Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates

• Published in: Molecular and biochemical parasitology Vol. 184; no. 2; pp. 82 - 89
• Main Authors: Caroselli, Elide E., Assis, Diego M., Barbiéri, Clara L., Júdice, Wagner A.S., Juliano, Maria A., Gazarini, Marcos L., Juliano, Luiz
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-08-2012
• Subjects: amastigotes; Amino Acid Sequence; Animals; Citrates - chemistry; Cricetinae; cysteine; Cysteine protease; Cysteine Proteinase Inhibitors - pharmacology; environmental factors; Fluorescent peptides; Hydrogen-Ion Concentration; hydrolysis; Leishmania; Leishmania - cytology; Leishmania - enzymology; Leucine - analogs & derivatives; Leucine - pharmacology; Mesocricetus; microscopy; Oligopeptidase B; Oligopeptides - chemistry; parasites; parasitology; Pepstatins - pharmacology; Peptidase; peptidases; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; pretreatment; promastigotes; Proteolysis; Protozoan Proteins - chemistry; Protozoan Proteins - metabolism; Salinity; salts; Serine Proteinase Inhibitors - pharmacology; Sulfates - chemistry; Temperature; Tosyllysine Chloromethyl Ketone - pharmacology; E64; Peptidase; DTT; TLCK; Oligopeptidase B; MCA; EDDnp; CPB 2.8 ΔCTE; pFF-MCA; CPB; Cysteine protease; PMSF; Abz; FRET; Fluorescent peptides
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2012.04.012

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► Leishmania amastigotes incorporate only peptides containing positively charged amino acids. ► Leishmania express cysteine peptidases adapted to different environments. ► Oligopeptidase B was detected in vivo inside the amastigotes. ► FRET peptide Abz-AGRRRAQ-EDDnp characteristically identify oligopeptidases B.
In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5–9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.