Amphibian peptides that inhibit neuronal nitric oxide synthase
The isolation of lesueurin from the skin secretion of the Australian Stony
Creek Frog
Litoria lesueuri
Jason Doyle
1
, Lyndon E. Llewellyn
1
, Craig S. Brinkworth
2
, John H. Bowie
2
, Kate L. Wegener
2
, Tomas Rozek
2
,
Paul A. Wabnitz
2
, John C. Wallace
3
and Michael J. Tyler
4
1
Australian Institute of Marine Science, Townsville MC, Queensland, Australia;
2
Departments of Chemistry,
3
Molecular Biosciences
and
4

M
concen-
trations): these are (a) the citropin/aurein type peptides
(of which lesueurin is a member), e.g. citropin 1.1
(GLFDVIKKVASVIGGL-NH
2
)(8.2l
M
); (b) the frenatin
type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG
GLFKPK-OH) (6.8 l
M
); and (c) the caerin 1 peptides, e.g.
caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH
2
)
(1.7 l
M
). From Lineweaver±Burk plots, t he mechanism of
inhibition is revealed as noncompetitive with respect to the
nNOS substrate arginine. When the nNOS i nhibition tests
with the t hree p eptides outlined above w ere c arried out in the
presence of increasing concentrations of Ca
2+
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
2+

caerin 1.1 G LLSVLGSVAKHVLPHVVPVIAEHL-NH
2
;
citropin 1.1 GLFDVIKKVASVIGGL-NH
2
;aurein1.2
GLFDIIKKIAESF-NH
2
.
Aurein 1.2 contains only 13 a mino-acid residues, and is
the smallest peptide from an anuran reported to have
signi®cant antibiotic activity. The aurein peptides have also
been shown to exhibit anticancer activity in tests carried ou t
by the N ational Cancer Institute ( NCI, Washington DC,
USA) [12].
The s olution s tructures o f t he antibiotic (and anticancer
active as appropriate) peptides shown above have been
investigated by NMR spectroscopy. In tri¯uoroethanol/
water mixtures, caerin 1.1 adopts two well-de®ned helices
(Leu2 to Lys11 and from Val17 to His24) separated by a
hinge region of less-de®ned helicity and greater ¯exibility,
with hydrophilic and hydrophobic residues occupying well-
de®ned zones [ 13]. The central hinge region is n ecessary for
optimal antibiotic activity [13]. Similar NMR studies of
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: [email protected]
Abbreviations: FAD, ¯ avin adenine dinucleotide, oxidized form;
FMN, ¯avin mononucleotide; IC

or neuromodulators [22], at least partly because they s how
structural similarity to the human brain endomorphins (e.g.
YPWG-NH
2
) [23]. The sequences of two tryptophyllins are
as follows: tryptophyllin L1, FPWL-NH
2
; tryptophyllin
L2, pEFPWL-NH
2
.
Even more unusual are the marsh frogs o f the Limno-
dynastes genus. These produce only minute amounts of
anionic skin peptides, none of which are post-translationally
modi®ed, or s how neur opeptide or antimicrobial activity
[24,25]. A particular example, dynastin 1 (from Limnodyn-
astes interioris) [ 24] has t he sequence GLLSGLGL-OH.
In this paper, we describe the isolation, sequence deter-
mination, and activities of t wo bioactive p eptides f rom the
skin glands of the stony creek frog Litoria l esueuri. We have
not studied a member of the L. lesueuri 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 t hat i nhibits the formation of nitric oxide by
neuronal n itric oxide synthase (nNOS). We next examined
other p eptides isolated earlier from a nurans of t he genus
Litoria. This has led to the discovery of three types of
amphibian peptides that inhibit nNOS.
MATERIALS AND METHODS

)1
. T he eluant was
monitored by ultraviolet absorbance at 214 nm using an ICI
LC-1200 variable wavelength detector (ICI Australia,
Melbourne, Australia). The r esultant HPLC trace shows
two major peaks (Fig. 1). The two fractions were collected,
concentrated and d ried in vacuo. T he ®rst major fraction
(Fig. 1B) (50 lg) contained the known neuropeptide caeru-
lein 1.1, identi®ed b y HPLC a nd mass spectrometry [27].
The second major f raction (Fig. 1C) contained 10 lgofa
new peptide, called l esueurin.
Sequence determination of lesueurin
Electrospray mass spectrometry. Electrospray mass spec-
tra were determined u sing a F innigan LCQ ion t rap mass
spectrometer. Puri®ed fractions from the HPLC s eparation
were dissolved in m ethanol/water (1 : 1 , v/v) a nd infused
into the electrospray source at 8 lLámin
)1
. Electro spray
conditions were as follows: n eedle potential 4.5 kV, tube
lens 60 V, heated capillary 200 °C and 30 V, sheath gas ¯ow
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 a s
described p reviously [29] using an applied Biosystem 492
Fig. 1. H PLC separation of the glandular secretion of Litoria lesueuri.

)1
against any of
these organisms.
Anticancer activity testing. Synthetic lesueurin showed no
activity below 10
)4
M
in the Ô60-human tumour line testing
programÕ of the US NCI (Washington) [12].
Neuronal nitric oxide synthase inhibition. Inhibition of
nNOS was measured by monitoring the conversion of
[
3
H]arginine to [
3
H]citrulline. In brief, this involved incuba-
tion of a homogenate o f r at cerebella (which had endogenous
arginine removed b y ion exchange chromatography) i n a
reaction buffer (33 m
M
Hepes, 0.65 m
M
EDTA, 0.8 m
M
CaCl
2
,3.5 lgámL
)1
calmodulin, 670 l
M

rulline. After repeated vortexing, this slurry was centrifuged
at 1200 g for 10 min, and 70 lL of supernatent was removed
and the [
3
H]citrulline measured b y scintillation c ounting.
Peptides selected for f urther examination t o d etermine the
mechanism of i nhibition were assayed in t he same reaction
buffer as used for initial screening except that it contained
30 n
M
[
3
H]arginine supplemented with 0.3±13.3 m
M
argi-
nine. Peptide concentrations used are given in the legend t o
Fig. 4.
Data analysis for nNOS studies. Peptide inhibition curves
were ®tted using the curve-®tting routin e of
SIGMAPLOT
(SPSS, Chicago, IL, USA) with the variation o f the Hill
equation: fmol [
3
H]citrulline production  1/(1 + [inhibi-
tor]/IC
50n
), where I C
50
is the c oncentration a t w hich the
peptide causes 50% inhibition and n is the slope of the curve

of the enzyme (5 lL; 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 av eraged, a djusted t o the change in A per
minute and corrected for background A (negative control).
Percent of control w as then calculated as (A
405 test sample
/
A
405 positive control
) ´ 100.
RESULTS
L. lesueuri, usually called either Lesueur's Frog or the
Stony Creek Frog, has varied do rsal colouration ranging
fromyellowtobrown,withablackheadstripefromthe
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 t wo
distinct populations of this frog, one con®ned 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].
AsinglespecimenofL. 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

GLLDIL KKV GKVA- NH
2
.
Lesueurin h as no antibiotic activity (at minimum
inhibitory concentration values below 100 lgámL
)1
)
against the nine bacteria we use in our test regime. Also,
lesueurin exhibited no cytotoxity at concentrations less
than 10
)4
M
against any of the 60 human tumour lines in
the NCI test regime. Lesueurin was, however, shown to
inhibit nNOS with an IC
50
of 16.2 l
M
(Table 1) . This
Fig. 2. Ele ctrospray mass spectrum (MS/MS)
of the MH
+
ion of lesueurin. Masses shown in
this spectrum are nominal m asses. Data from
B fragmentations give sequence information
from the C-terminal end of lesueurin (see
schematic arrows above the spectrum). D ata
from Y + 2 f ragmentations give sequence
information from the N-terminal end of the
peptide ( see schematic arrows below the

Trytophyllin L3.1
b
L. rubella [17] FPWP-NH
2
NA NA +1
Electrin 2.1
c
L. electrica [20] NEEEKVKWEFPDVP-NH
2
NA NA )2
Caeridin 3
d
L. caerulea [8] GLFDAIGNLLGGLGL-NH
2
NA NA 0
Maculatin 1.3
e
L. eucnemis
f
GLLGLLGSVVSHVLPAITQHL-NH
2
NA NA +1
Inhibitor Group 1
Lesueurin L. lesueuri
g
GLLDILKKVGKVA-NH
2
16.2 1.5 +3
Aurein 1.1 L. aurea [11] GLFDII KK I AES I-NH
2

2
1.7 3.7 +3
Caerin 1.9 L. chloris [8] GLFGVLGSI AKHVLPHVVPVIAEKL-NH
2
6.2 2.2 +2
a
Inactive at 13.3 lgámL
)1
.
b
Inactive at 33.3 lgámL
)1
.
c
Inactive at 66.7 lgámL
)1
.
d
Inactive at 133.3 lgámL
)1
.
e
Caused 46.4% inhibition at
31.4 l
M
. Full IC
50
determination was not possible because of solubility.
f
Brinkworth, C., Bowie, J.H., Wallace, J.C. & Tyler, M.J.

nNOS may retain endogenous calmodulin rendering it
unsuitable f or a Michaelis±Menten study of enzyme kinet-
ics, as was carried out with ar ginine.
An experimental procedure was used to gauge the
potential for selected peptides to inhibit nNOS by displacing
Ca
2+
calmodulin from the calmodulin b inding domain of
nNOS. In this p rocedure, the nNOS inhibition experiments
were carried out with citropin 1.1, frenatin 3, and caerin 1.9
with added Ca
2+
calmodulin to determine the in¯uence o f
the calmodulin on th e inhibition of nNOS. These experi-
ments measured nNOS activity in the presence of
142.9 lgámL
)1
of the a ctive p eptide (about 10-fold greater
than the IC
50
value of each peptide), with the Ca
2+
calmodulin concentration in the assay buffer increased 100-
Fig. 3. Inhibition of nNOS exempli®ed by (A)
lesueurin (circles) and frenatin 3 (squares), and
(B) citropin 1.1 (inverse triangles) and caerin
1.9 (triangles). These p ept ides represent each
of the groups of peptide inhibitors listed in
Table 1. Curves are drawn to the Hill equa-
tion, and the values f or the I C

Selected peptides were tested for t heir ability t o inhibit
calcineurin, another calmodulin dependent enzyme.
Lesueurin at 7 4 l
M
(4.6-fold g reat er tha n t he I C
50
against
nNOS) inhibited c alcineurin by 33%. C itropin 1 .1 reduced
calcineurin a ctivity b y 34% at 31 l
M
(3.8-fold h igher t han
the IC
50
against nNOS) but at double this concentration (i.e.
7.6-fold greater than its IC
50
against nNOS), calcineurin was
inhibited by 96%. Frenatin 3 inhibitied calcineurin by 38%
at 46 l
M
, a concentration that is 6.8-fold g reater than the
IC
50
against nNOS. Finally, caerin 1.9 inhibited c alcineurin
by 48.1% at 19.3 l
M
(4.8-fold more concentrated than its
IC
50
against nNOS).

inhibits the f ormation of the chemical messenger nitric
oxide.
Testing of other am phibian peptides indicated that there
are three well-de®ned groups of basic peptides that i nhibit
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,
signi®cant anticancer activity. These peptides are amphi-
pathic ahelices, 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
2
H group together with two lysine residues near the
C-terminus. T hese peptides show little or no antibiotic and/
or anticancer activity; (c) those caerins 1, particularly those
containing Phe3. These molecule s are also potent mem-
brane-active antibiotics, and NMR studies show they have
two a helical reg ions separated by a central ¯exible h inge
region [13]. How do these three seemingly unrelated groups
of peptides inhibit t he formation of n itric 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 th e most c omplex enzymes known
(for nNOS see [ 37,38]). By a complex sequence involving
binding sites for a number of cofactors including heme,
tetrahydrobiopterin, F MN, FAD and NADPDH, nNOS

(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 t hese plots is typical of
noncompetitive inhibition, and so these peptides are
unlikely to directly involve the arginine substrate site [33].
In its simplest de®nition, noncompetitive inhibition is when
an inhibitor binds at a site other than the active site,
changing the e nzyme±substrate af®nity.
These four peptides, lesueurin, citropin 1.1, frenatin 3
and c aerin 1 .9, a nd pr obably t he o ther active amphibian
peptides of inhibitor groups 1±3, m ust therefore inhibit t he
formation of nitric o xide by either blocking one or more of
the cofactor s ites on nNOS or by some chemical modi®ca-
tion reaction with nNOS that alters the activity of the
enzyme. A n obvious example o f b locking a cofactor s ite
would be if the amphibian peptide reacts with the regulatory
enzyme Ca
2+
calmodulin, t hus changing the t hree-dimen-
sional structure and preventing its attachment to the
calmodulin binding site on nNOS. There are examples of
small basic peptides, often a helices, being captured and
enclosed within Ca
2+
calmodulin, and as a consequence
changing the three-dimensional shape of the calmodulin
[45±48], but nNOS deactivation by these peptides has, to
Table 2. Se quen ce identity of lesueurin and aurein 1.1.
Peptide Sequence
Lesueurin

maximum r eduction of inhibition under t he experimental
conditions used is 50%.
The enzyme Ca
2+
calmodulin regulates not only nNOS
but also a number of other enzymes including calcineurin. If
the active amphibian pep tides are i ndeed interacting with
Ca
2+
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 concen trations lower t han t hose obtained f or
nNOS (Table 1). Even so, the f act that all four peptides
inhibit both n NOS and calcineurin enzymes, provides
credence to the proposal that the amphibian peptides are
affecting the Ca
2+
calmodulin interaction with nNOS. This
is an interesting observation because sequences of the
Ca
2+
calmodulin binding sites of nNOS and calcineurin are
quite different ( see below [51]), even though they have been
classi®ed a s belonging t o the s ame class o f enzymes [52].
The sequences are nNOS (rat, human) and calcineurin A
(rat, human), respectively are a s follows:
KRRAIGFKKLAEAVKFSAKLM
RKEIIRNKIRAIGKMARVFSVLR.
Currently, although three-dimensional structures o f c er-

2+
/calmodulin binding domain of nNOS, (c) w ith t he
sequence of Ca
2+
/calmodulin itself, and (d) with o ther
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 signi®cant homology of sequences
within each of the active groups shown in Table 1. Consider
group 1 (Table 1) as an example. All peptides of group 1
have a post-translational mo di®cation in that the
N-terminal group is -CONH
2
. Lesueurin has high homol-
ogy w ith some of the citropins and aureins. It i s s everal
residues shorter that aureins 2.2, 2.3, 2.4 and citropin 1.1, all
of which have IC
50
values more potent than that of
lesueurin. Aurein 1.1 has the same number of residues as
lesueurin but a l ower overall positive charge, and is not as
effective as lesueurin in inhibiting nNOS. The length of these
peptides seems t o b e a factor in determining t heir activity.
Another important feature conserved in this group of
peptides is the cen tral Lys-Lys pair and the b asic nature of
the peptides. It appears that the length of the peptide and
this pair of basic r esidues are important in determ ining the
magnitude of the nNOS inhibition. All three groups of

has many roles in animals and plays important roles in t he
nervous, muscular, cardiovascular and immune systems
[68]. In anurans, nitric oxide is already known to be involved
in sight [ 69], reproduction [70] and modulation o f gastric
acids [71]. It is possible that together with caerulein 1.1,
lesueurin has a role in e ither stress control and/or temper-
ature control.
The other scenario is that nNOS inhibitors are front line
defence c ompounds. W e have now identi®ed nNOS i nhib-
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
106 J. H. Bowie et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 c ytotoxic and antibiotic, and others w hich regulate the
neuronal isoform of the enzyme N OS. We propose t hat this
effect on nNOS is mediated by allosteric modulation of
arginine catalysis, through an effect on Ca
2+
calmodulin
binding to nNOS. We do not believe this is unexpected
bioactivity. The peptides that inhibit the formation of n itric
oxide from nNOS either p lay a role in the fundamental
physiology o f the animal, and/or are part o f t he defence

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