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- Experimental procedures
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- Supporting Information
Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic α-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.
Amphibians have rich chemical arsenals in the skin glands that form an integral part of their defence system, and also assist with the regulation of dermal physiological action [1,2]. In response to a variety of stimuli, host defence compounds are secreted from these specialized glands onto the dorsal surface and into the gut of the amphibian. Some of these compounds are antibacterial peptides, e.g. the bombinin, brevinin and magainin families of peptides [2–4]. The magainin peptides also show some anti-cancer activity . Some of the antibacterial peptides have attracted pharmaceutical interest .
The dermal secretions of some Australian tree-frogs (Litoria splendida, L. caerulea, L. gilleni, L. xanthomera, L. chloris and L. genimacula) collectively contain more than 30 antibacterial peptides [7–14]. The major wide-spectrum antibacterial agents from these tree-frogs belong to either the caerin or maculatin families of peptides. Particular examples are caerin 1.1 (one of 10 structurally related peptides from L. splendida, L. caerulea, L. gilleni, L. chloris and L. xanthomera) and maculatin 1.1 (from L. genimaculata). These peptides are, respectively, 25 and 21 amino acids in length: their sequences are related but maculatin 1.1 is missing four amino acid residues from the centre of a caerin 1-type sequence. The two peptides have the following sequences:
Caerin 1.1 GLLSVLGSVAKHVLPHVVPVIAEHL-NH2
Maculatin 1.1 GLFGVLAKVAAHVVPAIAEHF-NH2
The solution structures of both 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 . In contrast to caerin 1.1, maculatin 1.1 does not have such marked flexibility in its central region. In this case, NMR spectra [measured (a) in trifluoroethanol/water and (b) with the peptide incorporated into an artificial phospholipid micelle] show that the amphipathic α-helix of maculatin 1.1 is distorted by Pro15: the central axis of the helix preceding Pro15 makes an angle of approximately 20° to the axis of the helix following Pro15 .
Amphipathic peptides of this type are membrane-active antibacterial agents. 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, viz: aggregation of peptides is followed either by (a) penetration through the lipid bilayer via formation of a transmembrane helical bundle (the barrel-stave mechanism) or (b) formation of pores through the membrane as a result of the alignment of the peptides parallel to the membrane surface (the ‘carpet’ mechanism). As a result, disruption of normal membrane function occurs leading to lysis of the cell [17–22]. The magainin peptides (from the African clawed frog Xenopus laevis) are the best studied of such amphibian peptides. NMR studies show 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 [6,18,21–23]. As caerin 1.1 and maculatin 1.1 are also membrane-active basic peptides, then perhaps they react in a similar manner to the magainins. Although the solution structures of caerin 1.1 and maculatin 1.1 are quite different, the antibacterial activities of each peptide are very similar [15,16]. If they are synthetically modified to convert them into molecules with better defined α-helical structures, i.e. removal of the hinge of caerin 1.1 by conversion of both Pro residues to Ala, or distortion of maculatin 1.1 by conversion of Pro15 to Ala15, the spectrum of antibacterial activities is significantly reduced in each case.
In this paper we describe the isolation, sequence determination, and activities of the citropin peptides from the Blue Mountain tree-frog of Australia, L. citropa. This frog is found near flowing, rocky streams in heavily forested areas from the coast to the Dividing Ranges in eastern Victoria and New South Wales. The length of the adult frog is in the range 4.5–5.6 cm, and the frog can be distinguished by its brown dorsum, green flash colours near the head, and by a brown tympanum [24,25]. This tree-frog is unusual in that it has two types of skin glands, the granular glands on the dorsal surface and the large submental gland on the throat. Other Australian tree-frogs that we have studied have no submental gland, thus it is of interest to determine the role of this gland, for example, does it contain host defence peptides like the dorsal glands of other amphibians, and if so, whether these peptides are similar in activity and structure to the caerin 1 and maculatin 1 peptides.
- Top of page
- Experimental procedures
- Supplementary material
- Supporting Information
Citropins 1.1 and 1.2 are the major antibacterial and amphipathic peptides produced by both the dorsal and submental glands of L. citropa. These peptides are structurally dissimilar to the antimicrobial peptides of other tree-frogs of the genus Litoria that we have studied previously both in sequences and in length . They are the simplest, amphibian, wide-spectrum antibacterial peptides reported to this time. They differ only in the nature of the hydrophobic residues at positions 5, 6, 12 and 13 in their sequence, and all contain the hydrophilic residues Asp4, Lys7 and 8 and Ser11. There is however, one feature of L. citropa that is similar to other tree-frogs of the genus Litoria that we have studied, i.e. they all produce endoproteases that deactivate the major antibacterial peptides after they have been on the skin for some period (after about 10 min for L. citropa: within the 5–30 minute range for other frogs we have studied [13,14]). In the case of caerin 1.1 and maculatin 1.1, endoproteases remove the first two amino acid residues effectively deactivating each peptide [13,14], while citropins 1.1 and 1.2 are principally degraded by the removal of three residues, with peptides with two missing residues being formed in much smaller yield (see Tables 1 and 2 for sequence and activity data, respectively) (cf. also the degradation of the magainin peptides from X. laevis). The citropin peptides show little sequence similarity to other antibacterial peptides from frogs of the genus Litoria so far studied, but they do show an overall structural resemblance to the 17 residue uperin 3 antibacterial peptides that are produced by toadlets of the genus Uperoleia. For example, compare the sequences of uperin 3.6 (a major antibacterial peptide of Uperoleia mjobergii) and citropin 1.1.
Citropin 1.1 GLFDVIKKVASVIGGL-NH2
These two peptides have a very similar distribution of hydrophobic and hydrophilic amino acids, in particular the three conserved charged amino acids which are highlighted in bold. In addition to these amino acids, uperin 3.6 contains an additional Lys at residue 14. The spectrum of antibacterial activity of uperin 3.6 is very similar to those of citropin 1.1, 1.2 and 1.3 (see Table 2). The solution structure of uperin 3.6 has been investigated by NMR techniques , and like citropin 1.1, it is a well defined amphipathic α-helix along the entire length.
Although citropin 1.1 is almost entirely helical in the membrane-mimicking solvent TFE (Fig. 6), the length (16 amino acids) is too short to span the lipid bilayer. The potent antibacterial activity of citropin 1.1 cannot therefore arise from simple aggregation of the peptides in an orientation perpendicular to the bilayer plane to form a pore (e.g. via the barrel-stave mechanism). Other mechanisms for the lytic activity of the antibacterial citropin and uperin peptides must therefore be invoked, e.g. those involving the orientation of the peptides parallel to the membrane surface where they either aggregate in a carpet like manner  or diffuse continuously throughout the membrane . In both instances, destabilization of the membrane occurs which creates openings within the bilayer.
What are the roles of the citropin 2 and 3 peptides in the amphibian integument? The complex citropin 2 and 3 peptides (Table 1) have not shown significant antibacterial activity in our testing regime. The role of the basic citropin 2 peptides is not known at this time. The citropin 3 peptides are quite unusual in that (a) the sequence commences with Asp (the only other reported amphibian peptides that commence with Asp are hylambin  and kassinin ; both members of the tachykinin family of neuropeptides which bear no structural relationship to the citropins 2 and (b) they are anionic peptides. The anionic nature of the peptides suggests that they may be spacer peptides for the procitropin 1 peptides within the dorsal and submental glands (see  for a review of the biosynthesis of antibiotic peptides), although this proposal has not been confirmed.
Finally, the role of the four extra citropin 1 peptides from the dorsal gland secretion (I to L in Fig. 2 and Table 1), is, to date, a mystery. The four extra citropin 1 peptides are related structurally, two to citropin 1.1 and two to citropin 1.2. The last two residues of the citropins 1.1 and 1.2 have been replaced by Leu-Asp and two extra residues (either Ser-Pro-OH or Ser-Gln-OH) added. One of these peptides, citropin 1.1.3, has been tested for antibacterial activity, but such activity is insignificant (see Table 2). Perhaps these peptides are hormones or neuropeptides. If so, they bear no structural resemblance to any known amphibian hormone or neuropeptide: they are certainly not related to the potent amphibian caerulein or uperolein type neuropeptide vasodilators [1,2,45,46].