Inactivation of cathepsin B by oxidized LDL involves complex formation induced by binding of putative reactive sites exposed at low pH to thiols on the enzyme.
We recently showed that the poor degradation of apo B in oxidized (ox-) LDL by mouse peritoneal macrophages could be attributed to the inactivation of cathepsin B by ox-LDL. In this current study, we show that enzyme inactivation involves complex formation of ox-LDL with cathepsin B rather than the diffusion of reactive components from ox-LDL to the enzyme. Complex formation between ox-LDL and cathepsin B was far greater at pH 4.5 than at pH 7.4 and far greater with ox-LDL than with LDL. Even though complexes were also formed between ox-LDL and other proteins such as BSA, insulin, and LDL, ox-LDL bound up to 30 times more cathepsin B than BSA, when compared on a molar level and under the same conditions. Unlike ox-LDL alone, complexes of ox-LDL and BSA were unable to inactive cathepsin B, suggesting that BSA was sequestering reactive sites on ox-LDL. The interaction of ox-LDL with proteins such as cathepsin B appears to represent aldehydic modifications of apo B, since treatment of ox-LDL with the reductant NaBH4, which stabilizes such adducts, greatly decreased the binding of ox-LDL to BSA and prevented ox-LDL from inactivating cathepsin B. It is likely that thiols on cathepsin B or other proteins interact with reactive groups on ox-LDL, since BSA in which thiols were blocked with N-ethylmaleimide (NEM), failed to bind to ox-LDL. Moreover, NEM-treated BSA had no effect on the ability of ox-LDL to inactivate cathepsin B. Similar results were obtained with LDL modified with 4-hydroxynonenal (HNE). These data suggest that aldehydic adducts on ox-LDL that are unreactive at neutral pH, possibly HNE bound to apo B, become exposed at acidic pH and then covalently bind thiols on neighboring proteins such as cathepsin B in lysosomes, inducing crosslinking of proteins and enzyme inactivation.
O'Neil
J
J
Department of Cell Biology, Cleveland Clinic Foundation, Oh 44195, USA.
Hoppe
G
G
Sayre
L M
LM
Hoff
H F
HF
D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002417:Cattle; D005609:Free Radicals / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000447:Aldehydes; D001055:Apolipoproteins B; D005609:Free Radicals; D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; C099252:oxidized low density lipoprotein; D012710:Serum Albumin, Bovine; D002401:Cathepsin B