Dermaseptin DRG2

In addition to the highly specific cell-mediated immune system, vertebrates possess an efficient host-defense mechanism against invading microorganisms which involves the synthesis of highly potent antimicrobial peptides with a large spectrum of activity. A 34-residue cationic and amphiphatic peptide, designated dermaseptin I, was recently isolated from the skin of the arboreal frog Phyllomedusa sauvagii and was shown to exhibit microbicidal activity against various pathogenic microorganisms including bacteria, yeast, protozoa and filamentous fungi. In this study, we report the isolation and characterization of four novel antimicrobial peptides from frog skin through the combined use of an anti-dermaseptin enzyme immunoassay and an antifungal bioassay. The 28-34-residue antimicrobial peptides are cationic, containing 3-5 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. Based on their primary structure, all four peptides can be fitted to a class L amphipathic alpha helix which places all lysine residues on the polar side of the helix. The four antimicrobial peptides have high sequence similarity with dermaseptin I (53-94% similarity) suggesting that their respective genes are all members of the same family. In addition, pairwise sequence alignment of dermaseptin I and adenoregulin, a 33-residue cationic peptide recently isolated from frog skin and shown to enhance the binding of agonists to the A1 adenosine receptor, reveals 62% similarity (39% amino acid positional identity). Both peptides share a similar but non-identical spectrum of antimicrobial activity, being active against bacteria, yeast and filamentous molds. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic cells. These findings indicate that adenoregulin should be included in the dermaseptin family of peptides. In addition, tryptic digestion of purified pro-dermaseptin I liberated a 15-amino-acid peptide identified as the authentic C-terminus of dermaseptin I. These results are in accordance with the predicted sequences of pro-dermaseptins obtained through molecular cloning, in which the dermaseptin progenitor sequences are located at the extreme C-terminus of the precursors.

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.