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Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1–3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic α-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10−5−10−4 m range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.
Amphibians have rich chemical arsenals which form an integral part of their defence system, and also assist with the regulation of dermal physiological action. In response to a variety of stimuli, host defence compounds are secreted from specialized glands onto the dorsal surface and into the gut of the amphibian [1–4]. A number of different types of antibacterial peptides have been identified from the glandular skin secretions of Australian anurans, including the caerin peptides from green tree frogs of the genus Litoria[5,6], the maculatins from Litora genimacula[6,7], the citropins from the tree frog Litora citropa[6,8], and the uperins from floodplain toadlets of the Uperoleia genus [6,9]. Amongst the most active of these are caerin 1.1, maculatin 1.1, citropin 1.1 and uperin 3.6, which have the following sequences:
Caerin 1.1: GLLSVLGSVAKHVLPHVVPVIAEHL-NH2
Maculatin 1.1: GLFGVLAKVAAHVVPAIAEHF-NH2
Citropin 1.1: GLFDVIKKVASVIGGL-NH2
Uperin 3.6: GVIDAAKKVVNVLKNLF-NH2
The solution structures of these four peptides have been investigated by NMR spectroscopy. In trifluoroethanol/water mixtures, caerin 1.1 adopts two well-defined helices (Leu2 to Lys11 and from Val17 to His24) separated by a hinge region of less-defined helicity and greater flexibility, with hydrophilic and hydrophobic residues occupying well defined zones . The hinge region is necessary for antibiotic activity . Maculatin 1.1 has a structure similar to that of caerin 1.1 except that four residues are missing from the central hinge including the first Pro (of caerin 1.1). NMR solution and micelle studies indicate that maculatin 1.1 is an α-helix, but with a significant kink centred at Pro15 . In marked contrast, similar NMR studies of citropin 1.1  and uperin 3.6  show that these peptides adopt conventional amphipathic α-helical structures along their entire length in trifluoroethanol/water mixtures.
Amphipathic peptides of this type are membrane-active antibacterial agents [1–4]. Interaction occurs at the membrane surface with the charged (normally basic) peptide adopting an α-helical conformation and attaching itself to charged sites (normally anionic) on the lipid bilayer. Two potential mechanisms have been proposed for interaction of these peptides with membranes. Aggregation of peptides is followed either by: (a) penetration through the lipid bilayer via formation of a trans-membrane helical bundle (the barrel–stave mechanism); or (b) formation of pores through the membrane due to alignment of the peptides parallel to the membrane surface (the ‘carpet’ mechanism). In both scenarios, disruption of normal membrane function occurs leading to lysis of the cell [12–17]. The magainin peptides (from the African clawed frog Xenopus laevis) are the best studied of such amphibian peptides. NMR studies shows they form well-defined amphipathic α-helices in solution and when incorporated into an artificial phospholipid; the peptides are positively charged and interact readily with anionic phospholipids . The caerin, maculatin, citropin and uperin peptides are also membrane-active basic peptides: their precise action of membrane interaction is not known at this time.
In this paper we describe the isolation, sequence determination, and activities of the aurein peptides from the Green and Golden Bell Frog L. aurea, and the Southern Bell Frog L. raniformis. These frogs are closely related. L. aurea occurs in coastal regions of New South Wales, the normal habitat is large ponds containing abundant bulrushes near the bank. The dorsum of this frog is smooth, with a striking combination of green and gold colouring. The groin is a distinctive turquoise blue. The adult ranges from 57 to 108 mm in length [19,20]. The habitat of the Southern Bell Frog L. raniformis is large pools in southern New South Wales, throughout Victoria, northern Tasmania and the south-eastern area of South Australia. It has a more warty dorsum than the related L. aurea, and it can be distinguished from that species by the presence of a pale green mid-dorsal stripe. The groin is again turquoise, and the adult is from 55 to 104 mm in length [19–21].
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The aurein 1–3 peptides show wide-spectrum antibiotic and anticancer activity. Aurein 2.2, a major component of the skin secretion of L. aurea is the most potent of the aurein antibiotics, showing a spectrum of activities slightly less than those of caerin 1.1 and citropin 1.1 (Table 2). Aurein 3.1, an abundant antibiotic peptide from both L. aurea and L. raniformis, shows moderate antibiotic activity (Table 2). The aurein antibiotics show more activity towards gram-positive than gram-negative pathogens: this is normally the case with amphibian peptide antibiotics [1–4,6]. The amphipathic and α-helical aurein 1.2 (Fig. 8B) shows moderate antibiotic activity, and with only 13 amino-acid residues, is the smallest amphibian antibiotic peptide so far reported. Other small antibiotic peptides have been reported in the animal kingdom: an 11-residue synthetic modification of sapecin, from the flesh fly Sacrophage peregrina, a 12-residue synthetic modification of defensin from the beetle Allomynna dichotoma, and a 13-residue peptide from porcine bone marrow . Aurein 1.2 is also the smallest amphibian peptide to show both antibiotic and anticancer activity.
Aurein 1.2: GLFDI I KK I AESF-NH2
Aurein 2.1: GLLDIV KKVVGAFGSL-NH2
Aurein 3.1: GLFDIV KKI AGHI AGSI-NH2
Citropin 1.1: GLFDVI KKVASVIGGL-NH2
Uperin 3.6: GVI DAAKKVVNVLKNLF-NH2
From a comparison of their amino-acid sequences (see above), the aurein 1–3 peptides show features in common with the citropins 1  and the uperins 3 . The citropins and aureins are host defence peptides of related tree frogs of the genus Litoria, while the uperins fulfil the same antibiotic role for toadlets of the genus Uperoleia. This observation of some sequence correspondence between peptides from both the genus Litoria (family Myobatrachidae) and the genus Uperoleia (family Hylidae) is an example of evolutionary convergence , as both families are known to have diverged from common stock at least 60 million years ago . The peptides listed above all contain the hydrophilic residues Asp4, Lys7 and Lys8 (shown in bold type). In the case of citropin 1.1, synthetic modifications (replacing various residues with Ala) has shown that Lys7 and Lys8 are necessary for activity but that Asp4 is not . Indeed, the modified citropin 1.1 with Ala4 replacing Asp4 produces an antibiotic more active than the natural citropin 1.1. A change in hydrophobic residues can also significantly affect the activity: for example, compare the difference in antibiotic activities of aurein 1.1 and 1.2, where the only difference lies in the last residue (Ile13 or Phe13) (see Table 2).
Amphibian peptides like the caerins, maculatins, citropins, aureins and uperins are membrane active antibiotic species [1–4,6]. The biosynthesis of such peptides is understood: they are stored in inactive form (the pro-peptide) in the appropriate glands until some stimulus causes an endoprotease to hydrolyse the pro-peptide, with concomitant transfer of the active peptide onto the skin or into the gut . The active peptide then remains on the skin for some period (usually 5–30 min), after which time it is hydrolysed by a second endoprotease in order to destroy the activity of the natural peptide [6,57]. Such degradation products are found in the secretions of both L. raniformis and L. aurea (Tables 1 and 2). The abundant antibiotic peptides aurein 2.1, 2.4 and 3.3 are specifically degraded and deactivated by removal the first two amino-acid residues, whereas aurein 3.1 yields two degradation products, one missing the first two residues, the other missing the last three residues (see Tables 1 and 2).
The aurein antibiotics, with 13–17 amino-acid residues, are not long enough to penetrate through the bacterial membrane bilayer by the barrel stave mechanism [1–4,12–18]. In principle, the minimum requirement to span the 30 Å bilayer is an α-helical peptide 20 residues long. Interestingly, even though there are many peptide antibiotics which, in principle, should be able to penetrate through the bilayer by the barrel stave mechanism, few have been shown to do so. Antibiotic peptides that adopt an orientation within the membrane consistent with the barrel stave mechanism are alamethicin (20 residues ), cecropin p1 (31 residues ) and gramicidin (a 15-residue peptide (from Bacillus brevis) which forms a head to tail dimer when incorporating into micelles ).
Some peptides have been shown to have a membrane orientation consistent with the carpet mechanism, i.e. the peptides form a carpet on the surface of the membrane, causing the formation of pores in the membrane, followed by membrane disintegration (because of the high osmotic pressure of the cell), and ultimately, cell death. The following have been shown to orient in this way; dermaseptin b (34 residues ), magainin (22 residues ), and melittin (26, residues ). As there is no NMR evidence to suggest that aurein 1.2 is forming dimers or aggregating in solution, we anticipate that this peptide interacts with the bacterial lipid bilayer via an in-plane orientation. We will test this proposal in a future study using solid state NMR studies of 15N-labelled aurein 1.2 when incorporated into a model phospholipid. Similar studies are planned for the other major families of antibiotic peptides isolated from Australian anurans including the uperins 3, citropins 1, caerins 1 and maculatins 1.
The anticancer activity of the aureins 1–3 is not unexpected because the magainin peptides (from Xenopus laevis) are known to show both antibiotic and anticancer activity . The breadth of the activity of the aureins is the surprising feature: some of the peptides (e.g. aureins 1.2, 3.2 and 3.3) show activity against more than 90% of all human cancer cell types tested. The anticancer activity of the aureins is only moderate, with LC50 values in the 10−5−10−4 molar range (1 × 10−5 m is equivalent to 15 µg·mL−1). Aurein 2.2 is the best of the antibiotic agents, while aureins 1.2, 3.2 and 3.3 are the best anticancer agents (cf. Tables 2 and 3). There are clearly some subtle structural effects operating concerning the relative activities of the aureins 1–3, but in general terms, those aureins that show wide-spectrum antibiotic activity also show broad-spectrum anticancer activity.
The broad-spectrum anticancer activity together with the known membrane antibacterial activity of these peptides suggests that the anticancer activity is due to penetration and disruption of the membranes of the cancer cells. There are marked differences between the structures and chemical content of membranes of prokaryotic and eukaryotic cells  so such a proposal is interesting: perhaps these peptides are also cytotoxic to normal cells, which would limit any possible clinical or pharmaceutical application? Thus we have tested the effects of two of the most bioactive aurein peptides, 1.2 and 3.2, on red blood cells. They do not affect red blood cells at 100 µg·mL−1, the maximum concentration required to kill bacterial and cancer cells. The concentration of aurein 1.2 and 3.2 required to completely lyse red blood cells is much higher (1 mg·mL−1). This result is compatible with the proposal that the anticancer active and basic aurein peptides effect membrane disruption of cancer cells, as cancer cells have up to eight-fold more anionic phospholipids on their membrane surfaces than do normal cells [66–68]. We intend to study the anticancer activities of these peptides further by the attachment of antibodies for particular cancers, to direct the active peptide specifically towards those cancers.
Finally, the four aureins 4 and two aurein 5 peptides (Table 1) are unlike the other aureins in that they are neither post-translationally modified nor show antibiotic or anticancer activity. Their role in the amphibian integument has not been determined. Similar peptides have been found in the skin secretions of L. citropa and their roles are also unknown. However, it is of interest that aurein 5.2 has structural features in common with splendipherin (see below), the aquatic male sex pheromone of the magnificent tree frog L. splendida[69,70]. Perhaps one (or more) of the aureins 4 or 5 is an aquatic pheromone? We will test possible pheromone activity of these peptides in a future study: at present we do not have sufficient numbers of male and females of either species to carry out such behavioural studies.
Aurein 5.2 GLMSSIGKALGGLI VDVLKPKTPAS-OH
In conclusion, we have reported the sequences of 22 peptides from the skin glands of L. aurea and L. raniformis. Thirteen of these peptides show broad-spectrum antibiotic and anticancer activity. Aurein 1.2 is shown by NMR methods to adopt a stable amphipathic α-helical form in a solvent system which stabilizes helices in peptides with helical propensity [71,72]: this peptide with only 13 residues is the smallest anuran peptide yet reported to show both antibiotic and anticancer activity.