In vitro degradation of apo-, zinc-, and cadmium-metallothionein by cathepsins B, C, and D.
Metallothionein (MT) has been extensively studied over the past several years because of its probable role in endogenous metal homeostasis and cellular protection. A large body of knowledge now exists describing the physicochemical properties of MT as well as the mechanisms involved in MT induction. It has been well established that MT protects tissues from metal toxicity by chelating metals that would otherwise be available to interact with and disrupt vital cell functions. Information on the degradation of metal-saturated MT and the fate of the metals associated with it would be extremely important in predicting metal toxicity. Lysosomes have been targeted as a possible subcellular site for the turnover of MT; however, the susceptibility of MT to degradation by specific acidic proteases (i.e., cathepsins) has not been described. Therefore, the purpose of the present study was to examine the relative abilities of cathepsins B, C, and D to degrade Zn7-MT, Cd7-MT, and apo-MT in vitro. In so doing, the effects of metal species, degree of metal saturation, and pH on the degradation processes were evaluated. Time course experiments revealed that apo-MT was rapidly degraded by all three cathepsins. Cathepsin B degraded apo-MT approximately 36-fold more rapidly than cathepsin C and 45-fold more rapidly than cathepsin D. Therefore, under the in vitro conditions used in this study, the relative potency of the cathepsins tested was cathepsin B much much greater than cathepsin C greater than cathepsin D. In comparison, metal-saturated MT was more than 1000-fold more resistant to degradation by the cathepsins tested. In order to determine how much metal was needed to protect MT against degradation, apo-MT was reconstituted with increasing molar equivalents of Zn2+. The results suggest that as metal to apo-MT ratios increase, less apo-MT substrate is available to the protease and degradation decreases.
McKim
J M
JM
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City 66103.
Choudhuri
S
S
Klaassen
C D
CD
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002401:Cathepsin B / Q000494:pharmacology*; D020789:Cathepsin C; D002402:Cathepsin D / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007279:Injections, Subcutaneous; D008099:Liver / Q000378:metabolism; D008297:Male; D008668:Metallothionein / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013462:Sulfur Radioisotopes
D001059:Apoproteins; D002352:Carrier Proteins; D013462:Sulfur Radioisotopes; C004203:cadmium-binding protein; C017800:zinc-binding protein; D008668:Metallothionein; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D002401:Cathepsin B; D002402:Cathepsin D