Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis

Maria F. Lanfranco, Raúl Loayza-Muro, Daniel Clark, Regina Núñez, Amparo Iris Zavaleta Pesantes, Maribel Jimenez, Morten Meldal, Graham H. Coombs, Jeremy C. Mottram, Mario Izidoro, Maria A. Juliano, Luiz Juliano, Jorge Arévalo

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 (rCPB2.8ΔCTE) counterpart (76.36%) and has identical amino acid residues at the S1, catalytic triad and S1 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.

Original languageEnglish
Pages (from-to)91-100
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume161
Issue number2
DOIs
StatePublished - Oct 2008
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the INCO-DC Program (EU Contract No. ERBIC18CT 970225), United Nations University/Biotechnology Programme for the Latin America and the Caribbean (UNU/BIOLAC), the Danish National Research Foundation, the Swedish Agency for Research Cooperation with Developing Countries (SAREC), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Keywords

  • Cysteine protease B
  • Enzyme kinetic parameters
  • Fluorogenic peptides
  • Leishmania braziliensis
  • Peptide inhibitors
  • Recombinant protease

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