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 mansoni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present.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 expressed in Pichia pastoris. Autocatalytic processing to fully active C197 Sm32 occurred at acid pH. In contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. This was confirmed by mutation of N197 to C and re-expression in Pichia. The availability of recombinant active Sm32 allows detailed analysis of its catalytic mechanism and its function(s) in the biology of this important human parasite.
Caffrey
C R
CR
Department of Pathology, UCSF, VAMC, San Francisco, CA 94121, USA.
Mathieu
M A
MA
Gaffney
A M
AM
Salter
J P
JP
Sajid
M
M
Lucas
K D
KD
Franklin
C
C
Bogyo
M
M
McKerrow
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins; D012550:Schistosoma mansoni / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D018076:DNA, Complementary; D010940:Plant Proteins; D000099240:Asparaginyl Endopeptidase