Baculoviral expression and characterization of rodent cathepsin S

The cysteinyl proteinase cathepsin S is implicated as a key enzyme in the p rocessing of major histocompatability complex (MHC) class II molecules expr essed on antigen presenting cells and thus is a potential therapeutic targe t for modulation in immune system-based disease. We have identified a form of rat cathepsin S, similar to a published mouse form with an eight-amino a cid extended presequence relative to the human enzyme and the previously pu blished rat enzyme. In addition, we have expressed these mouse and rat prot eins in baculovirally infected Sf9 insect cells along with "humanized" form s truncated by eight residues at the amino-terminus. All forms of the roden t proteinases were overexpressed and milligram per litre amounts of functio nal enzyme could be isolated from the cells and/or the cell culture superna tant. Furthermore, addition of a carboxy-terminal hexahistidine purificatio n tag had no effect on the kinetic characteristics of any of the enzyme for ms against the Boc-Val-Leu-Lys-AMC peptide substrate (rat k(cat) S-1 simila r to 30; mouse k(cat) S-1 similar to 65). Differences were seen in the pote ncy of the generic cysteine proteinase inhibitor, E64, against the human, m ouse, or rat form of the enzyme (13.3 x 10(4), 43.2 x 10(4), and25 x 10(4)K (obs)/[1]M(-1)s(-1), respectively). Such data highlights the need for great er awareness of species variation in inhibitor potency. These reagents are vital for confirming inhibitor potency against the endogenous form of the e nzyme prior to evaluation of drug candidates in rodent model systems. (C) 2 001 Academic Press.