title: Baculoviral expression and characterization of rodent cathepsin S.
abstract: The cysteinyl proteinase cathepsin S is implicated as a key enzyme in the processing of major histocompatability complex (MHC) class II molecules expressed on antigen presenting cells and thus is a potential therapeutic target 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 acid extended presequence relative to the human enzyme and the previously published rat enzyme. In addition, we have expressed these mouse and rat proteins in baculovirally infected Sf9 insect cells along with "humanized" forms truncated by eight residues at the amino-terminus. All forms of the rodent proteinases were overexpressed and milligram per litre amounts of functional enzyme could be isolated from the cells and/or the cell culture supernatant. Furthermore, addition of a carboxy-terminal hexahistidine purification tag had no effect on the kinetic characteristics of any of the enzyme forms against the Boc-Val-Leu-Lys-AMC peptide substrate (rat k(cat) s(-1) approximately 30; mouse k(cat) s(-1) approximately 65). Differences were seen in the potency of the generic cysteine proteinase inhibitor, E64, against the human, mouse, or rat form of the enzyme (13.3 x 10(4), 43.2 x 10(4), and 25 x 10(4) K(obe)/[I] M(-1) s(-1), respectively). Such data highlights the need for greater awareness of species variation in inhibitor potency. These reagents are vital for confirming inhibitor potency against the endogenous form of the enzyme prior to evaluation of drug candidates in rodent model systems.
authors.affiliation: Medivir UK Ltd., Peterhouse Technology Park, 100 Fulbourn Road, Cambridge, CB1 9PT, United Kingdom. clive.mason@medivir.com
authors.forename: C S
authors.initials: CS
authors.lastname: Mason
authors.forename: M B
authors.initials: MB
authors.lastname: Lamers
authors.forename: I M
authors.initials: IM
authors.lastname: Henderson
authors.forename: T
authors.initials: T
authors.lastname: Monk
authors.forename: D H
authors.initials: DH
authors.lastname: Williams
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002401:Cathepsin B / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007313:Insecta; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D016415:Sequence Alignment; D014161:Transduction, Genetic
chemical_list: D004791:Enzyme Inhibitors; D002401:Cathepsin B