Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis.
Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.
Rebello
Karina M
KM
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), FIOCRUZ, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brazil.
Côrtes
Luzia M C
LM
Pereira
Bernardo A S
BA
Pascarelli
Bernardo M O
BM
Côrte-Real
Suzana
S
Finkelstein
Léa C
LC
Pinho
Rosa T
RT
d'Avila-Levy
Claudia M
CM
Alves
Carlos R
CR
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D057056:Cysteine Proteases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007892:Leishmania braziliensis / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D008565:Membrane Proteins; D015800:Protozoan Proteins; D057056:Cysteine Proteases; D007930:Leucine; C024974:E 64