Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing ...
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1 Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity L Marenah, P R Flatt, D F Orr, C Shaw and Y H A Abdel-Wahab School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland BT52 1SA, UK (Requests for offprints should be addressed to L Marenah; Email: l.marenah@ulster.ac.uk) Abstract Skin secretions of Rana saharica were evaluated for the 3519·3 Da, 4920·4 Da and 4801·2 Da respectively. These isolation and characterisation of novel insulinotropic peptides were found to be identical to brevinin-1E, peptides. Crude secretions obtained from young adult brevinin-2EC, esculentin-1 and esculentin-1B, which frogs by mild electrical stimulation of the dorsal skin belong to the group of antimicrobial peptides isolated surface were purified by reverse phase HPLC yielding from skin secretions of various Rana frog species. Prelimi- 80 fractions. In acute 20-min incubations with glucose nary studies on the mechanism underlying the insulino- responsive BRIN-BD11 cells, fractions 36–43, 46–54 and tropic actions of esculentins-1 and -1B suggested possible 57–63 significantly stimulated insulin release by 2- to involvement of both cyclic AMP–protein kinase A and 8-fold compared with 5·6 mM glucose alone. Pooled –C-dependent G-protein sensitive pathways. These data fractions in the latter two bands were rechromatographed indicate that the skin secretions of Rana saharica frogs to reveal 9 homogenous peaks, which elicited significant contain bioactive molecules with significant insulin- 1·3- to 3·5-fold increases in insulin release (P
2 L MARENAH and others · Novel insulin-releasing peptide
species have yielded peptides that are either identical or this was chromatographed on a Vydac 218TP510 semi-
structurally related to peptides synthesised in neuroendo- preparative C-18 column (251 cm) Grace Vydac
crine tissues of mammals (Erspamer et al. 1986, Roseghini (Hesperia, CA, USA). The column was equilibrated with
et al. 1988, 1989, Basir et al. 2000). 0·12% (v/v) TFA/water at a flow rate of 2 ml/min. Using
Rana saharica is a large sized frog being about 10–12 cm 0·1% (v/v) TFA in 70% acetonitrile/water, the concen-
in body length. The species is widely distributed in the tration of acetonitrile in the eluting solvent was raised to
bigger oases in the Sahara from Algeria across to Egypt 80% (v/v) over 80 min using linear gradients. Absorbance
(Frost 1985). This study describes the purification, struc- was monitored at 214 nm with collection of 2 ml fractions.
tural and biological characterisation of multiple peptides Fractions that showed major insulin releasing activity were
with insulin releasing activity from electrically stimulated pooled and rechromatographed using a Vydac 208TP54
skin secretions of Rana saharica frogs. Such peptides may analytical C-18 column (250·46 cm). The column was
be of therapeutic interest, as illustrated by the enthusiasm equilibrated with 0·12% (v/v) TFA/water at a flow rate of
for the clinical treatment of type 2 diabetes with 1 ml/min. Using 0·1% (v/v) TFA in 70% acetonitrile/
exendin-4 and related peptides isolated from the venom water, the concentration of acetonitrile in the eluting
of the lizard, Heloderma suspectum (Kolterman et al. 2003, solvent was raised to 30% (v/v) over 10 min and to 60%
Green et al. 2004). (v/v) over 40 min using linear gradients. Absorbance was
monitored at 214 nm and peaks were hand collected and
prepared for acute insulin release studies. The peaks show-
Materials and Methods ing insulin-releasing activity were pooled and further puri-
fied to a single homogenous peak using a Vydac 208TP54
Reagents analytical C-18 column (250·46 cm). The concentration
RPMI-1640 tissue culture medium, foetal bovine serum, of acetonitrile in the eluting solvent was raised to 15% (v/v)
penicillin and streptomycin were all purchased from over 5 min and to 80% (v/v) over 70 min using linear
Gibco (Paisley, Strathclyde, UK). Phorbol-12-myrisate- gradients. Absorbance was monitored at 214 nm.
13-acetate (PMA), forskolin, pertussis toxin and verapamil
were obtained from the Sigma Chemical Company Ltd Culture of insulin secreting cells
(Poole, Dorset, UK). High-performance liquid chroma-
tography (HPLC) grade acetonitrile was obtained from BRIN-BD11 cells were cultured in RPMI-1640 tissue
Rathburn (Walkerburn, Scotland). Sequencing grade culture medium containing 10% (v/v) foetal calf serum,
trifluoroacetic acid was obtained from Aldrich (Poole, 1% (v/v) antibiotics (100 U/ml penicillin, 0·1 mg/ml
Dorset, UK). All chemicals employed in the operation of streptomycin) and 11·1 mM glucose. The production and
the 491 Procise gas phase sequencer were supplied by characterisation of BRIN-BD11 cells have been described
Perkin Elmer Applied Biosystems (Warrington, Cheshire, elsewhere (McClenaghan et al. 1996). This robust, glucose-
UK). All other chemicals used were of the highest purity responsive cell line has been shown to respond to an
available. array of established insulinotropic peptides (McClenaghan
et al. 1996, O’Harte et al. 1998a,b, Abdel-Wahab et al.
1999). Cells were maintained in sterile tissue culture
Collection of skin secretions flasks (Corning Glass Works, Sunderland, UK) at 37 C
Four young captive bred Rana saharica were maintained in an atmosphere of 5% CO2 and 95% air using a
in terraria at 24 C under a 12 h light/12 h darkness cycle LEEC incubator (Laboratory Technical Engineering,
and were fed on crickets. The skin secretions were Nottingham, UK). In one experimental series, cells were
obtained from the frogs by gentle electrical stimulation cultured overnight with 25 µM forskolin, 10 nM PMA or
(4-ms pulse width, 50 Hz, 5 V) using platinum electrodes 0·1 µg/ml pertussis toxin prior to acute tests.
rubbed over the moistened dorsal skin surface for 10 s.
Secretions were washed off into a glass beaker, using Acute insulin release studies
deionised water. The resultant secretions were freeze-
dried in a Hetosicc 2·5 freeze dryer (Heto, UK). Approxi- Insulin release from BRIN-BD11 cells was determined
mately 50 mg, dry weight, of skin secretion was obtained. using cell monolayers (McClenaghan et al. 1996). The
This procedure was carried out in accordance with the cells were harvested with the aid of trypsin/EDTA
UK Animals (Scientific Procedures) Act 1986. It is a (Gibco), seeded into 24-multiwell plates (Nunc, Rosklide,
non-invasive technique causing no distress to the frog. Denmark) at a density of 1·5106 cells per well, and
allowed to attach overnight. Prior to the acute test, cells
were preincubated for 40 min at 37 C in a 1·0 ml Krebs
Purification of peptides Ringer bicarbonate buffer (115 mM NaCl, 4·7 mM KCl,
The lyophilised crude venom (20 mg) was dissolved in 1·28 mM CaCl2, 1·2 mM KH2PO4, 1·2 mM MgSO4,
0·12% trifluoroacetic acid (TFA)/water (2 ml) and 1 ml of 10 mM NaHCO3, 5 g/l bovine serum albumin, pH 7·4)
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via free accessNovel insulin-releasing peptide · L MARENAH and others 3 supplemented with 1·1 mM glucose. Test incubations post hoc test. Groups of data were considered to be were performed for three independent observations for significantly different if P
4 L MARENAH and others · Novel insulin-releasing peptide
Figure 1 (A) Reversed-phase HPLC of the crude venom of Rana saharica. The crude venom (20 mg) was
dissolved in 0·12% trifluoroacetic acid/water (2 ml) and 1 ml was applied to a semi-preparative Vydac C18
column as described in Materials and Methods. The dashed line shows the concentration of acetonitrile in
the eluting solvent. Bands 1 and 2 correspond to fractions eluting at 46–54 min and 57–63 min
respectively. (B) Effects of various semi-preparative C18 HPLC fractions of Rana saharica crude venom on
insulin secretion from BRIN-BD11 cells. Incubations were performed at 5·6 mM glucose using fractions
shown in (A). Values are the means S.E.M. for 3 separate observations. *PNovel insulin-releasing peptide · L MARENAH and others 5 Figure 2 (A) Reversed-phase HPLC purification of the pooled fractions of Rana saharica from bands 1 and 2 in Fig. 1A. Fractions were applied to an analytical Vydac C18 column as described in Materials and Methods. The dashed lines show the concentration of acetonitrile in the eluting solvent. Bands 3, 4, 5 and 6 correspond to peaks 1.8–1.12, 1.13–1.21, 2.2–2.7 and 2.12–2.22 respectively. (B) Effects of various semi-preparative C18 HPLC fractions of Rana saharica crude venom on insulin secretion from BRIN-BD11 cells. Incubations were performed at 5·6 mM glucose using fractions shown in (A). Values are the means S.E.M. for 3 separate observations. *P
6 L MARENAH and others · Novel insulin-releasing peptide
Figure 3 (A) Final reversed-phase HPLC purification of the pooled fractions of Rana saharica from bands 3, 4, 5 and 6 in Fig. 2A. Fractions
were applied to an analytical Vydac C18 column as described in Materials and Methods. Individual peaks were hand collected. The
dashed lines show the concentration of acetonitrile in the eluting solvent. (B) Effects of peptides isolated from Rana saharica venom on
insulin secretion from BRIN-BD11 cells. Incubations were performed at 5·6 mM glucose using peptide peaks shown in (A). Values are the
means S.E.M. for 3 separate observations. *PNovel insulin-releasing peptide · L MARENAH and others 7
Table 1 Homology search for peak 3.1, 3.4, 5.4 and 6.3 obtained from Rana saharica using
the GCG sequence analysis programme of Swiss-Prot FASTA database
Structure Identity
Peaks
3.1 GIFSKFGRKKIKNLLISGLKNVGKEVGMDVVRTGIDIAGCKIKGEC Esculentin-1
3.4 GIFSKLAGKKLKNLLISGLKNVGKEVGMDVVRTGIDIAGCKIKGEC Esculentin-1B
5.4 GILLDKLKNFAKTAGKGVLQSLLNTASCKLSGQC Brevinin-2EC
6.3 FLPLLAGLAANFLPKIFCKITRKC Brevinin-1E
Single letter code denote amino acids: A, Ala; R, Arg; N, Asn; D, Asp; C, Cys; E, Glu; Q, Gln, G, Gly; H,
His; X, Hyp; I, Ile; L, Leu; K, Lys; M, Met; F, Phe; P, Pro; S, Ser; T, Thr; W, Trp; Y, Tyr; V, Val.
Park et al. 1994, Conlon et al. 1999, Goraya et al. 2000). Determination of toxic effects of the isolated peptides
These cationic peptides exert antimicrobial properties on BRIN-BD11 cell viability, as assessed by vital neutral
against a wide variety of microorganisms including Gram- red staining, indicates that the observed secretory actions
positive and Gram-negative bacteria (Hancock & Lehrer cannot be simply attributed to cell lysis or toxicity. It
1998, Kwon et al. 1998). therefore follows that these peptides stimulate insulin
Figure 5 Acute effects of peaks 3.1 (esculentin-1) and 3.4 (esculentin-1B) from Rana saharica, forskolin
and PMA on insulin secretion from BRIN-BD11 cells cultured overnight in the absence (control) and the
presence of 25 M forskolin, 10 nM PMA or 0·1 g/ml pertussis toxin. Acute incubations were performed
at 5·6 mM glucose. Values are the means S.E.M. for 8 separate observations. **P8 L MARENAH and others · Novel insulin-releasing peptide
release through regulated pathways. Blockade of voltage- Anastasi A, Erspamer V & Endean R 1968 Isolation and amino acid
dependent Ca2+ channels with verapamil did not affect sequence of caerulein, the active decapeptide of the skin of Hyla
caerulea. Archives of Biochemistry and Biophysics 125 57–68.
secretory effectiveness of esculentin-1 and esculentin-1B Anastasi A, Erspamer V & Bucci M 1972 Isolation and amino acid
(peaks 3.1 and 3.4). Similarly, a powerful insulin response sequences of alytesin and bombesin, two analogous active
was observed using cells depolarised with 30 mM KCl, tetradecapeptides from the skin of European discoglossid frogs.
indicating a degree of independence from changes in ion Archives of Biochemistry and Biophysics 148 443–446.
permeability. However, down-regulation of cyclic AMP– Barthalmus GT 1994 Amphibian Biology, pp 382–410. Ed H Heatwole.
Chipping Norton, Oxfordshire: Surrey Beatty and Sons.
PKA- and –PKC-dependent pathways by overnight cul- Basir YJ, Floyd C, Knoop FC, Dulka J & Conlon JM 2000 Multiple
ture of BRIN-BD11 cells with forskolin (Altman et al. antimicrobial peptides and peptides related to bradykinin and
1987, Gromada et al. 1998) and PMA (Hii et al. 1987, neuromedin N isolated from skin secretion of the pickerel frog,
Yamatani et al. 1988, Persaud et al. 1989, Wolf et al. 1989) Rana palustris. Biochemica et Biophysica Acta 1543 95–105.
respectively, blocked the acute stimulatory effects of Chen, YE & Drucker, DJ 1997 Tissue-specific expression of unique
mRNAs that encode proglucagon-derived peptides or exendin 4 in
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esculentin-1 and -1B were inhibited by overnight culture Clark DP, Durell S, Maloy WL & Zasloff M 1994 Ranalexin. A
with pertussis toxin (Seaquist et al. 1992), indicating the novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin,
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stimulatory action. Additional studies are required to assess
Conlon JM, Halverson T, Dulka J, Platz JE & Knoop FC 1999
the actions of esculentin-1 and -1B on normal pancreatic Peptides with antimicrobial activity of the brevinin-1 family
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