Abstract
An antibacterial peptide with 16 amino acid residues was found in plasma of the freshwater crayfish, Pacifastacus leniusculus. This peptide, designated astacidin 1, was purified by cation-exchange column chromatography and reverse-phase high performance liquid chromatography. Astacidin 1 has a broad range of antibacterial activity, and it inhibits growth of both Gram-positive and Gram-negative bacteria. The primary sequence of astacidin 1 was FKVQNQHGQVVKIFHH-COOH. The molecular mass was 1945.2 Da, and no carbohydrate-linked amino acid residues could be found by mass spectrometry. A synthetic astacidin 1 resulted in similar activity as the authentic astacidin 1 against Gram-positive bacteria, whereas it had less or no activity against Gram-negative bacteria. Three amino-terminal-truncated synthetic peptides were made; they all showed low activity, suggesting that the amino-terminal part of astacidin 1 contributes to the antibacterial activity. The structure of astacidin 1 based on the CD results showed that it has a beta-sheet structure in citric acid buffer at pH 4, 6, and 8. Cloning of astacidin 1 shows that it is the carboxyl-terminal part of crayfish hemocyanin and that astacidin 1 is produced by a proteolytic cleavage from hemocyanin under acidic conditions. The processing and release of astacidin 1 from hemocyanin is enhanced when crayfish are injected with lipopolysaccharide or glucan.
Highlights
Antimicrobial peptides have become recognized as important components of the nonspecific host defense or innate immune system in a variety of organisms ranging from plants and insects to animals, including mollusca, crustaceans, amphibians, birds, fish, mammals, and humans [1,2,3]
The eluted fractions were assayed for their antibacterial activity against two bacterial strains, B. megaterium BM11 and E. coli D21
The antibacterial molecule was purified to homogeneity and is fully characterized at the level of its primary and secondary structure by a combination of reverse-phase chromatography, cation exchange chromatography, MALDI-TOF-MS, and CD spectrum
Summary
Animals—Freshwater crayfish, P. leniusculus, were purchased from Berga Kraftodling, Sodermanland, Sweden and were maintained in tanks with aerated water at 10 °C. For purification of antibacterial proteins from plasma, the frozen samples were thawed and trifluoroacetic acid was added to a final concentration of 0.1%. Astacidin 1 Has Antibacterial Activity was centrifuged at 16,000 ϫ g for 20 min, and the supernatant was diluted twice with 0.05% trifluoroacetic acid water and directly subjected to C-18 reverse-phase chromatography ( 2.7 ϫ 9 cm, Waters) equilibrated with 0.05% trifluoroacetic acid water. The sample was further purified to homogeneity by reverse-phase HPLC (Amersham Biosciences smart chromatography system) on C-18 column using acetonitrile/water/ 0.05% trifluoroacetic acid gradients of 0 – 60% acetonitrile in 60 min at a flow rate of 100 l/min. Bacteria grown in LB medium (peptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 1 g/distilled water 1 liter) was collected in the exponential phase of growth and resuspended with phosphate-buffered saline, pH 6.0, at a density of 1 ϫ 108 cells/ml. The membranes were prehybridized at 65 °C for 1 h in 5ϫ SSC (750 mM NaCl, 75 mM Na-citrate, pH 7.0), 5ϫ Denhardt’s solution (100ϫ Denhardt’s solution is 2% (w/v) bovine serum albumin,
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