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 (Paanenen, 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 alpha-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 [13C]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 13C-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.
Highlights
From the Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki and the :j:State Technical Research Center of Finland, Chemical Technology, FIN-02044 Espoo, Finland. Lipid droplets resembling those seen in the extracellular space ofthe arterial intima were generated in vitro when granule proteases of rat serosal mast cells degraded the apolipoprotein B-100 component of granule-bound low density lipoprotein (LDL), and the particles fused on the granule surface
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
This study shows that proteolysis of LDL with a-chymotrypsin renders the particles unstable and leads to formation of particles with increased size and with increased strength of binding to human aortic proteoglycans
Summary
Materials-a-Chymotrypsin, bovine serum albumin, E-aminocaproic acid, and soybean trypsin inhibitor were from Sigma. Affinity Chromatography ofProteolyzed LDL on Proteoglycan Affinity Columns-LDL was proteolyzed as described above, and 20-ILl samples of the incubation mixtures corresponding to 20 ILg of undegraded LDL were analyzed on the proteoglycan affinity columns by elution with a linear gradient ofNaCl (0-250 mM in 10 min) in buffer B. The sample was applied to a Bio-Gel A-15m column (2.5 X 50 em) equilibrated with saline (150 mM NaCl and 1 mM EDTA, pH 7.4) LDL eluted in two peaks, which were collected and pooled separately. Samples of the incubation mixtures (20 ILl) were analyzed by affinity chromatography on a 1-ml proteoglycan-HiTrap column (bold line). Dimethyl-lysine co-eluted from the column with histidine Another method was tried for analyzing the amino acids; the samples were analyzed as derivatives essentially as described by Chang et al [24]. X 15 em) column as described by Kritharides et al [28]
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