Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with α-amylase inhibitor properties

• Published in: Biochimica et biophysica acta Vol. 1810; no. 4; pp. 375 - 383
• Main Authors: Zottich, Umberto, Da Cunha, Maura, Carvalho, André O, Dias, Germana B, Silva, Nádia C.M, Santos, Izabela S, do Nacimento, Viviane V, Miguel, Emílio C, Machado, Olga L.T, Gomes, Valdirene M
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2011
• Subjects: acidification; alpha-amylase; alpha-Amylases - antagonists & inhibitors; Amino Acid Sequence; amino acid sequences; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; antifungal properties; antimicrobial peptides; Candida - drug effects; Candida albicans; Candida tropicalis; Coffea - chemistry; Coffea canephora; dose response; electrophoresis; fluorescence microscopy; Glucose - metabolism; Humans; in vitro studies; mammals; Molecular Sequence Data; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; plasma membrane; proteins; reversed-phase high performance liquid chromatography; seeds; Seeds - chemistry; yeasts
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2010.12.002

BACKGROUND: A growing number of cysteine-rich antimicrobial peptides (AMPs) have been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides, which include lipid transfer proteins (LTPs), play an important role in the protection of plants against microbial infection. METHODS: Peptides from Coffea canephora seeds were extracted in Tris–HCl buffer (pH 8.0), and chromatographic purification of LTP was performed by DEAE and reverse-phase HPLC. The purified peptide was submitted to amino acid sequence, antimicrobial activity and mammalian α-amylase inhibitory analyses. RESULTS: The purified peptide of 9kDa had homology to LTPs isolated from different plants. Bidimensional electrophoresis of the 9kDa band showed the presence of two isoforms with pIs of 8.0 and 8.5. Cc-LTP₁ exhibited strong antifungal activity, against Candida albicans, and also promoted morphological changes including the formation of pseudohyphae on Candida tropicalis, as revealed by electron micrograph. Our results show that Cc-LTP₁ interfered in a dose-dependent manner with glucose-stimulated, H⁺-ATPase-dependent acidification of yeast medium and that the peptide permeabilized yeast plasma membranes to the dye SYTOX green, as verified by fluorescence microscopy. Interestingly, we also showed for the first time that the well characterized LTP₁ family, represented here by Cc-LTP₁, was also able to inhibit mammalian α-amylase activity in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE: In this work we purified, characterized and evaluated the in vitro effect on yeast of a new peptide from coffee, named Cc-LPT1, which we also showed, for the first time, the ability to inhibit mammalian α-amylase activity.