title: Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes.
abstract: The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Darren R
authors.initials: DR
authors.lastname: Brooks
authors.forename: Paul M
authors.initials: PM
authors.lastname: Selzer
authors.forename: Hector L
authors.initials: HL
authors.lastname: Pandolfo
authors.forename: Wagner A S
authors.initials: WA
authors.lastname: Judice
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Morten
authors.initials: M
authors.lastname: Meldal
authors.forename: Sanya J
authors.initials: SJ
authors.lastname: Sanderson
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases