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Substrate specificity of schistosome versus human legumain determined by P1–P3 peptide libraries
- Source :
- Molecular and Biochemical Parasitology. 121:99-105
- Publication Year :
- 2002
- Publisher :
- Elsevier BV, 2002.
-
Abstract
- 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 positionalscanning 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. # 2002 Published by Elsevier Science B.V.
- Subjects :
- Proteases
Molecular Sequence Data
Peptide
Biology
Legumain
Substrate Specificity
law.invention
Pichia pastoris
Peptide Library
law
Animals
Combinatorial Chemistry Techniques
Humans
Amino Acid Sequence
Asparagine
Molecular Biology
Plant Proteins
chemistry.chemical_classification
Schistosoma mansoni
biology.organism_classification
Molecular biology
Endopeptidase
Cysteine Endopeptidases
Kinetics
chemistry
Biochemistry
biology.protein
Recombinant DNA
Parasitology
Peptides
Cysteine
Subjects
Details
- ISSN :
- 01666851
- Volume :
- 121
- Database :
- OpenAIRE
- Journal :
- Molecular and Biochemical Parasitology
- Accession number :
- edsair.doi.dedup.....f308c05627c5d9bd8a1de2451647fb4e