title: Differences in local conformation in human apolipoprotein B-100 of plasma low density and very low density lipoproteins as identified by cathepsin D.
abstract: The conformational changes of human apolipoprotein (apo) B-100 which accompany the conversion of plasma very low density lipoproteins (VLDL) to low density lipoproteins (LDL) were investigated by studying the accessibility of apoB-100 in LDL and VLDL to limited proteolysis with cathepsin D, an aspartyl proteinase involved in intracellular protein degradation. We characterized the proteolytic products of apoB-100 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by NH2-terminal sequence analysis to locate cleavage sites. The results identified at least 10 cleavage products generated from apoB-100 and showed differential accessibility of cleavage sites for cathepsin D in apoB-100 between LDL and VLDL. We identified a specific peptide region (residues 2660-2710), which is preferentially accessible to limited proteolysis by cathepsin D but inaccessible to limited proteolysis by 12 other enzymes tested. Within this peptide region, cathepsin D cleaved apoB-100 of LDL and VLDL preferentially at different sites, separated by 33-36 amino acids (2665-2666 or 2668-2669 (LDL) and 2701-2702 (VLDL]. In addition, we identified a cleavage site, located at residues 3272-3273, specific for cathepsin D, which is contained within the COOH-terminal enzyme-accessible peptide region (residues 3180-3280), which we have demonstrated using 12 endoproteases with various specificities. The previously identified NH2-terminal region (residues 1280-1320) appears to be resistant to limited cleavage by cathepsin D. However, a new site was revealed only approximately 66 kDA from the NH2 terminus. We conclude that differential accessibility and the shift of the novel scission site for cathepsin D by 33-36 amino acids indicate significant differences in local conformation at these sites in apoB-100 as VLDL are converted to LDL.
authors.affiliation: Cardiovascular Research Institute, University of California, San Francisco 94143-0130.
authors.forename: G C
authors.initials: GC
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Lau
authors.forename: R L
authors.initials: RL
authors.lastname: Hamilton
authors.forename: J P
authors.initials: JP
authors.lastname: Kane
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry*; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008077:Lipoproteins, LDL / Q000097:blood / Q000737:chemistry*; D008079:Lipoproteins, VLDL / Q000097:blood / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D002402:Cathepsin D