Abstract
We compared the effect of lipid composition and particle size of triglyceride-rich low density lipoprotein (LDL) upon apoprotein B conformation and binding to the LDL receptor. Three groups of triglyceride-rich LDL were studied: (a) LDL isolated from chronic hypertriglyceridemic individuals (HTG-LDL); (b) normal LDL made triglyceride-rich by in vitro incubation with triglyceride emulsion and the neutral lipid transfer protein (R-LDL); and (c) LDL from normolipidemic individuals made acutely hypertriglyceridemic by intravenous infusion of 10% Intralipid (IV-LDL). HTG-LDL was small and dense, whereas R-LDL and IV-LDL had normal size. HTG-LDL, but not R-LDL or IV-LDL, exhibited decreased binding to the LDL receptor on human skin fibroblasts in studies at 4 degrees C and reduced degradation at 37 degrees C. Apoprotein B conformation was assessed by circular dichroism and by analyzing the immunoreactivity of different monoclonal antibodies. HTG-LDL but not R-LDL or IV-LDL showed a change in the CD spectra and a consistent decrease in the immunoreactivity of monoclonal antibody 3F5 (2.5-fold) which recognizes an epitope adjacent to the receptor binding domain of apoprotein B. These findings suggest that in triglyceride-rich LDL, the relative content of neutral lipid in the core of LDL in the absence of changes in the size of the particle does not significantly affect apoprotein B conformation or its affinity for the LDL receptor.
Highlights
We compared the effect of lipid compositionand par- carrier of cholesterol in plasma, are associated with increased ticle size of triglyceride-rich low density lipoprotein risk of atherosclerosis [1].LDL exhibits awide heterogeneity (LDL) uponapoprotein B conformation and binding to in lipid composition, immunoreactivity, size and density in the LDL receptor
LDL, exhibited decreased binding to theLDL receptor tural analysis, the use of monoclonal antibodies (Mabs) on human skin fibroblasts in studies at 4 “C and re- against different epitopes of Apoprotein B-100 (apoB) has been useful in deterduced degradation at 37 “C. Apoprotein B conforma- mining the general structure, thebinding region to the LDL
LDL Composition-Compared with N-LDL, LDL from subjects with chronic hypertriglyceridemia had relative enrichment in protein (1.4-fold)and triglyceride (%fold) along with a relative decrease in phospholipid and free and esterified cholesterol (20, 30, and 25%, respectively) (TableI)
Summary
Na'? was obtained from Amersham Corp. 10 and 20% Intralipid was obtained from Kabivitrum, Alameda, CA. 6 mg of LDL esterified cholesterol was incubated with 60 mg of emulsion triglyceride and 300-400 mg protein of d > 1.21 g/ml plasma in a solution of 0.19 M NaCl, 1 mM EDTA, 100 kallikrein inhibitory units/ml aprotinin, 10 pg/ml leupeptin, 1pg/ml pepstatin, pH 8.25. SizelDensity of LDL-To assess LDL size and relative densities, nonequilibrium rate zonal ultracentrifugation was performed in d > 1.006 g/ml plasma from chronically hypertriglyceridemic subjects, from normolipidemic individuals after 6 hof intravenous infusion of. Cell Binding-To assess the affinity of different LDL for the LDL receptor, in most of experiments, competition studies were done in duplicate at 4 "C on monolayers of humanskin fibroblasts; the medium (pH 7.4) contained Dulbecco's modified Eagle's medium, 20 mM Hepes, 5% lipoprotein-deficient serum, 5 pg/ml Iz6I-LDL,and increasing amounts of experimental LDL [27, 28]. Saturation binding assays at 4 "C were done in duplicate using increasing amounts of 'Z51-experimental LDL in the presence and absence of a 50-fold excess of unlabeled LDL, and the total, nonspecific, and specific binding was calculated [27, 28]
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