Degradation of apolipoprotein B-100 by lysosomal cysteine cathepsins.
Although the degradation of cellular or endocytosed proteins comprises the normal function of lysosomal proteinases, these enzymes were also detected extracellularly during diseases such as atherosclerosis. Since lysosomal cysteine cathepsins were demonstrated to transform native LDL particles into a proatherogenic type, the following study was undertaken to characterize the modification of LDL particles and the degradation of apolipoprotein B-100 in more detail. LDL was incubated with cathepsins B, F, K, L, S, and V at pH 5.5 and under physiological conditions (pH 7.4) for 2 h to mimic conditions of limited proteolysis. Gel electrophoretic analysis of the degradation products revealed that cathepsin-mediated proteolysis of apolipoprotein B-100 is a fast process carried out by all enzymes at pH 5.5, and by cathepsin S also at pH 7.4. Electron microscopic analysis showed that cathepsin-mediated degradation of apolipoprotein B-100 rendered LDL particles fusion-competent compared to controls. N-Terminal sequencing of cathepsin cleavage fragments from apolipoprotein B-100 revealed an abundance of enzyme-specific cleavage sites located in almost all structurally and functionally essential regions. Since the cleavage sites superimpose well with results from substrate specificity studies, they might be useful for the development of cathepsin-specific inhibitors and substrates.
Linke
Martin
M
Department of Dentistry and UBC Center for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
Gordon
Ronald E
RE
Brillard
Michèle
M
Lecaille
Fabien
F
Lalmanach
Gilles
G
Brömme
Dieter
D
D000595:Amino Acid Sequence; D053299:Apolipoprotein B-100 / Q000737:chemistry / Q000378:metabolism*; D002403:Cathepsins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D004789:Enzyme Activation; D005782:Gels; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008077:Lipoproteins, LDL / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D053299:Apolipoprotein B-100; D005782:Gels; D008077:Lipoproteins, LDL; D002403:Cathepsins