Structural Domains of Human Apolipoprotein B-100

The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined.In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage.One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOHterminal region).In LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible.Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used.Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V 8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints.These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis.These three domains are connected by the two susceptible peptide regions.Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis.This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.Human apolipoprotein B-100 is one of the largest polypeptides known, consisting of 4536 amino acid residues as deduced from cDNA clones (1-3).Moreover, B-100 is the sole