparations used here contained 32-48% of
chondroitin-6-sulfate, 47-60% of chondroitin-4-sulfate and
4-8% of dermatan sulfate. The amount of the proteoglycans is
expressed in terms of their glycosaminoglycan content.
Preparation of Proteoglycan Affinity Column
Human
arterial proteoglycan monomers were coupled to a NHS-activated HiTrap
column (1 ml) according to the manufacturer's instructions. For
this purpose, 0.5-2.5 mg of proteoglycans were dialyzed against
the coupling buffer (0.2 M NaHCO and 0.5
M NaCl, pH 8.3) and coupled to the column at 25 °C for 2
h. The column was blocked with 0.5 M ethanolamine, pH 8.3,
containing 0.5 M NaCl. Under these conditions, 0.4-1.1
mg of the proteoglycans were coupled to the column. The columns were
equilibrated with buffer B (10 mM HEPES, 2 mM
CaCl
, 2 mM MgCl
, and 0.02%
NaN
, pH 7.4) before use.
Affinity Chromatography of Proteolyzed LDL on
Proteoglycan Affinity Columns
LDL was proteolyzed as described
above, and 20-µl samples of the incubation mixtures corresponding
to 20 µg of undegraded LDL were analyzed on the proteoglycan
affinity columns by elution with a linear gradient of NaCl (0-250
mM in 10 min) in buffer B. Chromatography was performed at a
flow rate of 1 ml/min. Proteins were detected by UV absorbance at 280
nm. The chromatographic apparatus consisted of two Applied Biosystems
solvent delivery system 400 pumps controlled by an Applied Biosystems
738 detector/gradient controller.
Modifications of
LDL (4 mg) was incubated for 24 h at 37 °C in 100 µl
of buffer A containing 200 µg of -Chymotrypsin-treated
LDL
-chymotrypsin. After
incubation, lysine or arginine residues of apoB-100 were modified by
treatment of 2 mg of the proteolyzed LDL with acetic anhydride
(21) or 1,2-cyclohexanedione (22).
Preparation of
LDL (100 mg) was incubated for 24 h at 37 °C in 13 ml of
buffer A containing 5 mg of C-Labeled LDL and
C-labeled
-Chymotrypsin-treated LDL for
NMR
-chymotrypsin. After incubation, the
sample was applied to a Bio-Gel A-15m column (2.5
50 cm)
equilibrated with saline (150 mM NaCl and 1 mM EDTA,
pH 7.4) LDL eluted in two peaks, which were collected and pooled
separately. For NMR analysis, amino groups of lysine residues of
apoB-100 of either native or
-chymotrypsin-treated LDL were
C-labeled by reductive methylation with
[
C]formaldehyde
(13, 14) . First,
0.1 M NaCNBH
was added to 50 mg of native LDL in
50 ml of saline to give a final concentration of 20 mM, and to
the two pooled peaks from the Bio-Gel A-15m gel filtration of the
-chymotrypsin-treated LDL (each corresponding to about 50 mg of
undegraded LDL in 50 ml of saline). After addition of 3.25
10
mol of H
CHO to the samples, the
mixtures were incubated at 4 °C for 18 h. The reactions were
stopped by dialysis against saline solution at 4 °C. The labeled
LDL solutions were concentrated in Amicon 30 concentrators with a
30-kDa cut-off membrane. The degree of methylation was determined by
amino acid analysis.
NMR Spectroscopy
Broadband proton-decoupled
C NMR spectra were measured from the LDL samples, which
contained 2.8 -16.5 mg of protein/ml in 0.79 ml of a solution
containing 150 mM NaCl, 1 mM EDTA, and 0.02%
NaN
, pH 7.4, and 10% of D
O for the spectrometer
field-lock.
C NMR spectra were obtained at 150.8 MHz with
a Varian Unity 600 NMR spectrometer. The spectral width was 250 ppm,
corresponding to 16k points in 0.219 s in all experiments. Relaxation
delay was 1.0 s and pulse length 8 µs. Total running times varied
from 4 to 45 h, depending on the sample. Proton decoupling was
performed with a GARP sequence
(23) . Prior to Fourier
transformations, free induction decays were zero filled to 32k and
weighted by 2 Hz line broadening. The spectra were recorded at 37
± 0.5 °C. Chemical shifts were referenced to 1,4-dioxane at
66.5 ± 0.05 ppm added as a 20% (v/v) aqueous solution in order
to prevent precipitation.
Amino Acid Analysis
The samples were added
dropwise to 20 volumes of cold ethanol/ether (2:1). After incubation at
-20 °C for 18 h, the samples were centrifuged at 1,500
g for 15 min at -10 °C, and the formed
pellets were treated twice with ether at -20 °C for 60 min.
Hydrolysis was carried out in hydrolysis tubes sealed under a vacuum
with 1 ml of 6 M HCl at 110 °C for 22 h, after which the
samples were lyophilized and dissolved in 1 ml of water. As an internal
standard, 25 µl of norvaline (final concentration 130
µM) was added to 75-µl aliquots of the samples. Amino
acid analysis was performed with Biochrom 20 (Pharmacia LKB
Biotechnology) using a lithium high performance column (0.46
20
cm). Dimethyl-lysine co-eluted from the column with histidine.
Therefore, another method was tried for analyzing the amino acids; the
samples were analyzed as derivatives essentially as described by Chang
et al.(24) . In this system, dimethyl-lysine co-eluted
with proline, but for the other amino acids the two methods gave
similar results. We therefore determined the amount of histidine in
each sample by the method of Chang et al., and used this value
to calculate the amount of dimethyl-lysine from ion-exchange
chromatograms.
Electron Microscopy of LDL
Samples were mixed 1:1
with 1% potassium phosphotungstate, pH 7.4, and dried on carbon-coated
grids
(25) . The negatively stained samples were viewed and
photographed in a JEOL 100CX electron microscope at the Department of
Electron Microscopy, University of Helsinki. The diameters of 250
randomly selected lipoprotein particles were measured from the electron
micrographs.
Other Assays
Protein was determined by the method
of Lowry et al.(26) , with bovine serum albumin as
standard. Glycosaminoglycans were determined by the method of Bartold
and Page (27). Cholesteryl linoleate was determined with reverse phase
HPLC, using an S5 ODS (0.3 15 cm) column as described by
Kritharides et al.(28) .
RESULTS
To study the effect of proteolytic degradation of LDL on LDL
particle size, 1.0 mg/ml of S-LDL/
H-LDL was
incubated with 0.1 mg/ml of
-chymotrypsin. After incubation at 37
°C for 24 h, the degree of proteolytic degradation was measured by
quantifying the
S-labeled trichloroacetic acid-soluble
degradation products. Under these conditions, 37% of the
S-labeled protein was degraded. The proteolyzed LDL sample
was then analyzed by gel filtration. Elution was monitored by measuring
the radioactivities of the collected fractions. The
H
elution profile of the proteolyzed LDL showed two peaks
(Fig. 1B): the material in peak I (fractions
30-40) eluted in the position corresponding to the void volume of
the column, and the material in peak II eluted in the position
corresponding to untreated LDL (Fig. 1A). The peak
fractions were analyzed with electron microscopy after negative
staining of the particles: the particles in peak II showed a size
distribution similar to native LDL, having a mean diameter of 23 nm
(±3.9 nm, median 23 nm), whereas the particles in peak I were
larger, their mean diameter being 38 nm (±8.8 nm, median 38 nm).
Thus, incubation of LDL with
-chymotrypsin leads both to
proteolytic degradation of apoB-100 and to LDL particle fusion.
Moreover, the degree of LDL fusion can be quantified by expressing the
radioactivity in peak I (fractions 30-40) containing the fused
LDL in percent of the total eluted radioactivity.
Figure 1:
Gel filtration of
-chymotrypsin-treated LDL. After incubation of 200 µg of
S-LDL/
H-LDL (830 dpm/µg protein; 5220
dpm/µg cholesterol linoleate) in 200 µl of buffer A with 20
µg of
-chymotrypsin at 37 °C for 24 h, the sample was
applied to a gel filtration system consisting of two Superose 6HR 10/30
columns connected in series, and eluted with buffer A at 0.5 ml/min.
Fractions of 500 µl were collected, and their radioactivities were
measured (panel B). Untreated
H-LDL (3600
dpm/µg cholesterol linoleate) served as a control (panel
A). The peak fractions were analyzed with electron microscopy as
described under ``Experimental Procedures.'' The bar under the inset in panel A represents 100
nm.
Next, we studied
the effect of the degree of apoB-100 degradation on LDL particle
fusion. S-LDL/
H-LDL was incubated with
-chymotrypsin at 37 °C for 24 h and, at various time points,
trypsin inhibitor was added to the incubation mixtures to prevent any
further degradation. After incubation, the degrees of apoB-100
degradation (Fig. 2A) and of particle fusion
(Fig. 2B) were determined. After proteolytic degradation
for 24 h, 30% of the apoB-100 was degraded, and 27% of the particles
had fused. It appeared that the higher the degree of apoB-100
degradation, the greater was the extent of fusion of LDL particles at
the end of the 24-h incubation (Fig. 2).
Figure 2:
Effect of
the degree of apoB-100 degradation on the amount of LDL fusion.
Incubation of 200 µg of S-LDL/
H-LDL (410
dpm/µg protein; 1600 dpm/µg cholesterol linoleate) took place
in 200 µl of buffer A containing 20 µg of
-chymotrypsin at
37 °C for 24 h. At the indicated time points, 200 µg of trypsin
inhibitor was added. The degree of degradation was measured as
described in ``Experimental Procedures'' and expressed as the
amount of trichloroacetic acid-soluble radioactivity. The amount of
fusion was determined by gel filtration as described under
``Experimental Procedures'' and is expressed as the ratio of
the amount of radioactivity eluting prior to native-sized LDL
(fractions 30-40) to the total amount of eluted radioactivity
(fractions 30-60) (see Fig. 1).
We next analyzed the
effect of proteolysis and fusion on binding of the particles to human
arterial proteoglycans. For this purpose, proteoglycans were isolated
from human aortas, and affinity columns were prepared by coupling the
proteoglycan monomers to NHS-activated HiTrap columns, as described
under ``Experimental Procedures.''
S-LDL/
H-LDL was incubated with
-chymotrypsin at 37 °C for 24 h and, at various time points,
trypsin inhibitor was added to the incubation mixtures. Native or
proteolyzed LDL was then applied to the proteoglycan column and eluted
with a linear NaCl gradient (0-250 mM in 10 min), and
elution was monitored with UV absorbance at 280 nm. Of the applied
native LDL (20 µg), 95% was bound to the column and eluted as a
single peak at 60 mM NaCl (Fig. 3, narrow
line). The elution profiles of the samples that had been
proteolyzed for various lengths of time (Fig. 3, bold
lines) revealed that the higher the degree of degradation and
fusion (see Fig. 2), the higher was the strength of LDL binding
to the proteoglycans, until a single peak emerged at 90 mM
NaCl when the LDL particles had been proteolyzed for 24 h
(Fig. 3, bottom panel). Determination of the
radioactivities in the fractions of this latter sample showed that 75%
of the applied [
H]cholesteryl linoleate became
bound to the column and eluted in a position corresponding to that
monitored with UV absorption. The unretained material emerging as the
initial peak in each chromatogram in Fig. 3consists of trypsin
inhibitor,
-chymotrypsin, and the released
S-labeled
peptide fragments. If the chromatography was performed with 250
mM NaCl (i.e. without a gradient), the native, and
the proteolyzed and fused LDL particles co-eluted from the column,
showing that separation of the particles during chromatography was not
due to differences in particle size (not shown). When the NaCl gradient
was changed from 0 to 250 mM in 10 min to 0-500
mM NaCl in 10 min, or to 0-250 mM in 20 min,
the elution profiles of the samples changed correspondingly (not
shown).
Figure 3:
Affinity chromatography of
-chymotrypsin-treated LDL on a human aortic proteoglycan column.
200 µg of
S-LDL/
H-LDL (540 dpm/µg
protein; 1300 dpm/µg cholesterol linoleate) was incubated in 200
µl of buffer A containing 20 µg of
-chymotrypsin at 37
°C for 24 h. At the indicated time points, 200 µg of trypsin
inhibitor was added. Double-labeled LDL was used to determine the
degrees of proteolysis and fusion (see Fig. 2). Samples of the
incubation mixtures (20 µl) were analyzed by affinity
chromatography on a 1-ml proteoglycan-HiTrap column (bold
line). Elution was carried out at 1.0 ml/min using a linear NaCl
gradient (0-250 mM in 10 min) in 10 mM HEPES, 2
mM CaCl
, and 2 mM MgCl
, pH
7.4. Elution was monitored by UV absorbance at 280 nm. In each panel
are shown the elution profile of untreated LDL (narrow line)
and the NaCl gradient (dotted
line).
Binding of native LDL to proteoglycans can be blocked by
removing the positive charge from the lysine or arginine residues in
the apoB-100 with acetic anhydride or 1,2-cyclohexanedione,
respectively
(29) . To examine whether these amino acid residues
were responsible for the observed increase in the strength of binding
of proteolyzed LDL to the proteoglycans, native and proteolyzed LDL
were treated with acetic anhydride or 1,2-cyclohexanedione. Both
modifications blocked the binding of both native and proteolyzed LDL to
the aortic proteoglycans (not shown).
values (8.9
and 10.5) can be studied with NMR spectroscopy
(13) . We used the
NMR method to gain information on the effects of proteolysis and fusion
on the two populations of lysine residues of apoB-100. For this
purpose, LDL was first incubated with
-chymotrypsin at 37 °C
for 24 h, then applied to a Bio-Gel A-15m gel filtration column, and
the peaks at 280 nm were collected. The two proteolyzed samples
(unfused and fused) and native LDL were treated with
[
C]formaldehyde to add
[
C]dimethyl groups to the lysine residues of
apoB-100. The size distributions of the particles were measured from
electron micrographs of negatively stained samples. The diameters of
the particles in the first peak were larger than those of native LDL,
whereas the diameters of the particles in the second peak were similar
to those of native LDL particles (). The retention times in
proteoglycan affinity column of both the unfused and the fused
proteolyzed particles to proteoglycans were increased, showing that
binding to the proteoglycans was strengthened (). The
protein and cholesteryl linoleate contents of the samples were also
measured, and it appeared that the ratio of protein to cholesteryl
linoleate had decreased in the unfused particles, and even more so in
the fused particles (). Thus, the fused particles were
either proteolyzed to a higher extent than the unfused particles, or
some of the apoB-100 fragments were released from the particles because
of rearrangement of the particle surface during fusion.
C-labeled native LDL showed lipid
resonances and, at 42.8 and 43.1 ppm,
[
C]dimethyl lysine resonances
(Fig. 4A), which is in accord with the results of
Lund-Katz et al.(13) . These resonances have been
characterized earlier, and at pH 7.6 active lysine residues are known
to have an average chemical shift of 43.1 ppm, whereas normal lysine
residues have a chemical shift of 42.8 ppm
(13) . The NMR spectra
of
C-labeled unfused and fused proteolyzed LDL show
similar resonances (Fig. 4, B and C,
respectively), whereas the NMR spectrum of unlabeled LDL lacks the
dimethyl lysine resonances (Fig. 4D). In Fig. 4,
the insets in each panel show enlargements of the
[
C]dimethyl lysine resonances. Integration of
the two resonances at 42.8 and 43.1 ppm gives the number of active and
normal lysine residues in the samples (). The numbers of
lysine residues were expressed in relation to the amount of cholesteryl
linoleate, the major component of the LDL core. One normal-sized LDL
particle contains 750 cholesteryl linoleate molecules, and when
calculated per 750 cholesteryl linoleate molecules, the total number of
methylated lysine residues was found to be decreased in both the
proteolyzed but unfused and the proteolyzed and fused particles. In
contrast, the number of active lysines in the native and the
proteolyzed but unfused particles was about the same (27 and 26,
respectively), and in the fused particles 42/750 cholesteryl linoleate
molecules. If the diameter of a spherical particle increases from 23 nm
(the diameter of an unfused LDL particle) to 43 nm, its volume
increases 6.5-fold. Thus, a fused particle with a diameter of 43 nm
results from coalescence of an average of 6.5 proteolyzed particles,
and the amount of cholesteryl linoleate in one fused particle must, on
average, be 6.5-fold higher than in the unfused particles. Accordingly,
the number of active lysine residues/fused particle should also be 6.5
times higher, i.e. 273 (6.5
42). The above results
revealed that the number of active lysine residues/particle does not
decrease during proteolysis, and increases when the proteolyzed
particles fuse.
Figure 4:
Proton-decoupled C NMR
spectra of native, proteolyzed but unfused, and proteolyzed and fused
LDL in which lysine residues were converted to dimethyl lysines by
reductive methylation. The spectra (150.8 MHz) were recorded at 37
± 0.5 °C as described in detail under ``Experimental
Procedures.'' Chemical shifts were referenced to aqueous
1,4-dioxane at 66.5 ± 0.05 ppm. The spectral widths were 250
ppm. Spectra of native LDL in which 95% of the lysine residues were
methylated (panel A), proteolyzed but unfused LDL (92% of
lysines modified) (panel B), proteolyzed and fused LDL (95% of
lysines modified) (panel C), and unlabeled LDL (panel
D). The insets in each panelare expansions of
the spectra showing dimethyl lysine resonances. Note that the vertical
expansions of the insets are different, so as to keep the
highest resonance on scale.
DISCUSSION
This study shows that proteolysis of LDL with
-chymotrypsin renders the particles unstable and leads to
formation of particles with increased size and with increased strength
of binding to human aortic proteoglycans. The strength of binding to
the proteoglycans was increased in both proteolyzed unfused and
proteolyzed fused particles, but to a lesser extent in the unfused. The
binding of proteolyzed LDL to proteoglycans was shown to be mediated by
both lysine and arginine residues. Most importantly, we found no
decrease in the number of active lysine residues, which are thought to
reside in the basic areas of apoB-100, the domains responsible for LDL
binding to proteoglycans. In fact, we actually observed an increase in
the number of active lysine residues if the proteolyzed particles
fused.
-chymotrypsin are
similar in specificity, it remains to be shown that the chymase present
in the exocytosed mast cell granules of human aortic
(32) and
coronary
(33) intima does actually produce fused LDL particles
with increased strength of binding to proteoglycans. Proteolytic fusion
of LDL, if it occurs in vivo, could lead to tight binding of
LDL to the extracellular matrix, and would also offer an explanation
for why the anchored LDL particles appear as large lipid droplets.
Table:
Analysis of native and
-chymotrypsin-treated LDL prepared for NMR
-chymotrypsin
at 37 °C for 24 h, the mixture was applied to a Bio-Gel A-15m
column (2.5
50 cm) and eluted with saline. The first two 280 nm
absorbing peaks were pooled separately (peaks I and II). LDL and the
two peaks were labeled with [
C]formaldehyde as
described under ``Experimental Procedures.'' The particle
diameters, retention times in a proteoglycan affinity column, and
protein and cholesteryl linoleate contents of LDL and of the two peaks
were determined as described under ``Experimental
Procedures.''
Table:
Effect of proteolysis and fusion on the numbers
of active and normal lysine residues in LDL particles
C]dimethyl-lysine resonances
shown in Fig. 4 in relation to the amount of cholesteryl linoleate in
each sample. Numbers of active lysine residues per particle are
calculated from the average diameters of the samples shown in Table I.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.