Abstract
We asked at what point in the metabolic cascade of very low density lipoproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the effect of hypertriglyceridemia on the proteolytic accessibility, secondary structure, and receptor-binding affinity of B-100 in LDL subspecies of varying density. Limited proteolysis with Staphylococcus aureus V8 protease and cathepsin D identified the density (about 1.033 g/ml) between two LDL subspecies, designated LDL-1 and -2, as the transition point during VLDL metabolism of both normolipidemic (N-) and hypertriglyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia greatly altered proteolytic accessibility of B-100 in the denser LDL subspecies. Specifically, B-100 in HTG-LDL exposed more cleavage sites than in N-LDL, including two novel sites, approximately 120 and approximately 130 kDa from the NH2 terminus in the small and dense subspecies (designated LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dichroic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HTG-LDL. Binding affinity for the LDL receptor of human fibroblasts decreased markedly with increasing density among HTG-LDL subspecies (by approximately 50% for LDL-4.5 or -5). We conclude that the changes in proteolytic accessibility observed between LDL-1 and -2 and in LDL-4, -4.5, or -5 indicate significant differences in local conformation of B-100 at specific peptide regions. The association of exposure of more cleavage sites, especially novel sites in the NH2-terminal regions, with greatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests that altered local conformation in B-100 apart from the putative receptor-binding domain might affect interaction with the receptor.
Highlights
ASSOCIATION O F ALTERED PROTEOLYTIC ACCESSIBILITY WITH DECREASED RECEPTOR BINDING OF Low Density Lipoproteins (LDL) SUBSPECIES FROM HYPERTRIGLYCERIDEMIC SUBJECTS*
We asked at what point in the metaboliccascadeof very pears to be thesole protein component of plasma low density low density lipoproteins (VLDL) to low density lipopro- lipoproteins (LDL) (1-7)
LDL are formed from very teins (LDL)the accessibility of proteolytic cleavagesites low density lipoproteins(VLDL)through intermediate density in B-100 changes, and we evaluated the effect of hyper- lipoproteins (IDL) via a metabolic cascade involvinglipolysis ary structure, andreceptor-binding affinity of B-100 in and loss of other apolipoproteins (6-9)
Summary
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. 18,PP. Binding affinity for the LDL cleavage by the same batteroyf enzymes (15).within receptor of human fibroblasts decreased markedly with the region of residues 2660-2710, cathepsin D cleavesB-100 of increasing density among HTG-LDL subspecies(by LDL and VLDL a t a different rate, buat lso preferen-. Faint bands of apoB-48 and apoE discrete subspecies, a s shown by analytical or density gradient ultracentrifugation (24-30) or gradient gel electrophoresis (3033) These LDL subspecies from both N- and HTG-subjects differ markedly in their size, density, chemical composition, and immunoreactivity (25-35). The chemical compositions ofLDL subspecies point in theVLDL to LDL metabolic cascadethe accessibility of were measured as described previousIy (41); the concentration of proproteolytic cleavage sites inB-100changes, and to effect of hypertriglyceridemia on the accessibility of evaluate the these sites tein was determined with hydrated bovine serum albumin as the standard, concentrationsofcholesteroland cholesteryl ester by a fluorometric enzymatic method, those of phospholipids as lipid-associatedphosphoto cleavage.
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