Chemical and biological characterization of the cytolytic protein enterolobin from seeds of enterolobium contortisiliqumm

Abstract

Enterolobin, a 55 KDa haemolytic protein from Enterolobium contortisiliquum seeds, was purified and further characterized. Its partial amino acid sequence was determined by using both manual and automatic methods of sequence determination. A number of 271 residues could be placed by means of overlaps. A computational search of these overlapped peptides against the PM database revealed that some residues of one peptide had short sequence matches to the bacterial pore-forming aerolysins from Aeromonas hydrophyla and Aeromonas sobria and to colicin lb from Escherichia coli. A subsequent search for similar sequence patterns stored in the PROSITE database showed that the predicted cytolytic site of enterolobin and the aerolysins have similarities to the pattern of the tonB-dependent receptors of bacteria. This pattern occurs not only in these membrane receptor proteins, but also in colicins and other pore- formers and transport proteins. Hydropathy profile analyses made for aerolysins and the predicted cytolytic sites demonstrated their overall hydrophilic character with some short stretches of hydrophobic regions. Studies on the kinetics of haemolysis of normal red cells by enterolobin in the presence of possible effectors such as Ca(^2+), EDTA, galactose, choline, phosphatidylcholine, cholesterol, ricin and with trypsinised erythrocytes were performed. The analyses indicated that the membrane receptor for enterolobin was probably a protein, perhaps the band 3 anion-exchanger protein. Optical and electron microscopic observations of the effects of enterolobin and gold-enterolobin on the membranes of red cell and cancer cells were carried out, and revealed severely damaged membranes after contact with the cytolysin. Gold-enterolobin had a random distribution around holes on the red cell membrane. Bioassays showed that enterolobin was toxic to larvae of the insect Callosobruchus maculatus but not to those of Spodoptera litorallis. Assays of in vitro proteolysis using larval gut enzymes showed that only S. litorallis proteases could digest enterolobin. The mechanism of toxicity of enterolobin did not involve any damage to the microvillar membranes of the epithelial gut cells of C. maculatus as shown by electron microscopy.