Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris

Asparaginyl endopeptidases, or legumains, are a recently identified family of cysteine-class endopeptidases. A single gene encoding a Schistosoma mans oni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has bee n reported, but by sequence homology it would be expected to yield an inact ive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present. Both gene products were ex pressed in Pichia pastoris. Autocatalytic processing to fully active C197 S m32 occurred at acid pH. in contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. Thi s was confirmed by mutation of N197 to C and re-expression in Pichia. The a vailability of recombinant active Sm32 allows detailed analysis of its cata lytic mechanism and its function(s) in the biology of this important human parasite. (C) 2000 Federation of European Biochemical Societies.