Proteases involved in the metabolic degradation of human interleukin-1beta by rat kidney lysosomes.
The in vitro metabolic degradation of human interleukin (IL)-1beta was studied using lysates of rat kidney lysosomes, and proteases involved in the degradation were identified. In the study of IL-1beta degradation, fluorescein isothiocyanate (FITC)-labeled IL-1beta was used as a substrate. The maximal degradation of IL-1beta occurred at pH 3.0, and the reaction was proportional to the lysosomal protein concentration and time of incubation. The degradation was stimulated by the addition of L-cysteine. The reaction was not inhibited by phenylmethanesulfonyl fluoride or EDTA, indicating that serine proteases or metalloproteases do not play a major role in the degradation process. N-Ethylmaleimide, leupeptin and E-64, inhibitors of thiol protease, inhibited the degradation of IL-1beta, by 59%-70%. Pepstatin A, an inhibitor of carboxyl protease, inhibited the degradation by 58%. Combinations of thiol and carboxyl protease inhibitors nearly completely inhibited the degradation. Bio-Gel P-10 gel filtration chromatography of in vitro reactants confirmed the ability of lysosomal proteases to degrade IL-1beta and revealed four to five peaks of degradation products. Taken together, these results indicate that thiol protease and carboxyl protease play an important role in the IL-1beta degradation process by kidney lysosomes. Leupeptin and E-64 dose dependently inhibited both cathepsin B and cathepsin L activities, and pepstatin A strongly inhibited cathepsin D activity in rat kidney lysosomes. The present results suggest that cathepsin B, cathepsin L, and cathepsin D in kidney lysosomes are involved in the metabolic degradation of human IL-1beta.
Kudo
S
S
Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Japan. s_kudo@research.otsuka.co.jp
Miyamoto
G
G
Kawano
K
K
D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D007375:Interleukin-1; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D016650:Fluorescein-5-isothiocyanate