Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis.
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.
Lanfranco
Maria F
MF
Laboratory of Molecular Biology of Trypanosomatids, Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, Lima 100, Peru.
Loayza-Muro
Raúl
R
Clark
Daniel
D
Núñez
Regina
R
Zavaleta
Amparo I
AI
Jimenez
Maribel
M
Meldal
Morten
M
Coombs
Graham H
GH
Mottram
Jeremy C
JC
Izidoro
Mario
M
Juliano
Maria A
MA
Juliano
Luiz
L
Arévalo
Jorge
J
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
D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases