Antimicrobial activity of synthetic magainin peptides and several analogues.

Abstract

We have previously reported the isolation of two broad-spectrum antimicrobial peptides ("magainins") from the skin of the African clawed frog Xenopus laevis. These natural peptides are active against many species ofbacteria and fungi and also induce osmotic lysis of protozoa. In this report we demonstrate that synthetic magainin peptides appear to be indistinguishable from the natural products with respect to chromatographic properties and biological activity. These studies demonstrate conclusively that the magainin peptides are potent antimicrobial substances.We have previously reported the isolation of two broadspectrum antibiotic peptides called "magainins" from the skin of the African clawed frog Xenopus laevis (1 In this report we demonstrate that the exceptionally broad range of antimicrobial activity exhibited by the magainin peptides recovered from Xenopus skin can be reproduced with synthetic analogues of the corresponding peptides. We show that the natural and synthetic peptides appear to be indistinguishable with respect to both biological and chromatographic properties. In addition, through the synthesis of several truncated analogues, we provide some insight into the structural requirements for the antimicrobial activity of the magainin peptides.MATERIALS AND METHODS Purification of Magainin Peptides. Magainin peptides were purified from the skin of the ventral surface of a female X. laevis as previously described (1).Assay of Magainin Antibacterial Activity. The standard assay has been described (1).Amino Acid Sequencing. Peptides were sequenced on an Applied Biosystems gas-phase protein sequencer (model 470A; Applied Biosystems, Foster City, CA) by the Edman degradation procedure. A standard version 3.0 program was followed, and all reagents used in the instrument were supplied by the manufacturer.Carboxyl-Terminal Analysis of Peptides. Between 2 and 4 nmol of peptide was incubated with 1 ,g of carboxypeptidase Y (122 units/mg; Sigma) in 55 ,ul of 0.1 M pyridine/acetate (pH 5.5) for 15 min at 21°C. Reactions were stopped by the addition of 50 .ul of 1 M acetic acid, and the solutions were then lyophilized to dryness. Amino acids were subsequently analyzed on a Beckman model 121 MB analyzer.Solid-Phase Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer using the manufacturer's reagents. Deprotection and cleavage from the resin by anhydrous HF followed standard procedures (3, 4). Methionine sulfoxides were reduced by incubation of the peptides in the presence of dithiothreitol (5). Peptides were purified to homogeneity by HPLC as described in Fig. 1. RESULTS As we have reported previously, several chromatographically distinct peptides with antimicrobial activity can be recovered from Xenopus laevis skin (1). The purification that resulted in successful recovery of antimicrobial activity involved homogenization of the skin of an anesthetized female Xenopus in acetate buffer at pH 4.0, step elution chromatography on carboxymethylcellulose, followed by s...

Magainin 2 amide and analogues Antimicrobial activity, membrane depolarization and susceptibility to proteolysis

“…The carboxy-terminus of magainin 2 is not amidated [2], whereas that of Mag A and of all peptides tested in the present paper, is. We investigated whether Mag A was also much more active antimicrobially than magainin 2 amide.…”

“…The magainin [1,2] peptides (also designated PGS [3]), isolated from the skin of Xenopus laevis, exhibit broad-spectrum antimicrobial activity. More potent analogues of the 23-residue magainin 2 peptide were obtained by substitutions of alanine for glycine or serine, and an amidation at the Cterminus, which resulted in enhanced a-helical structure and antimicrobial activity [4].…”

“…More potent analogues of the 23-residue magainin 2 peptide were obtained by substitutions of alanine for glycine or serine, and an amidation at the Cterminus, which resulted in enhanced a-helical structure and antimicrobial activity [4]. Although natural magainin 2 has a free carboxyl-terminus [1][2][3], the amidated form has been used for many studies [5][6][7][8][9].…”

Antimicrobial peptides from amphibians

“…Caerin 1.1 identified from the magnificent tree frog Litoria splendida and green tree frog Litoria caerulea, has an IC 50 value of -10 -6 M against all the major human cancer types (44). Citropin 1.1, gaegurins, magainin 2 and analog peptides of magainins are selectively cytotoxic to human cancer cells (17,79,89,90).…”

“…Magainins were identified from skin of Xenopus laevis in 1987 (16,17). Since then, a large number of AMPs with diversified structures and activities have been isolated from amphibians.…”