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Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

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Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Avenida Brasil, 4365, Manguinhos, Rio de Janeiro 21040-900, Brazil
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Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunoparasitologia, Avenida Brasil, 4365, Manguinhos, Rio de Janeiro 21040-900, Brazil
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Fundação Oswaldo Cruz, Centro de Desenvolvimento Tecnológico em Saúde, Avenida Brasil, 4365, Manguinhos, Rio de Janeiro 21040-900, Brazil
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(6), 1315; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20061315
Received: 8 February 2019 / Revised: 8 March 2019 / Accepted: 11 March 2019 / Published: 15 March 2019
(This article belongs to the Section Biochemistry)
Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 252 ± 20 µmol.min−1 mg of protein−1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min−1 mg of protein−1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes. View Full-Text
Keywords: Leishmania (Viannia) braziliensis; protease; serine proteinases; subtilisin-like; HiTrap benzamidine column; Z-FR-AMC; Suc-AFK-AMC Leishmania (Viannia) braziliensis; protease; serine proteinases; subtilisin-like; HiTrap benzamidine column; Z-FR-AMC; Suc-AFK-AMC
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MDPI and ACS Style

Santos-de-Souza, R.; Monteiro de Castro Côrtes, L.; dos Santos Charret, K.; Cysne-Finkelstein, L.; Alves, C.R.; Souza-Silva, F. Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression. Int. J. Mol. Sci. 2019, 20, 1315. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20061315

AMA Style

Santos-de-Souza R, Monteiro de Castro Côrtes L, dos Santos Charret K, Cysne-Finkelstein L, Alves CR, Souza-Silva F. Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression. International Journal of Molecular Sciences. 2019; 20(6):1315. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20061315

Chicago/Turabian Style

Santos-de-Souza, Raquel, Luzia Monteiro de Castro Côrtes, Karen dos Santos Charret, Léa Cysne-Finkelstein, Carlos R. Alves, and Franklin Souza-Silva. 2019. "Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression" International Journal of Molecular Sciences 20, no. 6: 1315. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20061315

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