(Received for publication, July 22, 1996, and in revised form, November 27, 1996)
From the Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki, Finland
Decorin is a small dermatan sulfate-rich proteoglycan which binds to collagen type I in vitro and in vivo. In atherosclerotic lesions the contents of low density lipoprotein (LDL), decorin, and collagen type I are increased, and ultrastructural studies have suggested an association between LDL and collagen in the lesions. To study interactions between LDL, decorin, and collagen type I, we used solid phase systems in which LDL was coupled to a Sepharose column, or in which LDL, decorin, or collagen type I was attached to microtiter wells. The interaction between LDL and decorin in the fluid phase was evaluated using a gel mobility shift assay. We found that LDL binds to decorin by ionic interactions. After treatment with chondroitinase ABC, decorin did not bind to LDL, showing that the glycosaminoglycan side chain of decorin is essential for LDL binding. Acetylated and cyclohexanedione-treated LDL did not bind to decorin, demonstrating that both lysine and arginine residues of apoB-100 are necessary for the interaction. When collagen type I was attached to the microtiter plates, only insignificant amounts of LDL bound to the collagen. However, if decorin was first allowed to bind to the collagen, binding of LDL to the decorin-collagen complexes was over 10-fold higher than to collagen alone. Thus, decorin can link LDL with collagen type I in vitro, which suggests a novel mechanism for retention of LDL in collagen-rich areas of atherosclerotic lesions.
Deposition of low density lipoproteins (LDL)1 in the extracellular matrix of the arterial intima is an important step in the development of atherosclerosis (1). LDL interacts with the various components of the extracellular matrix. These components include proteoglycans (PG), elastin, and collagen. Binding of LDL to PG has been characterized in detail (2). Interaction between LDL and elastin has also been reported (3). In contrast, interaction between native LDL and collagen has received little attention (4, 5).
Collagen is the major connective tissue component of atherosclerotic arteries, comprising up to 40% of the total protein in fibrous plaques and 60% in advanced lesions (6). Most of the collagen (50-75%) in a normal artery or in a diseased intima is of type I (7-9). Immunofluorescence microscopy has located LDL along collagen fibers in atherosclerotic lesions (10, 11). Electron microscopic and immunoelectron microscopic studies have also suggested an association between LDL and collagen in the arterial intima (12-14).
Decorin (15) is a small PG having a core protein with a molecular mass of 45 kDa and a single dermatan sulfate-rich side chain. Decorin has been shown to bind to and modify the fibrillar structure of collagen type I (16, 17), and importantly, to co-localize with collagen type I in primary atherosclerotic plaques (18).
The present study concerns the interaction between native LDL and collagen type I in the absence and presence of decorin. In the absence of decorin, there was no significant interaction between LDL and collagen type I. However, when decorin was first allowed to bind to the immobilized collagen, significant amounts of LDL were bound to the decorin-collagen complexes formed. Thus, decorin can link LDL with collagen type I.
[1,2-3H]Cholesteryl linoleate,
t-butoxycarbonyl-L-[35S] methionine,
N-hydroxysuccinimidyl ester (35S labeling
reagent), and N-succinimidyl[2,3-3H]propionate
(3H-Bolton-Hunter reagent) were from Amersham; acid-soluble
collagen type I from calf skin, -amino caproic acid, bovine serum
albumin (BSA), and heparin from Sigma; EDTA, SDS, and acetic anhydride from Merck; CM-Sepharose, DEAE-Sepharose, HiTrap Q-columns, Hi-Trap N-hydroxysuccinimidyl ester-activated column, Superose 6 HR
10/30 columns, and dextran sulfate from Pharmacia Biotech; Celite 545 (acid-washed) and 1,2-cyclohexanedione from Fluka; chondroitinase AC
and ABC, chondroitin-6-sulfate, and dermatan sulfate from Seikagaku, and microtiter plates (Combiplate 8, Enhanced Binding) from Labsystems (Helsinki, Finland). NuSieve GTG-agarose was from FMC BioProducts, phenylmethylsulfonyl fluoride from Boehringer Mannheim, the Schrynel nylon filter from Zürcher Beuteltuchfabrik AG, a 5-µm
NH2 (0.3 × 25 cm) column from Spherisorb, and
Dulbecco's phosphate-buffered saline from Life Technologies, Inc. The
cholesteryl ester transfer protein was a kind gift from Drs. C. Ehnholm
and M. Jauhiainen, National Public Health Institute, Helsinki,
Finland.
Proteoglycans were isolated from bovine fetal skin
essentially as described (19). Briefly, PG were extracted from the skin at 4 °C with 7.8 M urea, 0.15 M NaCl, 5 mM EDTA, and 25 mM Tris, pH 6.5, in the
presence of 5 mM -aminocaproic acid, 0.2 mM
phenylmethylsulfonyl fluoride, and 0.02% (w/v) NaN3
(extraction buffer). After extraction, the mixture was filtered through
a Schrynel 48-µm nylon filter and centrifuged at 100,000 × g for 60 min. The supernatant was batch-adsorbed with
CM-Sepharose. The CM-Sepharose was removed by centrifugation at
4 °C, and the supernatant was adsorbed with DEAE-Sepharose. The
Sepharose was packed into a column (5 × 30 cm), and the bound
material was eluted with extraction buffer containing 1 M
NaCl. The peaks at 280 nm were collected, diluted with the extraction
buffer without NaCl to give a final NaCl concentration of 0.15 M, and loaded on a HiTrap Q column (5 ml) equilibrated with
the extraction buffer. PG were eluted with a linear gradient of
0.15-1.0 M NaCl in the extraction buffer (120 ml) at a
flow rate of 2 ml/min, dialyzed against water, and lyophilized. The disaccharide composition of the PG was analyzed by high performance liquid chromatography using a 5-µm NH2 column after
treatment of the PG with chondroitinase ABC and AC (20). The PG
preparation contained 68% of dermatan sulfate, 3% of chondroitin
6-sulfate, and 29% of chondroitin 4-sulfate. The composition of the PG
preparation was analyzed by N-terminal sequencing. A fraction
corresponding to 6 µg of total protein was subjected to Edman
degradation, using a Procise 494 protein-sequencing system. Released
phenylthiohydantoin-derivatives were quantified using model 610 software. This revealed that the sample contained over 90% of decorin
and less than 10% of biglycan, and no other sequences were found. The
isolated decorin was found to have an anticoagulant capacity in
citrated human plasma, as previously characterized (21).
35S-Decorin was prepared by labeling the core protein of
decorin with 35S labeling reagent by the Bolton-Hunter
procedure (22). Chondroitinase ABC-treated decorin was prepared by
incubating 60 µg of 35S-decorin with 75 milliunits of
chondroitinase ABC in 160 µl of Dulbecco's phosphate-buffered saline
containing 100 µg/ml BSA at 37 °C for 18 h. The amounts and
concentrations of decorin are expressed in terms of protein.
Human LDL (d = 1.019-1.050 g/ml) and HDL3 (d = 1.125-1.210 g/ml) were isolated from plasma of healthy volunteers by sequential ultracentrifugation (23). The LDL was labeled with [3H]cholesteryl linoleate ([3H]CL-LDL) as described previously (24). Apolipoprotein B-100 (apoB-100) of LDL and apolipoproteins of HDL3 were tritiated by the Bolton-Hunter procedure (22) with N-succinimidyl[2,3-3H]propionate to yield 3H-LDL and 3H-HDL3, respectively. LDL was acetylated by treatment of [3H]CL-LDL with acetic anhydride (25). Cyclohexanedione-LDL was prepared by treatment of [3H]CL-LDL with 1,2-cyclohexanedione (26). The amounts and concentrations of LDL are expressed in terms of protein.
Adsorption of Proteins to Microtiter WellsA suspension of collagen was obtained by dissolving 1 mg/ml collagen type I in 0.1 M acetic acid followed by dialysis against 67 mM phosphate buffer, pH 7.2, for 2-3 days at 4 °C (27). For collagen binding experiments, microtiter wells were coated with 50 µl of either a suspension of collagen type I (50 µg/ml) or of BSA (5 mg/ml) in 67 mM phosphate buffer, pH 7.2, by incubation at 37 °C overnight (27). For decorin binding experiments, microtiter wells were coated with 50 µl of either decorin (10 µg/ml) or BSA (5 mg/ml) in Dulbecco's phosphate-buffered saline by incubation at 37 °C overnight. For LDL binding experiments, microtiter wells were coated with 50 µl of either LDL (100 µg/ml) or BSA (5 mg/ml) in Dulbecco's phosphate-buffered saline by incubation at 37 °C for 2 h. Nonspecific binding sites in the wells were blocked by incubation at room temperature for 1 h with 100 µl of buffer A (50 mM NaCl, 2 mM CaCl2, 2 mM CaCl2, 10 mM HEPES, pH 7.4, and 5 mg/ml BSA).
Microtiter Well Binding AssayBinding of radiolabeled LDL or HDL3 to microtiter wells coated with decorin, binding of radiolabeled decorin to microtiter wells coated with LDL, and binding of radiolabeled LDL to microtiter wells coated with collagen type I was studied by incubating the compound to be tested in 50 µl of buffer A at room temperature for 2 h in the respective wells. In some experiments various amounts of sulfated polysaccharides were added with radiolabeled LDL. Wells coated with BSA served as controls for nonspecific binding. Unbound compounds were removed by aspirating the incubation medium, and the wells were then rinsed three times with 100 µl of buffer A. The bound compounds were then detached by incubating the wells twice with 100 µl of 10% SDS at room temperature for 30 min, the SDS solutions were pooled, and the liberated compounds were quantified by scintillation counting. In some experiments, collagen-coated or BSA-coated microtiter wells were first incubated in the absence or presence of 35S-decorin (50 µl) for 2 h, after which the wells were washed, and the ability of 3H-LDL to bind to the decorin-collagen complexes formed was studied as described above.
Affinity Chromatography of Decorin on LDL Affinity ColumnLDL was coupled to a N-hydroxysuccinimidyl ester-activated HiTrap column (1 ml) according to the manufacturer's instructions. Decorin and 35S-decorin (20 µl samples) were analyzed on the LDL affinity column by elution with a linear gradient of NaCl (0-250 mM in 10 min) in buffer containing 2 mM CaCl2, 2 mM MgCl2, and 10 mM HEPES, pH 7.4, at a flow rate of 1 ml/min. Elution of decorin was monitored at 280 nm. In some experiments, 500-µl fractions were collected and analyzed for their radioactivities. Chromatography was performed with a SMART system from Pharmacia.
Gel Mobility Shift AssayAffinity of LDL for decorin was measured by the gel mobility shift assay, essentially as described previously by Camejo et al. (28). Briefly, 0.4 µg of 35S-decorin was mixed with 0-50 µg of LDL in 20 µl of buffer containing, 140 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.2. After incubation for 1 h at 37 °C, 3 µl of glycerol was added, and 10-µl aliquots from each tube were applied to slots in 0.5% agarose submarine gels. The gels, approximately 3 mm in thickness, were made of NuSieve-agarose dissolved in buffer containing 2 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.4. The gels were electrophoresed for 2 h at 60 V, fixed overnight in 0.1% cetylpyridinium bromide, dried, and autoradiographed.
Other AssaysProtein was determined by the procedure of Lowry et al. (29) with bovine serum albumin as standard.
To study the ability of
LDL to bind to decorin, 3H-LDL was added to microtiter
wells coated with decorin. As shown in Fig. 1A, increasing the concentrations of LDL led
to binding of progressively increasing amounts of LDL to the
immobilized decorin. No such increase in LDL binding was observed in
the albumin-coated wells. When 3H-HDL3 was
used, only insignificant binding to decorin was observed (Panel
B).
To study the interaction between decorin and LDL, 10 µg of
35S-decorin was applied to an LDL affinity column and then
eluted with a linear NaCl gradient. As shown in Fig. 2,
96% of the applied decorin bound to the LDL, and the bound decorin was
released from the LDL at approximately 100 mM NaCl in a
NaCl gradient, suggesting that decorin was bound to LDL via ionic
interactions. In accord with this result, we found that, in the
microtiter well binding assay, increasing concentrations of NaCl
progressively decreased the binding of 35S-decorin to
LDL-coated microtiter wells (not shown).
To find out whether LDL and decorin would interact in physiologic ionic
conditions, we studied the ability of LDL to retard the electrophoretic
mobility of decorin on agarose gel after LDL and decorin had been
incubated in a buffer with an ionic strength of 0.15. Fig.
3 is an autoradiograph of an agarose gel electrophoresis of 35S-decorin incubated with increasing amounts of LDL. It
is evident that LDL was able to retard the electrophoretic mobility of
decorin, showing that LDL and decorin had interacted in the physiologic ionic conditions used.
To study whether LDL binds to the core protein or to the
glycosaminoglycan (GAG) side chain of decorin, we treated
35S-decorin with chondroitinase ABC to yield a decorin
preparation having the core protein but lacking the GAG chain, and then
compared the abilities of native and chondroitinase ABC-treated
35S-decorin to bind to LDL-coated microtiter wells. As
shown in Fig. 4, chondroitinase ABC-treated decorin
lacked the ability to bind to LDL, suggesting that LDL binds to the GAG
side chain of decorin. To confirm this finding we studied the ability
of LDL to retard the electrophoretic mobility of chondroitinase
ABC-treated 35S-decorin. We found that similar
concentrations of LDL that were able to retard the electrophoretic
mobility of LDL (shown in Fig. 3) had no effect on the electrophoretic
mobility of the decorin core protein (not shown), demonstrating that
the GAG chain is indeed essential for binding of decorin to LDL.
Treatment of LDL with chondroitinase ABC did not affect the interaction
between LDL and decorin (not shown).
To study the role of the positively charged amino acids of apoB-100 in
the interaction between LDL and decorin, [3H]CL-LDL was
modified either by acetylation or cyclohexanedione treatment to block
the lysine and arginine residues of apoB-100, respectively. We found
that either modification abolished the binding of LDL to decorin (Fig.
5). Thus, both the lysine and the arginine residues of
apoB-100 are essential for the interaction between LDL and decorin, a
result consistent with previous studies on the interaction between LDL
and PG of other types (30).
To assess the degree of specificity in the interaction between LDL and
decorin, we compared the abilities of soluble sulfated polysaccharides
and decorin to inhibit the binding of 3H-LDL to immobilized
decorin. As expected, all studied sulfated polysaccharides were able to
inhibit the binding (Fig. 6). More specifically, dextran
sulfate and heparin, two molecules with the highest negative charge
densities, were most effective, and chondroitin 6-sulfate, dermatan
sulfate, and decorin which have lower negative charge densities, were
less effective. These results suggest that the other tested negatively
charged molecules bind to the same regions in apoB-100 of LDL as
decorin.
Interaction between LDL and Collagen Type I in the Presence of Decorin
To study the interaction between LDL and immobilized
collagen type I in the absence or presence of decorin, collagen-coated microtiter wells were incubated with 35S-decorin, the wells
were washed, and the ability of 3H-LDL to bind to collagen
or decorin-collagen complexes formed was assessed. As shown in Fig.
7A, in the absence of decorin the ability of
LDL to bind to immobilized collagen was negligible. However, when
collagen-coated wells had been incubated with decorin, which led, on
average, to binding of 100 ng of decorin to collagen per well, binding
of LDL to the decorin-collagen complexes formed at each LDL
concentration tested was more than 10-fold higher than binding to
collagen alone. Finally, we studied the ability of 3H-LDL
to bind to immobilized collagen as a function of the amount of decorin
bound to collagen (Fig. 7B). It appeared that the greater the amount of decorin bound to collagen, the greater was the amount of
LDL bound to the decorin-collagen complexes.
The present results show that decorin can link LDL and collagen type I in vitro. In this linkage, the positively charged amino acids of apoB-100 bind to the negatively charged GAG chain of decorin, and, as shown by others (16, 31), the core protein binds to collagen type I. The novelty of this "sandwich" is the association between LDL and collagen via a proteoglycan.
LDL is known to bind to various glycosaminoglycans and proteoglycans (2). This (relatively weak) interaction is electrostatic, and depends on the binding of some of the positively charged amino acids of two types, lysine and arginine, of apoB-100 to the negatively charged sulfate groups of the GAG (30). The segments of apoB-100 that are involved in the binding between LDL and GAG have been identified (32). When we compared different GAG and sulfated polysaccharides for their ability to block the binding of LDL to immobilized decorin, we found that soluble heparin and dextran sulfate were more effective than decorin, dermatan sulfate, and chondroitin 6-sulfate, which were equally effective. This is in good agreement with previous work showing that the strengths of binding of GAG and sulfated polysaccharides to LDL in descending order are: heparin > dermatan sulfate > heparan sulfate > chondroitin 4-sulfate (33).
Of the sulfated polysaccharides listed above, the two with the highest affinity for LDL are dextran sulfate and heparin, which also have the highest degree of sulfation. Indeed, the degree of sulfation of a GAG is known to be critical for LDL binding. Thus, LDL binds to chondroitin 6-sulfates that have oversulfated regions, but not to a chondroitin 6-sulfate that lacks regions of oversulfation (34). Camejo et al. (34) finding that proliferating smooth muscle cells produced GAGs with increased chain length that showed increased affinity for LDL, proposed that LDL binding depends on the chain length of the GAG (28). Vijayagopal et al. (35) showed that cholesterol-enriched smooth muscle cells also produced PG of increased size that had a stronger affinity for LDL. However, in that report the degree of sulfation of the GAGs was also studied, and was found to be increased. It is thus possible that the strength of binding of GAG to LDL is more intimately related to the increased sulfation than to the increased length of the GAG, a possibility which is consistent with a previous suggestion by Sambandam et al. (34).
The interaction between decorin and collagen type I has been studied extensively, and recently a scheme of interaction has been proposed (36). Is decorin unique in its ability to link LDL with collagen type I? Decorin belongs to a family of structurally related extracellular matrix proteoglycans and glycoproteins which also includes biglycan (37), fibromodulin (38), lumican (39), keratocan (40), and chondroadherin (41). There is strong evidence that, of these molecules, decorin (42) and fibromodulin (43) bind to collagen type I, but whether biglycan does so is unclear (44). Furthermore, only decorin and biglycan have chondroitin/dermatan sulfate chains that potentially bind LDL, whereas the other molecules listed either lack the GAG chain or have a keratan sulfate chain which, in arteries, is not sulfated or only lightly sulfated (45). Therefore, theoretically, only decorin and biglycan can link LDL with collagen type I. However, biglycan is located pericellularly and decorin is located in collagen-rich areas of the arterial intima (18), where, potentially, it can link LDL with collagen type I. Thus, in light of present knowledge, the only small PG capable of linking LDL with collagen type I is decorin.
The arterial intima has been shown to contain, besides collagen type I, also collagen types III, IV, V, VI, and VIII (46). Of these, decorin binds to types I (43), V (47), and VI (48) in vitro, and co-localizes with collagen types I and III in atherosclerotic plaques (18). Whether all these collagen types are important for retention of lipid in the atherosclerotic plaque is not known. Interestingly, two other molecules in atherosclerotic lesions, fibronectin (49, 50) and thrombospondin (51), have also been shown to bind decorin.
The arterial intima contains, besides decorin, a large variety of proteoglycans (52). These include basement membrane PG (e.g. perlecan), cell surface heparan sulfate PG (e.g. the syndecan family and glypicans), versican, small dermatan sulfate-rich PG (decorin, biglycan), small keratan sulfate PG (fibromodulin, lumican, and keratocan), and heparan sulfate PG on elastin. The findings that, of the pig (53) and human (34) aortic PG, the fractions that most effectively interacted with LDL were those containing the small dermatan sulfate-rich PG (i.e. decorin and biglycan) support the idea that LDL binds to decorin even in the presence of other PG in the arterial intima.
Taken together, the current finding that decorin links LDL with collagen type I, and the previous demonstrations that the amounts of both collagen (6) and decorin (18) are increased in arterial lesions, suggest that the collagen fibers in the lesions, when covered with decorin, are capable of retaining significant amounts of LDL.
The excellent technical assistance of Päivi Hiironen is gratefully acknowledged. We thank Dr. Tom Krusius for helpful advice in isolating decorin.