Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties

• Published in: Physiologia plantarum 2011-07, Vol.142 (3), p.233-246
• Main Authors: Diz, Mariângela S., Carvalho, Andre O., Ribeiro, Suzanna F. F., Da Cunha, Maura, Beltramini, Leila, Rodrigues, Rosana, Nascimento, Viviane V., Machado, Olga L. T., Gomes, Valdirene M.
• Format: Article
• Language: English
• Subjects: Acetonitrile; alpha -Amylase; alpha-Amylases - antagonists & inhibitors; alpha-Amylases - metabolism; Antifungal activity; Antifungal Agents - pharmacology; Biological and medical sciences; C.D; Candida tropicalis; Capsicum - drug effects; Capsicum - metabolism; Capsicum - ultrastructure; Capsicum annuum; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Carrier Proteins - pharmacology; Carrier Proteins - ultrastructure; Cell Membrane Permeability - drug effects; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Circular Dichroism; Colletotrichum; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Exudates; Fundamental and applied biological sciences. Psychology; Fungi - drug effects; Fungi - growth & development; Humans; Isoelectric points; Lipid transfer proteins; Long-term potentiation; Microbial Sensitivity Tests; Plant physiology and development; Plant Proteins - metabolism; Plant Proteins - ultrastructure; Plasma membranes; propanol; protein purification; Protein Transport - drug effects; pseudohyphae; Seeds; Seeds - drug effects; Seeds - metabolism; Seeds - ultrastructure; Solvents; Species Specificity; Staining and Labeling; Vesicles; Western blotting
• ISSN: 0031-9317; 1399-3054
• DOI: 10.1111/j.1399-3054.2011.01464.x

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9‐kDa LTP from Capsicum annuum seeds that we call Ca‐LTP1. Ca‐LTP1 was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein's biological activity. Bidimensional electrophoresis of the 9‐kDa band, which corresponds to the purified Ca‐LTP1, showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α‐helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca‐LTP1 from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca‐LTP1 indicated that it was mainly localized within dense vesicles. In addition, isolated Ca‐LTP1 exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca‐LTP1 also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca‐LTP1 is able to inhibit mammalian α‐amylase activity in vitro.