(4-24)-ply(a) and xenopsin

The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism spectroscopy. Increasing the pD to 10 induced the formation of predominantly alpha-helical secondary structures, with some beta-sheet. In the presence of negatively charged liposomes (dimyristoylphosphatidylglycerol), the peptides folded into alpha-helical secondary structures with some beta-sheet structure evident. On the other hand, in the presence of zwitterionic phospholipids (dimyristoylphosphatidylcholine), the spectra were identical to those in aqueous solution. For some magainins, the interaction with charged liposomes was modulated by the presence of cholesterol; cholesterol was found to promote the formation of beta-sheet structures, as evidenced by the appearance of amide I bands at 1614 and 1637 cm-1. Differences in structure were observed between the amidated and nonamidated forms of some peptides. From the data, a mechanism of antimicrobial action of the magainin family of peptides is proposed.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Dimyristoylphosphatidylcholine; Fourier Analysis; Liposomes; Magainins; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptides; Phosphatidylglycerols; Protein Conformation; Sequence Homology, Nucleic Acid; Spectrophotometry, Infrared; Xenopus laevis; Xenopus Proteins

From genomic libraries of Xenopus laevis, parts of the genes coding for the precursors of the skin peptides GLa (peptide with amino-terminal glycine and carboxy-terminal leucinamide), xenopsin and levitide have been isolated and sequenced. The gene for prepropeptide GLa comprises four exons, separated by relatively small introns. The gene for preproxenopsin is composed of five exons, of which all but the last one have been analyzed. This is a large gene encompassing at least 25,000 base pairs. In addition, two exons of the gene for preprolevitide have been isolated. A comparison of these genes reveals the presence of a homologous exon. This exon contains 161 bp, starts one base pair prior to the initiation codon and encodes a signal peptide and part of a pro region with processing sites. In addition, the two genes for preprocaerulein analyzed previously [Vlasak et al. (1987) Eur. J. Biochem. 169, 53-58] also contain a similar exon. This demonstrates the existence of a homologous export exon in genes encoding the precursors of different skin peptides.

Topics: Animals; Antimicrobial Cationic Peptides; Base Sequence; Ceruletide; Cloning, Molecular; DNA; Exons; Genes; Genetic Code; Introns; Molecular Sequence Data; Oligopeptides; Peptides; Protein Sorting Signals; RNA; Sequence Homology, Nucleic Acid; Skin; Xenopus laevis; Xenopus Proteins

We observed a striking sequence similarity between precursors for promagainin and procaerulein type I (excluding the caerulein peptide region). Additional comparisons of the promagainin precursor with those of other procaeruleins, proxenopsin, and peptide-Gly-Leu-amide revealed that all possess one or more copies of a structurally similar spacer module, from which an amphiphilic spacer peptide is cleaved. Promagainin yields the magainins, spacer peptides with antimicrobial activity; we suggest other spacer peptides may have similar activity. We propose that the genes for the four kinds of hormones were derived from a common ancestral gene through gene and exon duplications and that the procaerulein and proxenopsin genes are mosaic genes in which the original 3'-ends were replaced by exon shuffling.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Ceruletide; Hormones; Molecular Sequence Data; Oligopeptides; Peptides; Protein Precursors; Sequence Homology, Nucleic Acid; Skin; Software; Xenopus laevis; Xenopus Proteins

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.

Topics: Amino Acid Sequence; Amino Acids; Animals; Antimicrobial Cationic Peptides; Ceruletide; Chromatography, High Pressure Liquid; Mass Spectrometry; Oligopeptides; Peptide Fragments; Peptides; Protein Precursors; Skin; Xenopus laevis; Xenopus Proteins

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fast atom bombardment mass spectrometry (FAB-MS). High performance liquid chromatography (HPLC) of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH and PGLa. Fractions from the HPLC were analyzed directly by FAB-MS to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of FAB-MS, manual Edman degradation, enzymatic digestions and amino acid analyses, identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2,700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by FAB-MS. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from PGLa and the spacer regions in the precursors to xenopsin and caerulein.

Topics: Amides; Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Ceruletide; Chromatography, High Pressure Liquid; Mass Spectrometry; Molecular Weight; Oligopeptides; Peptides; Pyrrolidonecarboxylic Acid; Skin; Xenopus laevis; Xenopus Proteins