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

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

doi:10.1016/j.molbiopara.2008.06.005

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

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

Research output: Contribution to journal › Article › peer-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 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.

Original language	English
Pages (from-to)	91-100
Number of pages	10
Journal	Molecular and Biochemical Parasitology
Volume	161
Issue number	2
DOIs	https://doi.org/10.1016/j.molbiopara.2008.06.005
State	Published - Oct 2008

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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10.1016/j.molbiopara.2008.06.005

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Lanfranco, M. F., Loayza-Muro, R., Clark, D., Núñez, R., Zavaleta, A. I., Jimenez, M., Meldal, M., Coombs, G. H., Mottram, J. C., Izidoro, M., Juliano, M. A., Juliano, L., & Arévalo, J. (2008). Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis. Molecular and Biochemical Parasitology, 161(2), 91-100. https://doi.org/10.1016/j.molbiopara.2008.06.005

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

keywords = "Cysteine protease B, Enzyme kinetic parameters, Fluorogenic peptides, Leishmania braziliensis, Peptide inhibitors, Recombinant protease",

author = "Lanfranco, {Maria F.} and Ra{\'u}l Loayza-Muro and Daniel Clark and Regina N{\'u}{\~n}ez and Zavaleta, {Amparo I.} and Maribel Jimenez and Morten Meldal and Coombs, {Graham H.} and Mottram, {Jeremy C.} and Mario Izidoro and Juliano, {Maria A.} and Luiz Juliano and Jorge Ar{\'e}valo",

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), Funda{\c c}{\~a}o de Amparo {\`a} Pesquisa do Estado de S{\~a}o Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Cient{\'i}fico e Tecnol{\'o}gico (CNPq).",

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Lanfranco, MF, Loayza-Muro, R, Clark, D, Núñez, R, Zavaleta, AI, Jimenez, M, Meldal, M, Coombs, GH, Mottram, JC, Izidoro, M, Juliano, MA, Juliano, L & Arévalo, J 2008, 'Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis', Molecular and Biochemical Parasitology, vol. 161, no. 2, pp. 91-100. https://doi.org/10.1016/j.molbiopara.2008.06.005

TY - JOUR

T1 - Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis

AU - Lanfranco, Maria F.

AU - Loayza-Muro, Raúl

AU - Clark, Daniel

AU - Núñez, Regina

AU - Zavaleta, Amparo I.

AU - Jimenez, Maribel

AU - Meldal, Morten

AU - Coombs, Graham H.

AU - Mottram, Jeremy C.

AU - Izidoro, Mario

AU - Juliano, Maria A.

AU - Juliano, Luiz

AU - Arévalo, Jorge

N1 - 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).

PY - 2008/10

Y1 - 2008/10

N2 - 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 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.

AB - 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 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.

KW - Cysteine protease B

KW - Enzyme kinetic parameters

KW - Fluorogenic peptides

KW - Leishmania braziliensis

KW - Peptide inhibitors

KW - Recombinant protease

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U2 - 10.1016/j.molbiopara.2008.06.005

DO - 10.1016/j.molbiopara.2008.06.005

M3 - Artículo

C2 - 18639590

AN - SCOPUS:49249120868

SN - 0166-6851

VL - 161

SP - 91

EP - 100

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

IS - 2

ER -

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

Abstract

Bibliographical note

Keywords

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