Leishmania ( Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

Abstract

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania ( Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium–polyacrylamide gel electrophoresis (SDS–PAGE). The surface iodinated protein profiles showed two major polypeptides of 65–60 and 50–47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS–PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65–60, 52, and 50–47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65–60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. ( L.) amazonensis. Index Descriptors and Abbreviations: cell-surface protein, Leishmania ( L.) amazonensis; proteinases; promastigotes, trypanosomatid, virulence; BSA, bovine serum albumin; CRD, cross-reacting determinant; DTT, dithiothreitol; E-64, trans-epoxysuccinyl l-leucylamido-(4-guanidino) butane; EDTA, ethylenediaminetetraacetic acid; GPI, glycosylphosphatidylinositol; kDa, kilodalton; LPG, lipophosphoglycan; PLC, phospholipase C; PMSF, phenylmethylsulphonyl fluoride; SDS–PAGE, dodecyl sulfate sodium–polyacrylamide gel electrophoresis; VSG, variant surface glycoprotein