Proline Conformation-Dependent Antimicrobial Activity of a Proline-Rich Histone H1 N-Terminal Peptide Fragment Isolated from the Skin Mucus of Atlantic Salmon

Abstract

A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 was identified as the dominant antimicrobial peptide in skin mucus from Atlantic salmon (Salmo salar). The peptide (termed salmon antimicrobial peptide [SAMP H1]) was purified to homogeneity by a combination of reversed-phase and cation-exchange chromatographies. By Edman degradation of the deacetylated peptide and by sequencing of the PCR-amplified DNA that encodes the peptide, the complete amino acid sequence was determined to be AEVAPAPAAAAPAKAPKKKAAAKPKKAGPS. The theoretical molecular weight of N-terminally acetylated SAMP H1 was calculated to be 2,836, which is the same as that determined by matrix-assisted laser desorption ionization mass spectrometry. The peptide was active against both gram-negative and -positive bacteria. The N-terminal acetyl group was not necessary for activity since deacetylation did not reduce the activity. A synthetic peptide whose sequence was identical to that of the isolated fragment was initially inactive but could be activated by binding it to a cation-exchange column. Treatment of the synthetic peptide when it was bound to the exchange column with peptidylproline cis-trans-isomerase increased the amount of active peptide, indicating that isomerization of the proline peptide bond(s) was necessary for activation of the synthetic peptide. Comparison of the active and inactive forms by circular dichroism and chromatographic analyses suggests that the active form, both the natural and the synthetic forms, is more structured, condensed, and rigid than the inactive form, which has a more nonstructured conformation. This work shows for the first time the importance of proline isomers in the activity of an antimicrobial peptide.

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, Anguilla japonica

“…In addition, the mucus which contains a variety of antimicrobial components such as AMPs, lysozyme, proteases, and lectins could therefore play a protecting role against the environment [13,14]. The antimicrobial peptides isolated from skin mucus against infectious pathogens have been demonstrated previously from Misgurnus anguillicaudatus [10,32], Pardachirus marmoratus and Pardachirus pavoninus [24,42], Pleuronectes americanus [8], Salmo salar [29], Oncorhynchus mykiss [16,17], Morone chrysops and Morone saxatilis [28], Hippoglossus hippoglossus L. [5], Parasilurus asotus [33] and Myxine glutinosa [41], which supports the important role of epidermal mucus in protecting fish from infectious pathogens. So the mucus was chosen as the source of purification for antibacterial peptide.…”

“…Ebran et al extracted the 43-67 kDa proteins and a 45 kDa protein from skin mucus of A. anguilla were hydrophobic molecules [11,12]. However, the Pardaxins P-2 [24], Parasin I [42], Pleurocidin [8], SAMP H1 [29], moronecidin [28] and hipposin [5] were amphipathic molecules. In addition to three Cys residues, the 20 N-terminal sequences of the AJN-10 also possess two Gly residues, two Asn and two Thr, which are all the hydrophilic residues.…”

“…However, there are many lower MW antibacterial peptides (1.3-5.5 kDa) than the AJN-10, such as misgurin isolated from Misgurnus anguillicaudatus [10,32], Pardaxin I & II and Pardaxins P-2 from Pardachirus marmoratus and Pardachirus pavoninus [24,42], pleurocidin from Pleuronectes americanus [8], SAMP H1 from Salmo salar [29], moronecidin from Morone chrysops and Morone saxatilis [28], hipposin from Hippoglossus hippoglossus L. [5], Parasin I from Parasilurus asotus [33] and myxinidin from Myxine glutinosa [41], except for few showing little higher MW (6.7-9.8 kDa), such as MAAP from Misgurnus anguillicaudatus, Oncorhyncin II & III from Oncorhynchus mykiss [10,16,17]. The molecular mass-analysis and BLAST result of the 20 N-terminal amino acid sequences further confirmed that the AJN-10 was a novel antibacterial peptide.…”

“…It is well established that skin secretions of fish contain a number of innate humoral defence factors, including complement factors [47], lectins [3], proteases [20] and lysozymes [21,38]. It is revealed that the surface mucosa of some fish also contains a range of AMPs, such as those with the molecular weight (MW)\10 kDa from Misgurnus anguillicaudatus [10,32], Pardachirus marmoratus and Pardachirus pavoninus [24,42], Pleuronectes americanus [8], Salmo salar [29], Oncorhynchus mykiss [16,17], Morone chrysops and Morone saxatilis [28], Hippoglossus hippoglossus L. [5], Parasilurus asotus [33] and Myxine glutinosa [41]. The majority of these peptides were amphipathic cation peptides, while few were anionic.…”

L-Amino Acid Oxidase, a Novel Antibacterial Protein in the Rockfish <i>Sebastes schlegeli</i>

“…Among them, antimicrobial agents are most likely to play an important role in frontline defense of fish, because fish inhabit an aquatic environment which is rich in pathogenic microorganisms. bacterial agents and have been found in fish skin mucus; parasin I (N-terminus of histone H2A, a 19-residue peptide) from catfish Parasilurus asotus (Park et al 1998), histone H2B-like protein from channel catfish Ictalurus punctatus (Robinette et al 1998), histone H2A and oncorhycin II (histone H1 C-terminal fragment, a 69-residue peptide) from rainbow trout Oncorhynchus mykiss (Fernandes et al , 2004Noga et al 2011), hipposin (histone H2A fragment, a 51-residue peptide) from Atlantic halibut Hippoglossus hippoglossus (Birkemo et al 2003), histone H2B from Atlantic cod Gadus morhua (Bergsson et al 2005), and SAMP H1 (N-terminus of histone H1, a 30-residue peptide) from Atlantic salmon Salmo salar (Lüders et al 2005). Moreover, antibacterial peptides derived from ribosomal and chromosomal proteins have been reported in the skin secretions of rainbow trout and Atlantic cod Fernandes et al 2003;Bergsson et al 2005).…”