RESUMO
Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.
Assuntos
Peptídeo Hidrolases/metabolismo , Trypanosomatina/enzimologia , Animais , Western Blotting , Caseínas/metabolismo , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Gelatina/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liase , Hemoglobinas/metabolismo , Metaloendopeptidases/metabolismo , Fosfatidilinositol Diacilglicerol-Liase/metabolismo , Proteínas de Protozoários , Soroalbumina Bovina/metabolismo , Simbiose/fisiologia , Trypanosomatina/fisiologiaRESUMO
In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.
Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Glucocorticoides/biossíntese , Mineralocorticoides/biossíntese , Oligossacarídeos/farmacologia , Fosfolipases Tipo C/metabolismo , Córtex Suprarrenal/metabolismo , Aldosterona/biossíntese , Animais , Bovinos , Corticosterona/biossíntese , Ativação Enzimática/efeitos dos fármacos , Glicolipídeos/química , Glicolipídeos/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liase , Hidrocortisona/biossíntese , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Masculino , Fosfatidilinositol Diacilglicerol-Liase , Polissacarídeos , Ratos , Ratos Sprague-Dawley , Trypanosoma cruzi/químicaRESUMO
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous null mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.