Inhibition of cathepsin L-like cysteine proteases by cytotoxic T-lymphocyte antigen-2 beta.
The protein sequence of cytotoxic T-lymphocyte antigen-2 beta (CTLA-2 beta) is 36% identical to the proregion of mouse cathepsin L (Denizot, F., Brunet, J.F., Roustan, P., Harper, K., Suzan, M., Luciani, M. F., Mattei, M. G., and Goldstein, P. (1989) Eur. J. Immunol. 19, 631-635). Here we report the expression, purification, and characterization of recombinant murine CTLA-2 beta. The protein was purified by consecutive gel-filtration, anion-exchange, and reverse-phase (C4) chromatography. Purified CTLA-2 beta exists in solution primarily as a dimer but also as a disulfide-linked tetramer as judged by size exclusion chromatography. Circular dichroism studies suggest that the dimeric form of the protein contains 8% alpha-helix, 67% beta-sheet, and 21% random coil and also indicates that there is a conformational change upon formation of the tetramer. The protein is a competitive inhibitor of certain cysteine proteases including papain (Ki = 25 nM), cathepsins L (Ki = 24 nM) and H (IC50 = 67 nM) but not cathepsin B. CTLA-2 beta forms a noncovalent complex with cathepsin L and has a stoichiometry of binding to papain of 1 mol of CTLA-2 beta/mol of papain. There is no homology between CTLA-2 beta and any of the known cysteine protease inhibitors, including the kininogens and cystatins. Therefore, CTLA-2 beta represents a novel class of cysteine protease inhibitor that is specific for the cathepsin L family of proteases.
Delaria
K
K
Miles, Inc., Pharmaceutical Division, West Haven, Connecticut 06516-4175.
Fiorentino
L
L
Wallace
L
L
Tamburini
P
P
Brownell
E
E
Muller
D
D
D000941:Antigens / Q000737:chemistry / Q000302:isolation & purification*; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D010450:Endopeptidases; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000941:Antigens; D015853:Cysteine Proteinase Inhibitors; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse