Guilherme Santana Vieira¹, Bruno Matheus Ferreira de Paula¹, Consuelo Medeiros Rodrigues de Lima¹, Fábio Morato de Oliveira², Marcelo Valle de Sousa¹, Wagner Fontes¹, Carlos Andre Ornelas Ricart¹

Enterolobin is a 53 kDa cytolytic protein isolated from the seeds of Enterolobium contortisiliquum. It is known for its haemolytic ability, can lyse different cell types, and exhibits pro-inflammatory activity. Its primary structure, previously determined by Edman degradation (Accession: P81007), comprises 485 amino acids and belongs to the aerolysin-like family of pore-forming proteins, whose activity depends on oligomerization. Given previous evidence of enterolobin oligomerization in solution and SDS-PAGE, we hypothesized that this structural feature is essential for its cytolytic function. In this study, we aim to validate enterolobin sequence using mass spectrometry, characterize its oligomeric properties, and explore novel biological activities. The protein was purified using saline extraction (0.9%), ammonium sulfate precipitation (F1), batch purification on DEAE-cellulose (F2), and Mono Q anion-exchange chromatography via FPLC (F3). Purity and activity were evaluated by SDS-PAGE and haemolysis assays. Fraction F2 was tested for cytotoxic on bacteria, murine macrophages, human neutrophils, and various cancer cell lines, including leukemia, glioblastoma, breast, and prostate cancer. While enterolobin did not reduce viability of bacterial or macrophage, it induced reactive oxygen species (ROS) production in neutrophils and reduce the viability of all tested cancer cells, assays suggest that this reduction is due to apoptosis. BN-PAGE and SDS-PAGE analyses of F3 confirmed concentration and pH dependent oligomerization. SDS-PAGE revealed not only the expected 53 kDa monomer but also higher molecular weight bands. These were excised and subjected to in-gel tryptic digestion followed by LC-MS/MS analysis. All the bands produced the same peptides, indicating that higher bands are enterolobin oligomers. Surprisingly, sequence coverage using PEAKS software was limited to 10% when matched against the enterolobin Edman enterolobin sequence. In contrast, database searches revealed 74% identity with an agglutinin domain-containing protein from Acacia crassicarpa (Accession: A0AAE1JWN9), both of which share a C-terminal aerolysin-like domain. However, global alignment between enterolobin and this protein was only 27%. Further enzymatic digestions are currently underway to improve sequence coverage and resolve the complete primary structure of enterolobin.

Agradecimentos: This work was supported by Federal District Research Support Foundation (FAPDF), University of Brasília (UnB) and CNPQ