©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteolysis and Fusion of Low Density Lipoprotein Particles Strengthen Their Binding to Human Aortic Proteoglycans (*)

Katariina Paananen , Juhani Saarinen , Arto Annila (1), Petri T. Kovanen (§)

From the (1) Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki State Technical Research Center of Finland, Chemical Technology, FIN-02044 Espoo, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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)() 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) .

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 -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 pKvalues, 8.9 and 10.5 (13) . The unusually low pKof 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.


EXPERIMENTAL PROCEDURES

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 NaSO, 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 -Chymotrypsin-treated LDL

LDL (4 mg) was incubated for 24 h at 37 °C in 100 µl of buffer A containing 200 µg of -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 C-Labeled LDL and C-labeled -Chymotrypsin-treated LDL for NMR

LDL (100 mg) was incubated for 24 h at 37 °C in 13 ml of buffer A containing 5 mg of -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 HCHO 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 DO 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).

The two lysine populations of apoB-100 having different pKvalues (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.

The NMR spectrum of the 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.

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 -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

After incubation of 100 mg of LDL in 8 ml of buffer A containing 5 mg of -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

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 [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.



FOOTNOTES

*
This work was supported in part by the Academy of Finland and Magnus Ehrnrooth Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: LDL, low density lipoprotein; apo, apolipoprotein; HPLC, high performance liquid chromatography; dpm, disintegrations/min; ppm, parts/million.


ACKNOWLEDGEMENTS

We are grateful to Dr. Nisse Kalkkinen for help in amino acid analysis. The excellent technical assistance of Minna Ahlstedt is gratefully acknowledged.


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