CONFORMATIONAL DIFFERENCES IN HUMAN APOLIPOPROTEIN B-100 AMONG SUBSPECIES OF LOW-DENSITY LIPOPROTEINS (LDL) - ASSOCIATION OF ALTERED PROTEOLYTIC ACCESSIBILITY WITH DECREASED RECEPTOR-BINDING OF LDL SUBSPECIES FROM HYPERTRIGLYCERIDEMIC SUBJECTS

We asked at what point in the metabolic cascade of very low density li poproteins (VLDL) to low density lipoproteins (LDL) the accessibility of proteolytic cleavage sites in B-100 changes, and we evaluated the e ffect of hypertriglyceridemia on the proteolytic accessibility, second ary structure, and receptor-binding affinity of B-100 in LDL subspecie s of varying density. Limited proteolysis with Staphtylococcus 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 hypertri glyceridemic (HTG-) subjects at which accessibility to protease attack changed in three peptide regions of B-100. Hypertriglyceridemia great ly altered proteolytic accessibility of B-100 in the denser LDL subspe cies. Specifically, B-100 in HTG LDL exposed more cleavage sites than in N-LDL, including two novel sites, similar to 120 and similar to 130 kDa from the NH2 terminus in the small and dense subspecies (designat ed LDL-4, -4.5 or -5, d = 1.048-1.062 g/ml). Analysis of circular dich roic spectra indicated no difference in helical content between B-100 in N- and HTG-LDL but showed a greater content of beta-structure in HT G-LDL. Binding affinity for the LDL receptor of human fibroblasts decr eased markedly with increasing density among HTG-LDL subspecies (by si milar to 50% for LDL-4.5 or -5). We conclude that the changes in prote olytic 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 clea vage sites, especially novel sites in the NH2-terminal regions, with g reatly decreased receptor-binding affinity in LDL-4.5 or -5 suggests t hat altered local conformation in B-100 apart from the putative recept or-binding domain might affect interaction with the receptor.