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.
Highlights
Differences in Local Conformationin Human Apolipoprotein B-100 of Plasma Low Density and Very LowDensity Lipoproteins as Identified by Cathepsin D*
Plasma very low density lipoproteins (VLDL) to low Under normal circumstances, LDL are formed fromvery low density lipoproteins (LDL) were investigated by studdye-nsity lipoproteins (VLDL),which contain apoB-100 as the ing theaccessibility of apoB-100 inLDL and VLDL to major protein component, via a catabolic cascade involving limited proteolysis with cathepsinD, an aspartyl pro- lipolysis and loss of other apolipoprotein species [8].During teinase involved in intracellular protein degradation
Electron Microscopy-To determine whether limited proteolysis under the incubation conditions used disturbed the integrity of the lipoprotein particles, negative stains of at least three pairs of VLDL and LDL were examined by electron microscopy in three conditions
Summary
Electron Microscopy-To determine whether limited proteolysis under the incubation conditions used disturbed the integrity of the lipoprotein particles, negative stains of at least three pairs of VLDL and LDL were examined by electron microscopy in three conditions (see "ExperimentalProcedures"). Other minor band of approximately 170 kDa was identified indirectly as the COOH-terminal fragment based on the observation that it was absent in the unreduced SDS-PAGE pattern (Fig. 4), similar to thecorresponding -170-kDa band ever, the appearance of these additional bands was less complicated or was not detectable a t all when cathepsin D digests of short incubation were analyzed, i.e. during theinitial attack by cathepsin D, such as within 20 min of digestion, as shown in Fig. 4 (lane v"). To identify the cleavage products of apoB-100 in LDL and VLDL generated by cathepsin D and to locate their cleavage sites our approach was to determine the NH2-terminal sequences directly on theapoB-100 fragments.After separation by SDS-PAGE the individual bands in the cleavage pattern generated from the digestion of LDL These results indicated that apoB-100 of VLDL, like apoB-100 of LDL, was accessible to limited cleavage by cathepsin D at three peptide regions but with very different accessibility. With mobility corresponding to apparent molecular mass of kDa fragments, one time 11amino acid residues were identiapproximately 310 kDa and theminor band of approximately fied out of 12 cycles, and otherexperiments gave 9,8, 6, and Cathepsin D Identifies Conformational ChangienApoB-100
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