title: Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis.
abstract: The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high percentage identity with its Leishmania mexicana CPB2.8 (rCPB2.8DeltaCTE) counterpart (76.36%) and has identical amino acid residues at the S(1), catalytic triad and S'(1) subsites. Nevertheless, when the kinetics of substrate hydrolysis was measured using a combinatorial library of internally quenched fluorescent peptides based upon the lead sequence Abz-KLRSSKQ-EDDnp, significant differences were obtained. These results suggest that the differences in substrate utilization observed between the L. mexicana and L. braziliensis CPs must be related to amino acid modifications outside the core of the active site cleft. Moreover, a potent inhibitor with Pro at P1 and high affinity for L. braziliensis recombinant CPB showed less affinity to L. mexicana CPB 2.8, which preferred Phe, Leu, and Asn at the same position.
authors.affiliation: Laboratory of Molecular Biology of Trypanosomatids, Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 100, Peru.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Lanfranco
authors.forename: Raúl
authors.initials: R
authors.lastname: Loayza-Muro
authors.forename: Daniel
authors.initials: D
authors.lastname: Clark
authors.forename: Regina
authors.initials: R
authors.lastname: Núñez
authors.forename: Amparo I
authors.initials: AI
authors.lastname: Zavaleta
authors.forename: Maribel
authors.initials: M
authors.lastname: Jimenez
authors.forename: Morten
authors.initials: M
authors.lastname: Meldal
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Mario
authors.initials: M
authors.lastname: Izidoro
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Jorge
authors.initials: J
authors.lastname: Arévalo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007892:Leishmania braziliensis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases