ABSTRACT
Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of â¼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.
Subject(s)
Immunologic Factors/pharmacology , Macrophages, Peritoneal/drug effects , Plant Lectins/pharmacology , Amino Acid Sequence , Animals , Brassica , Cattle , Cells, Cultured , Chromatography, Affinity , Chromatography, Gel , Drug Evaluation, Preclinical , Goats , Hemagglutination , Horses , Humans , Hydrogen-Ion Concentration , Immunologic Factors/chemistry , Immunologic Factors/isolation & purification , Macrophage Activation , Macrophages, Peritoneal/metabolism , Male , Mice, Inbred BALB C , Phagocytosis/drug effects , Plant Lectins/chemistry , Plant Lectins/isolation & purificationABSTRACT
The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11-46 µM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.
