Báo cáo Y học: Amphibian peptides that inhibit neuronal nitric oxide synthase The isolation of lesueurin from the skin secretion of the Australian Stony Creek Frog Litoria lesueuri docx

269, 100-109 2002 © FEBS 2002 Amphibian peptides that inhibit neuronal nitric oxide synthase The isolation of lesueurin from the skin secretion of the Australian Stony Creek Frog Litor

Eur J Biochem 269, 100-109 (2002) © FEBS 2002

Amphibian peptides that inhibit neuronal nitric oxide synthase

The isolation of lesueurin from the skin secretion of the Australian Stony

Creek Frog Litoria /esueuri

Jason Doyle’, Lyndon E Llewellyn’, Craig S Brinkworth”, John H Bowie”, Kate L Wegener”, Tomas Rozek?, Paul A Wabnitz”, John C Wallace? and Michael J Tyler*

"Australian Institute of Marine Science, Townsville MC, Queensland, Australia; *Departments of Chemistry, *Molecular Biosciences and *Environmental Biology, The University of Adelaide, South Australia

Two neuropeptides have been isolated and identified from

the secretions of the skin glands of the Stony Creek Frog

Litoria lesueuri The first of these, the known neuropeptide

caerulein 1.1, is a common constituent of anuran skin

secretions, and has the sequence pEQY(SO3) TGWMDF-

NH) This neuropeptide is smooth muscle active, an anal-

gaesic more potent than morphine and is also thought to be

a hormone The second neuropeptide, a new peptide, has

been named lesueurin and has the primary structure

GLLDILKKVGKVA-NH5 Lesueurin shows no signifi-

cant antibiotic or anticancer activity, but inhibits the for-

mation of the ubiquitious chemical messenger nitric oxide

from neuronal nitric oxide synthase (nNOS) at ICs

(16.2 tm), and is the first amphibian peptide reported to

show inhibition of nNOS As a consequence of this activity,

we have tested other peptides previously isolated from

Australian amphibians for nNOS inhibition There are three

groups of peptides that inhibit nNOS (Cso at um concen-

trations): these are (a) the citropin/aurein type peptides

(of which lesueurin is a member), e.g citropin 1.1

(GLFDVIKK VASVIGGL-NH.,) (8.2 HM); (b) the frenatin type peptides, e.g frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 um); and (c) the caerin | peptides, e.g caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH;>) (1.7 tm) From Lineweaver—Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca** calmodulin, the inhibition dropped by ~ 50% in each case In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca** calmodulin It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca? * calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS

Keywords: amphibians; Litoria lesueuri; neuropeptides;

nNOS inhibition; Ca? * calmodulin interaction

Amphibians have rich chemical arsenals that 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

bio-active peptides have been identified from the glandular

skin secretions of Australian anurans of the Litoria genus,

including (a) neuropeptides of the caerulein family [5-8],

and (b) wide-spectrum antibiotics, e.g the caerin peptides

Correspondence to J H Bowie, Department of Chemistry,

The University of Adelaide, South Australia, 5005

Fax: + 61 08 83034358, Tel.: + 61 08 83035767,

E-mail: john.bowie@adelaide.edu.au

Abbreviations: FAD, flavin adenine dinucleotide, oxidized form;

FMN, flavin mononucleotide; ICs9, concentration (of peptide) which

causes 50% inhibition; MS/MS, mass spectroscopy/mass spectro-

scopy; NADPH, nicotinamide adenine nucleotide phosphate, reduced

form; NCI, National Cancer Institute (Washington); nNOS, neuronal

nitric oxide synthase

(Received 18 June 2001, revised 8 October 2001, accepted 23 October

2001)

from green tree frogs of the genus Litoria [6-8], the citropins from the tree frog Litoria citropa [9,10], and the aureins from the bell frogs Litoria aurea and Litoria raniformis [11] Amongst the most active of these are neuropeptide caeru-

lein 1.1, and the antibiotics caerin 1.1, citropin 1.1 and

aurein 1.2: caerulein 1.1 pEQGY(SO3;)TGWMDF-NH>; caerin 1.1 GLLSVLGSVAKHVLPHVVPVIAEHL-NH>;

citropin 1.1 GLFDVIKKVASVIGGL-NH>; aurein 1.2

GLFDITKKIAESF-NH3

Aurein 1.2 contains only 13 amino-acid residues, and is the smallest peptide from an anuran reported to have significant antibiotic activity The aurein peptides have also been shown to exhibit anticancer activity in tests carried out

by the National Cancer Institute (NCI, Washington DC, USA) [12]

The solution structures of the antibiotic (and anticancer active aS appropriate) peptides shown above have been investigated by NMR spectroscopy In trifluoroethanol/ water mixtures, caerin 1.1 adopts two well-defined helices (Leu2 to Lysl1 and from Vall7 to His24) separated by a hinge region of less-defined helicity and greater flexibility, with hydrophilic and hydrophobic residues occupying well- defined zones [13] The central hinge region is necessary for optimal antibiotic activity [13] Similar NMR studies of

citropin 1.1 [9] and aurein 1.2 [11] show that these peptides

adopt conventional amphipathic «helical structures, a

feature commonly found in membrane-active agents

[14.8] Interaction occurs at the membrane surface with

the charged, and normally basic peptide adopting an

a helical conformation and attaching itself to charged, and

normally anionic sites on the lipid bilayer This ultimately

causes disruption of normal membrane function leading to

lysis of the bacterial or cancer cell [14—16]

Many Australian anurans that we have studied conform

to the model outlined above in that they have a variety of

host defence peptides in skin glands including a neuropep-

tide that acts on smooth muscle, and at least one powerful

wide-spectrum antibiotic peptide such as those described

above [8] However there are some species of anuran that

divert markedly from this scenario For example, the Red

Tree Frog Litoria rubella {17—19], and the related species

Litoria electrica [20] excrete neither antimicrobial peptides

nor neuropeptides such as caerulein Instead, they release

large quantities of small peptides, called tryptophyllins [21],

onto their skin, which are thought to be neurotransmitters

or neuromodulators [22], at least partly because they show

structural similarity to the human brain endomorphins (e.g

YPWG-NH) [23] The sequences of two tryptophyllins are

as follows: tryptophyllin L1, FPWL-NH,; tryptophyllin

L2, pEFPWL.-NH:

Even more unusual are the marsh frogs of the Limno-

dynastes genus These produce only minute amounts of

anionic skin peptides, none of which are post-translationally

modified, or show neuropeptide or antimicrobial activity

[24,25] A particular example, dynastin 1 (from Limnodyn-

astes interioris) |24] has the sequence GLLSGLGL-OH

In this paper, we describe the isolation, sequence deter-

mination, and activities of two bioactive peptides from the

skin glands of the stony creek frog Litoria lesueuri We have

not studied a member of the L /esueuri complex of frogs

previously, and we have now found that it does not produce

antimicrobial peptides, unlike the majority of the other

members of the genus Litoria Instead, it produces a

neuropeptide that inhibits the formation of nitric oxide by

neuronal nitric oxide synthase (nNOS) We next examined

other peptides isolated earlier from anurans of the genus

Litoria This has led to the discovery of three types of

amphibian peptides that inhibit nNOS

MATERIALS AND METHODS

Preparation of skin secretions

L lesueuri was held by the back legs, the skin moistened

with deionized water, and the granular dorsal glands

situated on the back were stimulated by means of a

bipolar electrode of 21G platinum attached to a Palmer

Student Model electrical stimulator The electrode was

rubbed gently in a circular manner on the particular gland

(under study) of the animal, using 10 V and a pulse

duration of 3 ms [26] The resulting secretion was washed

from the frog with deionized water (SO mL), the mixture

diluted with an equal volume of methanol, centrifuged,

filtered through a Millex HV filter unit (0.45 um), and

lyophilized This work conforms with the Code of Practice

for the Care and Use of Animals for Scientific Purposes

(1990) and the Prevention of Cruelty to Animals Act

(1985), and was approved by the University of Adelaide Animal Ethics Committee

HPLC separation of peptide material HPLC separation of the skin secretion was achieved using a VYDAC C18 HPLC column (Sum, 300 A, 4.6 x 250 mm) (Separations Group, Hesperia, CA, USA) equilibrated with 10% acetonitrile/aqueous 0.1% trifluoroacetic acid The lyophilized mixture (0.5 mg) was dissolved in deionized water (20 uL) and injected into the column The elution profile was generated using a linear gradient produced by an ICI DP 800 Data Station controlling two LC1100 HPLC pumps, increasing from 10 to 75% acetonitrile over a period

of 60 min at a flow rate of 1 mL-min™’ The eluant was monitored by ultraviolet absorbance at 214 nm using an ICI LC-1200 variable wavelength detector (ICI Australia, Melbourne, Australia) The resultant HPLC trace shows two major peaks (Fig 1) The two fractions were collected, concentrated and dried in vacuo The first major fraction (Fig 1B) (50 ug) contained the known neuropeptide caeru- lein 1.1, identified by HPLC and mass spectrometry [27] The second major fraction (Fig 1C) contained 10 ug of a new peptide, called lesueurin

Sequence determination of lesueurin Electrospray mass spectrometry Electrospray mass spec- tra were determined using a Finnigan LCQ 1on trap mass spectrometer Purified fractions from the HPLC separation were dissolved in methanol/water (1 : 1, v/v) and infused into the electrospray source at 8 uL-min™’ Electrospray conditions were as follows: needle potential 4.5 kV, tube lens 60 V, heated capillary 200 °C and 30 V, sheath gas flow

30 p.s.i Mass spectra were acquired with the automatic gain control on, a maximum time of 400 ms, and averaging over three microscans Mass spectrometric sequencing was carried by the MS/MS method using B and Y + 2 fragmentations [28]

Amino-acid sequencing Automated Edman sequencing of lesueurin was performed by a standard procedure as described previously [29] using an applied Biosystem 492

B

Lm nt —

12 20 mins 28

Fig 1 HPLC separation of the glandular secretion of Litoria lesueuri

B, caerulein 1.1; C, lesueurin A, nonpeptide material.

102 J H Bowie et al (Eur J Biochem 269)

procise sequencer equipped with a 900-A data analysis

module The best results were obtained using a disc of

immobilon film treated with bioprene in ethanol, onto

which the peptide was absorbed from aqueous acetonitrile

(90%) The disc was pierced several times with a razor blade

to aid the flow of solvent

Preparation of synthetic lesueurin

Lesueurin was synthesized (by Mimotopes, Clayton, Vic-

toria, Australia) using L-amino acids via the standard

N-o-Fmoc method (full details including protecting groups

and deprotection have been reported recently [30]) Syn-

thetic lesueurin was shown to be identical to the natural

lesueurin by electrospray mass spectrometry, and HPLC

Bioactivity assays

Antimicrobial testing Synthetic lesueurin was tested for

antibiotic activity by the Microbiology Department of the

Institute of Medical and Veterinary Science (Adelaide,

Australia) by a standard method [31] The method used

involved the measurement of inhibition zones (produced by

the applied peptide) on a thin agarose plate containing the

microorganisms under study The microorganisms used in

this procedure were Bacillus cereus, Escherichia coli, Leuco-

nostoc lactis, Listeria innocua, Micrococcus luteus, Pasteu-

rella multocida, Staphylococcus aureus, Staphylococcus

epidermidis and Streptococcus uberis Lesueurin showed no

activity at MIC values below 100 ugmL"! against any of

these organisms

Anticancer activity testing Synthetic lesueurin showed no

activity below 10~* m in the ‘60-human tumour line testing

program’ of the US NCI (Washington) [12]

Neuronal nitric oxide synthase inhibition Ynhibition of

nNOS was measured by monitoring the conversion of

[H]arginine to [*H]citrulline In brief, this involved incuba-

tion of a homogenate of rat cerebella (which had endogenous

arginine removed by ion exchange chromatography) in a

reaction buffer (33 mm Hepes, 0.65 mm EDTA, 0.8 mm

CaCh, 3.5 pgmL calmodulin, 670 um B-NADPH, 670 tm

dithiothreitol, pH 7.4) containing 20 nm [*H]arginine (NEN

Life Sciences, Boston, MA, USA) The nNOS inhibitor,

N®-nitro-L-arginine (1 mm) was used to measure back-

ground radioactivity Reactions were terminated after

10 min with 50 uL of 0.3 mM EGTA An aliquot (50 pL) of

this quenched reaction mixture was transferred to 50 pL of

500 mm Hepes (pH 5.5) AGSOW-X8 (Na~ form) resin

(100 pL) was added to separate [H]arginine from [HIcit-

rulline After repeated vortexing, this slurry was centrifuged

at 1200 gfor 10 min, and 70 uL of supernatent was removed

and the [*H]citrulline measured by scintillation counting

Peptides selected for further examination to determine the

mechanism of inhibition were assayed in the same reaction

buffer as used for initial screening except that it contained

30 nm [Hlarginine supplemented with 0.3-13.3 mm argi-

nine Peptide concentrations used are given in the legend to

Fig 4

Data analysis for nNOS studies Peptide inhibition curves

were fitted using the curve-fitting routine of sIGMAPLOT

© FEBS 2002

(SPSS, Chicago, IL, USA) with the variation of the Hill

equation: fmol [*H]citrulline production = 1/(1 + [inhibi-

tor]/ICson), where ICsp is the concentration at which the

peptide causes 50% inhibition and 7 is the slope of the curve and can be considered as a pseudo Hill coefficient [32] Lineweaver—Burk plots [33] were generated using sIGMA- PLOT (SPSS, Chicago, IL, USA)

Calcineurin assay This assay was performed following the manufacturer’s protocol [34] with minor modifications Calcineurin was diluted to 0.036 U-pL! in enzyme dilution buffer (50 mm Tris, 0.5 mgmL™ BSA, pH 7.4) Peptides (5 wL) were assayed in duplicate in a reaction mixture containing 16 mm p-nitrophenylphosphate, 0.4 mgmL™! BSA, 0.8 mm NiCh, 4 uemL7! calmodulin in Tris buffer (40 mm, pH 7.4) The reaction was started with the addition

of the enzyme (5 nL; 0.1 U) All samples were assayed at

30 °C with positive controls containing water in the place of the test sample and negative controls containing no enzyme Absorbance (A) readings at 405 nm were taken after 30 min with readings averaged, adjusted to the change in A per minute and corrected for background A (negative control)

Percent of control was then calculated as (A405 test sample/

1aos positive control) x 100

RESULTS

L lesueuri, usually called either Lesueur’s Frog or the Stony Creek Frog, has varied dorsal colouration ranging from yellow to brown, with a black head stripe from the snout to the tympanum [35] The animal ranges from 37

to 63 mm in length, and is often found in the vicinity of rocky streams in coastal regions from north of Queens- land to eastern Victoria It is reported that there are two distinct populations of this frog, one confined to north- eastern Queensland, the other in New South Wales and Victoria Whether these are two different subspecies of

L lesueuri or two different species has not yet been determined [36]

A single specimen of L lesueuri, collected at Atherton, Queensland, was used in this study The electrical stimula-

tion method [26] was used to elicit secretion from the

granular skin glands The animal was not harmed in this study Less than 0.5 mg of material was obtained following work up of the secretion, and this contained less than 100 pg

of peptide material This is an unusually small amount of peptide material It is normal for a species of the genus Litoria to contain 5 mg of peptide material in the glandular

secretion; some frogs, such as Litoria splendida, secrete more

than 100 mg of peptide material [8] HPLC separation (Fig 1) revealed two major peptide components The first, and major component was identified as caerulein 1.1 from its electrospray mass spectrum and HPLC behaviour [27] This potent smooth muscle agent, analgaesic and hormone

is often a major peptide component of species of the genus Litoria [8]

The minor component is a 13-residue peptide that we have called lesueurin Only 10 ug of this material was available for study The MS/MS of the MH™ ion of lesueurin is shown in Fig 2 As this mass spectrum does not differentiate between isomeric Leu or Ile or isobaric Lys and Asn, we have confirmed the identity of these four residues using the automated Edman procedure [29] The automated

Fig 2 Electrospray mass spectrum (MS/MS)

of the MH“ ion of lesueurin Masses shown in

this spectrum are nominal masses Data from

B fragmentations give sequence information 4

from the C-terminal end of lesueurin (see 2

1264

1335

1352

schematic arrows above the spectrum) Data a

from Y + 2 fragmentations give sequence

information from the N-terminal end of the +00

peptide (see schematic arrows below the

spectrum) For a review of fragmentations of

MH_™ ions of peptides see [28] This method

does not distinguish between isomeric Leu and

Ile (nominal mass 113) and isobaric Lys and

Gln (nominal mass 128) These residues have

been differentiated using the automated

399

-^ 3

¥

512

728

841

Edman procedure [29] The correct residues 350 460

are shown in this figure

it

mí

Table 1 nNOS Inhibition by amphibian peptides Amino-acid sequences, source species and naming convention used where a peptide has been named previously Peptides with minimal or no effect are highlighted with respective concentration used listed in footnotes below All peptides in Group | are amphipathic « helices in solution [10,11] Caerin | peptides in solution have «helices from residues I-11 and 18-25, with a flexible hinge region including residues 12-17 [13]

ICso Hill Net

Inactive or weakly active peptides

Rubellidin 3.1” L rubela [L7] IEFFT-NH> NA NA 0

Electrin 2.1° L electrica [20] NEEEK VK WEFPDVP-NH> NA NA —2 Caeridin 39 L caerulea [8Ì GLFDAIGNLLGGLGL-NH> NA NA 0 Maculatin 1.3° L eucnemis ' GLLGLLGSVVSHVLPAITQHL-NH> NA NA +] Inhibitor Group |

Lesueurin L lesueuri ® GLLDILKKVGKVA-NH> 16.2 1.5 +3 Aurein 1.1 L aurea [11] GLFDII KK I AES I-NH> 33.9 2.0 +] Citropin 1.1 L citropa [10] GLFDVIKK VASVIGGL-NH> 8.2 1.6 +2 Aurein 2.2 L aurea [11] GLFDIVKKVVGALGSL-NH, 4.3 2.5 +2 Aurein 2.3 L aurea [11] GLFDIVKK VVGIAGSL-NH> 1.8 1.7 +2 Aurein 2.4 L aurea [11] GLFDIVKKVVGTLAGL-NH,> 2.1 3.1 +2 Inhibitor Group 2

Not named L dahlii' GLLGSIGNAIGAFIANKLKP-OH 3.2 2.2 +3 Frenatin 3 L infrafrenata [8] GLMSVLGHAVGNVLGGLFKPKS-OH 6.8 1.4 +3 Splendipherin L splendida [67] GLVSSIGKALGGLLADVVKSKGQPA-OH 8.5 1.3 +3 Inhibitor Group 3

Caerin 1.1 L splendida [6] GLLGVLGSIAKHVLPHVVPVIAEHL-NH> 36.6 1.4 +] Caerin 1.10 L chloris [8] GLLSVLGSVAKHVLPHVVPVIAEKL-NH> 41.0 0.6 +2 Caerin 1.6 L chloris [8] GLFSVLGAVAKHVLPHVVPVIAEKL-NH, 8.5 1.7 +2 Caerin 1.8 L chloris [8] GLFKVLGSVAKHLLPHVVPVIAEKL-NH> 1.7 3.7 +3 Caerin 1.9 L chloris [8] GLFGVLGSI AKHVLPHVVPVIAEKL-NH> 6.2 2.2 +2

* Inactive at 13.3 ugmL™! ° Inactive at 33.3 ng'mL”” © Inactive at 66.7 ugmL' ¢ Inactive at 133.3 ugmL7! ° Caused 46.4% inhibition at

31.4 um Full ICs) determination was not possible because of solubility ' Brinkworth, C., Bowie, J.H., Wallace, J.C & Tyler, M.J unpublished work ° Present paper

Edman procedure identifies all but the last amino acid in the

lesueurin sequence This residue, C-terminal Ala-NHb, 1s

clearly identified by mass spectrometric fragmentation data

(Fig 2) A synthetic sample of lesueurin was prepared, and

shown to be identical with natural lesueurin using HPLC

and mass spectrometry The sequence of lesueurin 1s

GLLDILKKVGKVA-NH:

Lesueurin has no antibiotic activity (at minimum inhibitory concentration values below 100 ugmL~”) against the nine bacteria we use in our test regime Also, lesueurin exhibited no cytotoxity at concentrations less than 107* M against any of the 60 human tumour lines in

the NCI test regime Lesueurin was, however, shown to

inhibit nNOS with an ICsp of 16.2 um (Table 1) This

104 J H Bowie et al (Eur J Biochem 269)

discovery of previously unreported pharmacological

activity of an amphibian peptide prompted us to test for

similar bioactivity of amphibian peptides that we have

investigated previously Table | lists the sequences of the

natural peptides tested, both active and inactive towards

nNOS The solubility of these peptides and the maximum

tolerable amount of the peptide solvent in the nNOS

assay affected the maximum concentration tested for

several of the peptides Those peptides found to inhibit

nNOS from the primary screen were then titrated to

determine their ICs, parameters These results are given in

Table 1 together with the slope of the inhibition curves

and various physical properties Three examples of the

inhibition curves, including that generated by lesueurin,

are shown in Fig 3

Four model peptides were selected from the inhibitor

groups (Table 1) to further examine the mode of action by

which they were inhibiting nNOS Lesueurin and citro-

pin 1.1 (both from inhibitor group 1), frenatin 3 Gnhibitor

70 ———

© FEBS 2002

group 2) and caerin 1.9 Gnhibitor group 3) all produced Lineweaver—Burk plots consistent with a noncompetitive mode of inhibition (Fig 4) with respect to the nNOS substrate arginine Thus inhibition 1s not mediated by direct action upon the arginine-catalysing site Rat cerebellar nNOS may retain endogenous calmodulin rendering it unsuitable for a Michaelis-Menten study of enzyme kinet- ics, aS was carried out with arginine

An experimental procedure was used to gauge the potential for selected peptides to inhibit nNOS by displacing Ca** calmodulin from the calmodulin binding domain of nNOS In this procedure, the nNOS inhibition experiments

were carried out with citropin 1.1, frenatin 3, and caerin 1.9

with added Ca** calmodulin to determine the influence of the calmodulin on the inhibition of nNOS These experi- ments measured nNOS activity in the presence of 142.9 ug-mL*"’ of the active peptide (about 10-fold greater than the ICs value of each peptide), with the Ca** calmodulin concentration 1n the assay buffer increased 100-

Fig 3 Inhibition of nNOS exemplified by (A) lesueurin (circles) and frenatin 3 (Squares), and (B) citropin 1.1 (inverse triangles) and caerin 1.9 (triangles) These peptides represent each

of the groups of peptide inhibitors listed in Table 1 Curves are drawn to the Hill equa-

TT

@®

0.01 0.1 1 10 100 0.01 0.1 10 100

[peptide] (uM)

tion, and the values for the ICs) and slope of the curve are given in Table 1

Fig 4 Lineweaver—Burk plots derived from changes in enzyme velocities in the presence of increasing amounts of the peptides citropin 1.1 (A), lesueurin (B), caerin 1.9 (C) and frenatin 3 (D) Enzyme kinetics data obtained in the absence of peptide are depicted by filled cir- cles, and represent the uninhibited reaction Open circles show the effect of the citropin 1.1, lesueurin, caerin 1.9 and frenatin 3 at 4.1, 24.7, 5.1 and 6.1 uM, respectively Filled inverse triangles show the effect when the concentra-

1/[arginine (uM)]

tion of citropin 1.1, lesueurin, caerin 1.9 and

frenatin 3 are increased to 8.2, 49.4, 10.3 and 12.2 uM, respectively.

fold to 350 ugmL7' For citropin 1.1, the inhibition

decreased from 94 to 58% in the presence of Ca * calmod-

ulin The results for frenatin 3 and caerin 1.9 are 83-52%,

and 84-53%, respectively

Selected peptides were tested for their ability to inhibit

calcineurin, another calmodulin dependent enzyme

Lesueurin at 74 um (4.6-fold greater than the [C59 against

nNOS) inhibited calcineurin by 33% Citropin 1.1 reduced

calcineurin activity by 34% at 31 um (3.8-fold higher than

the ICs) against nNOS) but at double this concentration (i.e

7.6-fold greater than its ICsp against nNOS), calcineurin was

inhibited by 96% Frenatin 3 inhibitied calcineurin by 38%

at 46 tM, a concentration that is 6.8-fold greater than the

ICsq against nNOS Finally, caerin 1.9 inhibited calcineurin

by 48.1% at 19.3 um (4.8-fold more concentrated than its

ICso against nNOS)

DISCUSSION

Lesueurin is the name we have given to a new 13-residue

basic peptide isolated from L lesueurii The sequence

shows reasonable homology to that of aurein 1.1, an

antimicrobial and anticancer peptide isolated from

L aurea [11] (Table 2)

There are some critical differences in the hydrophilic

residues of the two peptides with lesueurin having Lys11

whereas aurein 1.1 has Glull Even so, we predicted that

lesueurin should show similar antimicrobial and anticancer

activity to that of aurein 1.1 Surprisingly, lesueurin neither

exhibits antibiosis below 100 ng:mL””, or cytotoxity below

10M ïn the 60 human tumour lines in the NIC test

program Thus, L lesueuri, as with only a minority of

species of the genus Litoria (see introduction), has no host

defence antibiotic against microbial pathogens To discover

whether lesueurin has other biological properties, it was

subjected to the bioactive molecule discovery program of

the Australian Institute of Marine Science It effectively

inhibited nNOS with an ICs value of 16.2 HM with a slope

of 1.5 This is the first instance of an amphibian peptide that

inhibits the formation of the chemical messenger nitric

oxide

Testing of other amphibian peptides indicated that there

are three well-defined groups of basic peptides that inhibit

nNOS, and the results are summarized in Table 1 These

are: (a) the aurein/citropin group of peptides, of which

lesueurin is a member Most of these (lesueurin is a notable

exception) are membrane active peptides, which show

potent antibiotic acitivity, and in the case of the aureins,

significant anticancer activity These peptides are amphi-

pathic « helices, as evidenced by solution NMR studies on

aurein 1.2 [11] and citropin 1.1 [9]; (b) the frenatin 3 type

peptides, molecules that are characterized by a C-terminal

CO>H group together with two lysine residues near the

C-terminus These peptides show little or no antibiotic and/

or anticancer activity; (c) those caerins |, particularly those

Table 2 Sequence identity of lesueurin and aurein 1.1

Peptide Sequence

Lesueurin GLLDILKKVGKVA-NH>

Aurein 1.1 GLFDITKKIAEST-NH>

containing Phe3 These molecules are also potent mem- brane-active antibiotics, and NMR studies show they have two ahelical regions separated by a central flexible hinge region [13] How do these three seemingly unrelated groups

of peptides inhibit the formation of nitric oxide by nNOS? The three nitric oxide synthases, namely neuronal, endothelial and inducible, are highly regulated enzymes responsible for the synthesis of the signal molecule nitric oxide They are amongst the most complex enzymes known (for nNOS see [37,38]) By a complex sequence involving binding sites for a number of cofactors including heme, tetrahydrobiopterin, FMN, FAD and NADPDH, nNOS converts arginine to citrulline, releasing the short-lived but reactive radical nitric oxide [39,40] Nitric oxide synthases are composed of two domains: (a) the catalytic oxygenase domain that binds heme, tetrahydrobiopterin and the substrate arginine, and (b) the electron-supplying reductase

domain that binds NADPH, FAD and FMN Communi-

cation between the oxygenase and reductase domains is determined by the regulatory enzyme calmodulin that interacts at a specific site between the two domains In the

cases of the nNOS and eNOS isoforms, the calmodulin is

regulated by intracellular Ca?” , but not for iNOS [41-44] Dimerization of the oxygenase domain is necessary for catalytic activity [39,40]

The significant departure of the Hill slope of all of the inhibition curves from unity was the first indication that these peptides are not acting directly upon the arginine substate site of nNOS [33] A Hill slope = 1 would indicate

an interaction between a single active enzyme element with a single substrate As Hill slopes > | are obtained, some other interaction must be causing the inhibition of nNOS To confirm this suspicion and to elucidate the general mech- anism of inhibition, an analysis was conducted of the kinetics of the nNOS inhibition reaction at different inhibitor concentrations Lineweaver—Burk plots are shown

in Fig 4 for representatives of each of the inhibitor groups

listed in Table 1, namely, lesueurin as the original nNOS inhibitor found, citropin 1.1, another member of inhibitor

group 1, frenatin Gnhibitor group 2) and caerin 1.9 (inhibitor group 3) The fact that the regression lines plotted for each inhibitory peptide shown in Fig 4 all intercept at a common point on the X-axis of these plots is typical of noncompetitive inhibition, and so these peptides are unlikely to directly involve the arginine substrate site [33]

In its simplest definition, noncompetitive inhibition is when

an inhibitor binds at a site other than the active site,

changing the enzyme-substrate affinity

These four peptides, lesueurin, citropin 1.1, frenatin 3 and caerin 1.9, and probably the other active amphibian peptides of inhibitor groups 1—3, must therefore inhibit the formation of nitric oxide by either blocking one or more of the cofactor sites on nNOS or by some chemical modifica- tion reaction with nNOS that alters the activity of the enzyme An obvious example of blocking a cofactor site would be if the amphibian peptide reacts with the regulatory enzyme Ca** calmodulin, thus changing the three-dimen- sional structure and preventing its attachment to the calmodulin binding site on nNOS There are examples of small basic peptides, often «helices, being captured and enclosed within Ca?* calmodulin, and as a consequence changing the three-dimensional shape of the calmodulin [45-48], but nNOS deactivation by these peptides has, to

106 J H Bowie et al (Eur J Biochem 269)

our knowledge, not been tested There are also examples

where certain small peptides (one of which mirrors part of

the sequence of the calmodulin binding site of rat nNOS)

preferentially interact with Ca** calmodulin, impeding the

interaction of Ca? calmodulin with its binding site on

nNOS, and, as a consequence, preventing the formation of

nitric oxide (ICs values at um concentrations) [49,50]

The possibility that the mechanism of nNOS inhibition

by the amphibian peptides does involve complex formation

of peptides with Ca?‘ calmodulin is supported by the

following experiments The inhibition of nNOS by selected

peptides (citropin 1.1, frenatin 3 and caerin 1.9) is reduced

by the addition of Ca? ‘calmodulin to the assay buffer The

maximum reduction of inhibition under the experimental

conditions used is 50%

The enzyme Ca*‘ calmodulin regulates not only nNOS

but also a number of other enzymes including calcineurin If

the active amphibian peptides are indeed interacting with

Ca** calmodulin, they should also inhibit the activity of

calcineurin The four model peptides lesueurin, citropin 1.1,

frenatin 3 and caerin 1.9, all inhibit the activity of calcineu-

rin, but at concentrations lower than those obtained for

nNOS (Table 1) Even so, the fact that all four peptides

inhibit both nNOS and calcineurin enzymes, provides

credence to the proposal that the amphibian peptides are

affecting the Ca” ' calmodulin interaction with nNOS This

is an interesting observation because sequences of the

Ca’ * calmodulin binding sites of nNOS and calcineurin are

quite different (see below [51]), even though they have been

classified as belonging to the same class of enzymes [52]

The sequences are nNOS (rat, human) and calcineurin A

(rat, human), respectively are as follows:

KRRAIGFKKLAEAVKFSAKIM

RKETIRNKIRAIGKMARVFSVLR

Currently, although three-dimensional structures of cer-

tain domains of nitric oxide synthase isoforms are known

[53-57], we are not aware of the three-dimensional structure

of the calmodulin binding sites of any isoform being

reported Thus, we cannot make meaningful comparisons of

common structural motifs that might underlie calmodulin

binding to the enzymes

Most nNOS inhibitors are small organic molecules that

are either analogues of the arginine substrate such as

N-nitro-L-arginine [58] or species that prevent calmodulin

conjugation such as tamoxifen [59] and melatonin [60]

Protein and peptidic inhibitors of nNOS are few Apart

from those we have mentioned above [45,46], caveolin-1, a

structural protein component of plasmalemmel caveolae,

inhibits endothelial NOS but not neuronal NOS or indu-

cible NOS [61] Residues 82-101 of caveolin-1 in the form of

glutathione S-transferase fusion proteins have been shown

to attenuate the activities of all three isoforms of NOS [62]

A novel 10-kDa protein inhibitor of nNOS, known as PIN,

destabilizes the native dimer form of nNOS preventing it

from functioning [63-65] Proteins such as presynaptic

density proteins-93 and -95 and synophin bind to nNOS via

a specialized domain called PDZ domain, which serves as

cellular localization signals, but these proteins do not

modulate nNOS activity [66] The basic amphibian peptides

that inhibit nNOS show little homology of sequence (a)

between the three active groups of peptides shown in

Table 1, (b) with the sequence of amino acid residues of the

Ca** /calmodulin binding domain of nNOS, (c) with the

© FEBS 2002

sequence of Ca*” /calmodulin itself, and (d) with other peptides or proteins which inhibit nNOS Thus we are unable, at this time, to predict from homology, any features

of the active amphibian peptides that allow them to inhibit nNOS

There is, however, a significant homology of sequences within each of the active groups shown in Table | Consider group | (Table 1) as an example All peptides of group 1 have a_ post-translational modification in that the N-terminal group is -CONH> Lesueurin has high homol- ogy with some of the citropins and aureins It is several

residues shorter that aureins 2.2, 2.3, 2.4 and citropin 1.1, all

of which have ICsy) values more potent than that of

lesueurin Aurein 1.1 has the same number of residues as lesueurin but a lower overall positive charge, and is not as effective as lesueurin in inhibiting nNOS The length of these peptides seems to be a factor in determining their activity Another important feature conserved in this group of peptides is the central Lys-Lys pair and the basic nature of the peptides It appears that the length of the peptide and this pair of basic residues are important in determining the magnitude of the nNOS inhibition All three groups of nNOS active amphibian peptides (Table 1) are unique new molecular probes for the functioning of nNOS We intend

to carry out further structural studies on each of the three groups of active peptides listed in Table 1, in order to probe the relationship between sequence, solution structure (determined by NMR) and nNOS inhibition

The final question to be addressed is what role do these active peptides have in the amphibian integument? The glandular secretion of the animal is exuded onto the skin (and into the gut) when the animal is stressed, sick, or under attack Logically, there are two possibilities, either the nNOS inhibitor is playing some regulatory role in the animal and/or it is acting as part of the primary host defence arsenal against predators

Consider the first possibility The nNOS used in this investigation is from rat Although the sequence of nNOS in anurans has not been determined, it is likely to be very similar to that of rat nNOS as the sequences of nNOS so far determined (from man, rat, rabbit and snail) are very similar [S51] We have shown that L lesueuri secretes two neuro- peptides onto its skin The first of these, caerulein 1.1 is a known host defence peptide of many anurans [14,8] It has

a multifaceted role including smooth muscle activity (at ngkg' body weight), which makes it a powerful toxin against predators, it has potent analgaesic properties and it

is also thought to be a hormone involved in the hibernation cycle of anurans [67] The other neuropeptide, lesueurin, inhibits the formation of nitric oxide by nNOS Nitric oxide has many roles in animals and plays important roles in the

nervous, muscular, cardiovascular and immune systems

[68] In anurans, nitric oxide is already known to be involved

in sight [69], reproduction [70] and modulation of gastric

acids [71] It is possible that together with caerulein 1.1,

lesueurin has a role in either stress control and/or temper- ature control

The other scenario is that nNOS inhibitors are front line defence compounds We have now identified nNOS inhib- itors as major skin peptides in 10 out of 12 studied species of frogs of the genus Litoria These compounds are all active at

micromolar-concentrations A predator ingesting even a

small amount of the anuran skin secretion could be seriously

affected if even part of its nitric oxide messenger capability is

reduced The predator could either be large, or small

(bacteria have recently been shown to contain NOS

[72-75])

In conclusion, most frogs of the genus Litoria secrete a

cocktail of bioactive peptides onto their skin, some of which

are cytotoxic and antibiotic, and others which regulate the

neuronal isoform of the enzyme NOS We propose that this

effect on nNOS is mediated by allosteric modulation of

arginine catalysis, through an effect on Ca? * calmodulin

binding to nNOS We do not believe this is unexpected

bioactivity The peptides that inhibit the formation of nitric

oxide from nNOS either play a role in the fundamental

physiology of the animal, and/or are part of the defence

arsenal to combat attack by predators, both small and large

ACKNOWLEDGEMENT

Amphibian experiments and peptide syntheses were funded by the

Australian Research Council

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