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 inact...

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Bibliographic Details
Published in:FEBS letters 2000-01, Vol.466 (2), p.244-248
Main Authors: Caffrey, Conor R., Mathieu, Mary A., Gaffney, Alan M., Salter, Jason P., Sajid, Mohammed, Lucas, Kimberley D., Franklin, Christopher, Bogyo, Matthew, McKerrow, James H.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Asparaginyl endopeptidase
Base Sequence
Catalysis
Cloning, Molecular
Cysteine Endopeptidases - genetics
Cysteine Endopeptidases - metabolism
DNA, Complementary
Enzyme Activation
Humans
Legumain
Molecular Sequence Data
Pichia - genetics
Plant Proteins
Recombinant expression
Schistosoma
Schistosoma mansoni - enzymology
Sequence Homology, Amino Acid
Sm32
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Summary: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been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present.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.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(99)01798-6