Caracterização bioquímica e atividades biológicas de quitinases laticíferas de Calotropis procera

Abstract

Calotropis procera is a latificer plant in the Apocynaceae family. Defensive proteins with activity against phytophagous insects and phytopathogenic fungi in latex have been described. Latex’s proteic fraction also performs pharmacological activities, such as selective cytotoxic effect in carcinogenic cells. From the proteic fraction effective against neoplastic cells, this work had the goal to identify and further characterize cytotoxic protein(s) biochemically, biologically, and evaluate their biotechnological prospects. Soluble proteins in latex (LP) have been fractioned in ion-exchange matrix, and the fraction capable of cytotoxicity was further fractioned in another ion-exchange matrix (Mono-Q) coupled to a medium-pressure system. P4 was identified as cytotoxic fraction, showing an IC50 of 2.2, 1.2 and 2.9 mg/mL for the cell lines HCT-116, Ovcar-8 and SF-295, respectively. Proteomic analysis of the cytotoxic fraction by two-dimensional electrophoresis allowed 15 spots to be identified, comprising acid proteins (pI among 4 and 6) with 30 kDa apparent molecular weight. All spots were identified as chitinases when evaluated by mass spectrometry, and showed intact mass of 27.4 kDa. The sample reacted positively to the Schiff reagent, suggesting glycosilations. The carbohydrate content was estimated at 12.8%. The amino-terminal sequence obtained (1QPVMNLEYPRYLNDINDYRDDNNYD28) revealed 50% similarity with Solanum lycopersicum, Oryza sativa and Nicotiana tomentosiformes chitinases, among others. The enzymes showed strong chitinolytic activity, with optimal pH varying between 5-6 and optimal temperature of 25 °C. The chitinolytic activity was diminished when treated with increasing concentrations of DTT (3, 10 and 30 mM), which suggests the presence of disulfide bonds stabilizing the enzyme. Circular dichroism analyses indicate a larger presence of alpha helices in the structure and that the isoforms has low resistance to pH and temperature variation. High-resolution images generated through atomic force microscopy suggested sample homogeneity and a hexameric configuration. The chitinases did not inhibit mycelial growth of phytopathogenic fungi Fusarium oxysporum and Colletotrichum gloeosporioides, however reduced the germination C. gloeosporioides spores. In consonancy the chitinases did not induce spore oxidative stress, but caused a slight change in membrane permeability of the evaluated spores. Laticifer chitinases (0.1% w/w) showed a strong deleterious effect on the phytophagous insect Callosobruchus maculatus. Effects produced a 57% survival reduction, larval weight reduction (7.8 mg ± 0.2 / 4.0 ± 0.8), adult insets emergence reduction (50%), in addition to prolonging the mean maturation time from 28 to 33 days. The chitinases (2 mg/Kg, i.v.) showed a strong anti-inflammatory activity, reducing 95% of neutrophil infiltration into the peritoneal cavity in mouse in an inflammation induced by carrageenan assay. L-NAME and Aminoguanidine, two inhibitors of nitric oxide synthase, reversed this effect, possibly indicating involvement of nitric oxide in the effects observed. This action was associated to a reduction of pro-inflammatory cytokines TNF-α and IL-1 levels within the peritoneal cavity and increased serum levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1. The conclusion is reached that many isoforms of chitinases coexist in C. procera latex. These proteins act possibly in defense against insects, though low action against fungi is shown. Laticifer chitinases were identified as latex proteins cytotoxic on neoplastic cells and they have even been able to modulate pro-inflammatory cytokines levels and nitric oxide in an acute inflammation model. C. procera laticifer chitinases represent interesting molecules for biotechnology prospection in plant defense and pharmacology.