Antimicrobial properties of peptides from Xenopus granular gland secretions

Previously, we described a family of novel broad spectrum antimicrobial peptides, magainins, from the skin of Xenopus laevis. In this report we show that at least two other Xenopus peptides, present in the skin and its secretions, PGLa and a peptide released from the xenopsin precursor, exhibit antimicrobial properties comparable to the magainins. The identification of these newer members provides insight into the structural diversity of vertebrate antimicrobial peptides.     

Solid-phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa-(4-24)] from skin secretion of Xenopus laevis

From the nucleotide sequence of clones isolated from a cDNA library constructed from skin of Xenopus laevis, the existence of PYLa, a peptide comprised of 24 amino acids, was predicted. This peptide was synthesized by solid-phase methods and purified to homogeneity with an overall yield of 61%. The synthetic peptide was used as reference substance to search for its natural counterpart in skin secretion of Xenopus. Two peptides were found which were very similar to PYLa except for the absence of the first three amino acids. These 21-amino-acid peptides, termed PGLa, can be generated from PYLa by cleavage after the single arginine residue present in the latter. The two forms of PGLa differ in their retention time on HPLC but have identical amino acid compositions and terminal sequences. Tryptic hydrolysis of synthetic PYLa after the single arginine yields exclusively PGLa with the shorter retention time on HPLC. The chemical difference between the two forms of PGLa is currently not known. The possible biological role of these newly discovered constituents of frog skin secretion is discussed.     

Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of host-defense peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families from the tetraploid frogs, Xenopus petersii (Peters' clawed frog) and Xenopus pygmaeus (Bouchia clawed frog), and the octoploid frog Xenopus lenduensis (Lendu Plateau clawed frog). Xenopsin-precursor fragment (XPF) peptides were not detected. The primary structures of the antimicrobial peptides from X. petersii demonstrate a close, but not conspecific relationship, with Xenopus laevis whereas the X. pygmaeus peptides show appreciable variation from previously characterized orthologs from other Xenopus species. Polyploidization events within the Xenopodinae (Silurana+Xenopus) are associated with extensive gene silencing (nonfunctionization) but unexpectedly the full complement of four PGLa paralogs were isolated from X. lenduendis secretions. Consistent with previous data, the CPF peptides showed the highest growth-inhibitory activity against bacteria with CPF-PG1 (GFGSLLGKALKIGTNLL.NH(2)) from X. pygmaeus combining high antimicrobial potency against Staphylococcus aureus (MIC=6 μM) with relatively low hemolytic activity (LC(50)=145 μM).     

Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis

The skin secretion of the frog Xenopus laevis has been fractionated by reverse-phase HPLC and the most polar components studied by fast-atom-bombardment mass spectrometry (FAB/MS). Esterification of the hydrophilic peptides with methanol and ethanol was employed to improve the sensitivity of the technique. A number of small, highly acidic peptides have been identified, and alcoholysis of the peptide bonds within a number of these permitted their sequencing by FAB/MS. The sequences confirmed that they originate from acidic spacer regions found in the precursors to peptide hormones, such as caerulein, which have already been found in the secretion. In addition, acidic peptides derived from the spaces of the precursor to the antimicrobial peptides, PGS (or the magainins) have been isolated. The release of these from the preprotein cannot be fully accounted for by documented processing mechanisms, suggesting that a novel type of cleavage site has been identified.     

Antimicrobial peptides in the stomach of Xenopus laevis.

Antimicrobial peptides are widely distributed in nature and appear to play a role in the host defense of plants and animals. In this study we report the existence of antimicrobial peptides in the stomach of the vertebrate Xenopus laevis, an animal previously shown to store high concentrations of antimicrobial peptides in its skin. Antimicrobial activity was detected in extracts of X. laevis stomach tissue and nine antimicrobial peptides were then purified. A novel 24-amino acid peptide, designated PGQ, was isolated from these extracts, and has the following amino acid sequence: GVLSNVIGYLKKLGTGALNAVLKQ. PGQ is relatively basic and has the potential to form an amphipathic alpha-helix. The other peptides isolated are members of the magainin family of antimicrobial peptides, and include magainins I and II, PGLa, xenopsin precursor fragment, and four caerulein precursor fragments. None of these peptides had been previously identified in tissues other than the skin. The purification of the peptides from stomach extracts and subsequent protein sequence analysis reveals that the peptides have undergone the same processing as their dermal counterparts, and that they are stored in their processed forms. Northern blot analysis indicates that the magainin family of peptides are synthesized in the stomach, and immunohistochemical studies demonstrate that magainin is stored in a novel granular multinucleated cell in the gastric mucosa of Xenopus. This study demonstrates that the magainin family of antimicrobial peptides is found in the gastrointestinal system of X. laevis and offers an opportunity to further define the physiological role of these defense peptides.     

Biogenic amines and active peptides in the skin of fifty-two African amphibian species other than bufonids

1. Extracts prepared from dried or fresh skins of 52 African amphibian species, other than bufonids, were subjected to chemical (colour reactions) and biological screening, to determine occurrence and contents of aromatic biogenic amines and peptides active on smooth muscle preparations and blood pressure. 2. Only indolealkylamines were detectable in the skins. They were represented by 5-hydroxytryptamine, its N-methylated derivatives and tryptamine. The indolealkylamines considered included the alkaloid trypargine, a carboline derivative resulting from the condensation of tryptamine with arginine. 3. The peptide families identified in skin extracts of the African frogs were as follows: caeruleins (caerulein, [Asn2, Leu5] caerulein), tachykinins (kassinin, [Glu2, Pro5] kassinin, hylambatin), bradykinins [( Hyp3] bradykinin), xenopsin, thyrotropin releasing hormone, peptide PYLa and the magainins I and II. The last five peptides have been so far identified only in the skin of Xenopus laevis, together with their precursors. 4. Since numerous other peptide molecules await isolation, elucidation of structure, and definition of possible biological activities, the array of peptides occurring in the skin of African amphibians, as in that of Australian and American amphibians, is destined to increase.     

Peptidomic analysis of skin secretions provides insight into the taxonomic status of the African clawed frogs Xenopus victorianus and Xenopus laevis sudanensis (Pipidae)

Peptidomic analysis was used to compare the distribution of host-defense peptides in norepinephrine-stimulated skin secretions from Xenopus victorianus Ahl, 1924 (also described as the subspecies X. laevis victorianus) and Xenopus laevis sudanensis Perret, 1966 with the previously determined distributions in Xenopus laevis (Daudin, 1802) and Xenopus petersii Bocage, 1895. Peptides belonging to the magainin, peptide glycine-leucine-amide (PGLa), and caerulein precursor fragment (CPF) families were purified by reversed-phase HPLC and characterized by electrospray mass spectrometry. Magainin-P2, PGLa-P1, CPF-P1, CPF-P2, and CPF-P3 previously isolated from X. petersii and structurally different from orthologous peptides from X. laevis, were identified in X. victorianus and X. laevis sudanensis skin secretions whereas the corresponding X. laevis peptides were absent. Magainin-1, identical in X. petersii and X. laevis, was also identified in the secretions. Xenopsin-precursor fragment (XPF) peptides, absent from X. petersii but present in X. laevis skin secretions, were not identified in the X. victorianus and X. laevis sudanensis secretions. The data indicate that X. victorianus and X. laevis sudanensis are more closely related to X. petersii than to X. laevis and support separate species status. The study illustrates the value of analysis of host-defense peptides in the evaluation of taxonomic and phylogenetic relationships between closely related frog species.     

Isolation of a dipeptidyl aminopeptidase, a putative processing enzyme, from skin secretion of Xenopus laevis

A dipeptidyl aminopeptidase has been purified to apparent homogeneity from skin secretion of Xenopus laevis. This enzyme is a glycoprotein with a molecular mass of about 98 kDa. It hydrolyzes a variety of dipeptidyl-p-nitroanilides and oligopeptides containing proline, alanine or glycine as the second amino acid and is inhibited by diisopropylfluorophosphate. The pH optimum was found to be around 8, while at pH 6, substrates were cleaved at about one-third of the maximal rate. This dipeptidyl aminopeptidase has the specificity required for the cleavage of amino-terminal extensions preceding the sequence of caerulein and xenopsin in their respective precursors.     

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 μM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 μM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 μM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca²⁺ concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

Two precursors of thyrotropin-releasing hormone from skin of Xenopus laevis. Each contains seven copies of the end product.

From the skin of Xenopus laevis, the cloned cDNAs of two precursors of thyrotropin-releasing hormone have been isolated and sequenced. These encode almost identical precursor polypeptides each containing seven copies of the end product flanked by typical prohormones processing signals. Northern blot analysis has corroborated the existence of two thyrotropin-releasing hormone precursor mRNAs of similar size as the cloned cDNA and demonstrated the existence of a third smaller species as well. Two or more mRNAs for this precursor are also present in the brain and eyes of X. laevis adults and tadpoles.     

Differences in the skin peptides of the male and female Australian tree frog Litoria splendida. The discovery of the aquatic male sex pheromone splendipherin, together with phe8 caerulein and a new antibiotic peptide caerin 1.10.

The skin secretions of female and male Litoria splendida have been monitored monthly over a three-year period using HPLC and electrospray mass spectrometry. Two minor peptides are present only in the skin secretion of the male. The first of these is the female-attracting aquatic male sex pheromone that we have named splendipherin, a 25 amino acid peptide (GLVSSIGKALGGLLADVVKSKGQPA-OH). This pheromone constitutes about 1% of the total skin peptides during the breeding season (January to March), dropping to about 0.1% during the period June to November. Splendipherin attracts the female in water at a concentration of 10-11-10-9 M, and is species specific. The second peptide is a wide-spectrum antibiotic of the caerin 1 group, a 25 residue peptide (GLLSVLGSVAKHVLPHVVPVIAEKL-NH2) named caerin 1.10. The neuropeptides of L. splendida are also seasonally variable, the change identical for both the female and male. During the period October to March, the sole neuropeptide present in skin secretions is caerulein [pEQDY(SO3)TGWMDF-NH2]; this is active on smooth muscle and is also an analgaesic. During the southern winter (June to September), more than half of the caerulein is hydrolysed to [pEQDYTGWMDF-NH2], a peptide that shows no smooth muscle activity. In place of caerulein, a new peptide, Phe8 caerulein [pEQDY(SO3)TGWFDF-NH2], becomes a major component of the skin secretion. Perhaps this seasonal change is involved in thermoregulation, that is, with the initiation and maintenance of the inactive (hibernation) phase of the animal.     

The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide

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.     

Is caerulein amphibian CCK?

The amphibian skin decapeptide caerulein is structurally related to the mammalian peptides gastrin and CCK, suggesting that the peptides might share a common evolutionary history. It has been suggested that caerulein is the amphibian counterpart of gastrin and CCK, and that the Amphibia do not possess authentic gastric and CCK. High Performance Liquid Chromatography (HPLC) in conjunction with radioimmunoassay using a caerulein-specific antiserum and C-terminal CCK antisera, was used to characterize CCK-and caerulein-like peptides in amphibian brain and gut. In the brain of Xenopus laevis, two CCK-like peptides were present, one of which was indistinguishable by HPLC from mammalian CCK8. No decapeptide caerulein was detected in the brain of Xenopus laevis or Rana temporaria. In the stomach of Xenopus and in the intestine of both species studied, CCK-like and caerulein-like peptides were present. The results indicate therefore that the Amphibia possess CCK8-like rather than caerulein-like peptides in brain. In contrast, stomach and intestine contain both CCK-like and caerulein-like peptides, but the latter are however distinguishable from the decapeptide found in skin.     

Evidence from peptidomic analysis of skin secretions that allopatric populations of Xenopus gilli (Anura:Pipidae) constitute distinct lineages

The International Union for Conservation of Nature (IUCN) Endangered Cape Platanna Xenopus gilli inhabits disjunct ranges at the tip of Cape Peninsula and near the town of Kleinmond on opposite sides of False Bay in the extreme southwest of Africa. Peptidomic analysis of host-defense peptides in norepinephrine-stimulated skin secretions from frogs from the Cape Peninsula range resulted in the identification of two magainins, two peptide glycine–leucine–amide (PGLa) peptides, two xenopsin-precursor fragment (XPF) peptides, nine caerulein-precursor fragment (CPF) peptides, and a peptide related to peptide glycine–glutamine (PGQ) previously found in an extract of Xenopus laevis stomach. The primary structures of the peptides indicate a close phylogenetic relationship between X. gilli and X. laevis but only magainin-1, PGLa and one CPF peptide are identical in both species. Consistent with previous data, the CPF peptides show the greatest antimicrobial potency but are hemolytic. There are appreciable differences in the expression of host-defense peptide genes in frogs from the population of animals sampled near Kleinmond as peptides corresponding to magainin-G2, XPF-G1, XPF-G2, and four CPF peptides, present in secretions from the Cape Peninsula frogs, were not identified in the skin secretions from Kleinmond frogs. Conversely, PGLa-G3, XPF-G3, and three CPF peptides were identified in the Kleinmond frogs but not in the Cape Peninsula animals. The data support the conclusion from morphometric analyses and comparisons of the nucleotide sequences of mitochondrial genes that the disjunct populations of X. gilli have undergone appreciable genetic, morphological, and phenotypic divergence.     

Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product

From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs.     

A comparison of host-defense peptides in skin secretions of female Xenopus laevis × Xenopus borealis and X. borealis × X. laevis F1 hybrids

Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (X_LB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (X_BL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1was present in low concentration. No peptides were identified in secretions of either X_LB or X_BL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis × X. muelleri F1 hybrids and no novel peptide expression.     

Biosynthesis of caerulein in the skin of Xenopus laevis: partial sequences of precursors as deduced from cDNA clones

The decapeptide caerulein represents one of the main constituents of the skin secretion of Xenopus laevis. Total mRNA was isolated from skin, transcribed into cDNA and inserted via GC-tailing into the plasmid pUC8. Among the transformants, 300 clones were selected at random and screened with a cDNA primed with the synthetic deoxynucleotide d(AGTCCATCCA), which is complementary to the mRNA region coding for the fragment Trp-Met-Asp-Phe of cerulein. Of nine strongly hybridizing clones, three were sequenced and these were found to contain inserts with very similar nucleotide sequences. The cloned cDNAs code for parts of two different caerulein precursors. These contain one or two copies of caerulein and five additional amino acids located between pairs of arginine residues. The extra glycine at the carboxy terminus is considered to serve as the signal for amidation, while the tetrapeptide Phe-Ala-Asp-Gly, linked to the amino end of caerulein in these precursors, must be cleaved by an unusual processing mechanism.     

A novel peptide designated PYLa and its precursor as predicted from cloned mRNA of Xenopus laevis skin

A variety of peptides closely related to mammalian hormones and neurotransmitters are secreted from amphibian skin. Using cDNA clones of mRNA isolated from skin of Xenopus laevis, we have been searching for precursors of some of these constituents. Here we present the sequences of parts of cloned mRNAs which code for precursors of a novel peptide. In the predicted polypeptides, pairs of basic residues flank a sequence of 25 amino acids terminating with glycine, the signal for the formation of a terminal amide. The predicted final product liberated from these precursors would be a peptide comprised of 24 amino acids starting with tyrosine and ending with leucine amide, which has therefore been designated PYLa. This peptide can form an amphipathic helix similar to that found in peptides with cytotoxic, bacteriostatic and/or lytic properties.     

Caerulein secretion by dermal glands in xenopus laevis

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.