1. Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds
Seiya Yokoyama, Kouji Kato, Atsuko Koba, Yuji Minami, Keiichi Watanabe, Fumio Yagi Peptides. 2008 Dec;29(12):2110-7. doi: 10.1016/j.peptides.2008.08.007. Epub 2008 Aug 20.
Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.
2. The chitin-binding capability of Cy-AMP1 from cycad is essential to antifungal activity
Seiya Yokoyama, Yuto Iida, Yousuke Kawasaki, Yuji Minami, Keiichi Watanabe, Fumio Yagi J Pept Sci. 2009 Jul;15(7):492-7. doi: 10.1002/psc.1147.
Antimicrobial peptides are important components of the host innate immune responses by exerting broad-spectrum microbicidal activity against pathogenic microbes. Cy-AMP1 found in the cycad (Cycas revoluta) seeds has chitin-binding ability, and the chitin-binding domain was conserved in knottin-type and hevein-type antimicrobial peptides. The recombinant Cy-AMP1 was expressed in Escherichia coli and purified to study the role of chitin-binding domain. The mutants of Cy-AMP1 lost chitin-binding ability completely, and its antifungal activity was markedly decreased in comparison with native Cy-AMP1. However, the antimicrobial activities of the mutant peptides are nearly identical to that of native one. It was suggested that the chitin-binding domain plays an essential role in antifungal, but not antimicrobial, activity of Cy-AMP1.
