1. Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression
Raquel Santos-de-Souza, Luzia Monteiro de Castro Côrtes, Karen Dos Santos Charret, Léa Cysne-Finkelstein, Carlos Roberto Alves, Franklin Souza-Silva Int J Mol Sci. 2019 Mar 15;20(6):1315. doi: 10.3390/ijms20061315.
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.
2. Serine protease activities in Leishmania (Leishmania) chagasi promastigotes
Raquel Elisa da Silva-López, Tatiana Resende dos Santos, José Andrés Morgado-Díaz, Marcelo Neves Tanaka, Salvatore Giovanni de Simone Parasitol Res. 2010 Oct;107(5):1151-62. doi: 10.1007/s00436-010-1983-y. Epub 2010 Jul 29.
The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.
3. A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis
Raquel Elisa da Silva Lopez, Salvatore Giovanni De Simone Z Naturforsch C J Biosci. 2004 Jul-Aug;59(7-8):590-8. doi: 10.1515/znc-2004-7-825.
Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.
