Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries.
Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.
Mathieu
Mary A
MA
Department of Pathology, UCSF, San Francisco, CA 94143, USA. mmathie@itsa.ucsf.edu
Bogyo
Matthew
M
Caffrey
Conor R
CR
Choe
Youngchool
Y
Lee
Jewok
J
Chapman
Harold
H
Sajid
Mohammed
M
Craik
Charles S
CS
McKerrow
James H
JH
D000595:Amino Acid Sequence; D000818:Animals; D020650:Combinatorial Chemistry Techniques; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins; D012550:Schistosoma mansoni / Q000201:enzymology*; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D019151:Peptide Library; D010455:Peptides; D010940:Plant Proteins; D000099240:Asparaginyl Endopeptidase