Download Cantharidin, another natural toxin that inhibits the

Volume 330, number 3, 283-286
0 1993 Federation of European Biochemical
FEBS 12983
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September 1993
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Cantharidin, another natural toxin that inhibits the activity of
serinelthreonine protein phosphatases types 1 and 2A
Richard E. Honkanen
Depurtrnent of Biochemistry, College of Medicine. University of South Alabarnu, Mobile, AL 36688, USA
Received 22 June 1993; revised version received 26 July 1993
Cantharidin,
a natural toxicant of blister beetles, is a strong Inhibitor of
cantharidin
inhibits the activity of the purified catalytic subunit of PPZA
,uM) and only inhibits the activity of protein phosphatase
type 2B (PP2B)
cell homogenates
indicate that cantharidin
also inhibits the native forms
available, may be useful as an additional probe for studying
Canthandin:
Serinejthreonine.
Protein:
protein phosphatases
types l(PPI) and 2A (PPZA). Like okadaic acid,
(IC,, = 0.16 PM) at a lower concentration
than that of PPl (IC,,, = 1.7
at high concentrations.
Dose+mhibition
studies conducted with whole
of these enzymes. Thus, cantharidin.
which 1s economical
and readily
the functions of serinelthreonine
protein phosphatases.
Phosphatase:
1. INTRODUCTION
Mylabris, the dried body of the Chinese blister beetle
(Mylabris phalerata or M. cichorii), can be traced back
more than 2,000 years as a traditional
medicine
in
China and is still used as a folk medicine in Asia today
[l]. The active constituent
of mylabris, cantharidin
(exo,
exo-2,3-dimethyl-7-oxabicyclo[2.2.l]heptane-2.3-dicarboxylic acid anhydride) (Fig. l), is produced by as many
as 1500 different species of blister beetles, with the
Spanish fly Cantharis vesicatoria probably
being the
best known source [l-3]. Cantharidin
also has a history
of medical usage in Europe where it was first isolated
in the early 1800’s as the active principle of a blister
beetle extract [3]. Although
occasionally
utilized as a
topical vesicant for the removal of warts [4], cantharidin
was considered as obsolete and too toxic for use as an
internal medicine by the early 1900’s. Nonetheless, folklore surrounding
the purported
properties of ‘Spanish
fly’ as an aphrodisiac
and abortifacient
continues
to
result in human poisonings
today, with 26-50 mg estimated as a lethal dose for humans [5]. Livestock toxicosis due to the consumption
of feed containing
blister
beetles also continues to present a problem for ranchers
Inhibitor:
Dephosphorylation:
Toxin
years. Early studies indicated
that cantharidin
interfered with mitochondrial
respiration
[ 12,131 or induced
the activation of an acantholytic
factor [S]. Cantharidin
was then shown to bind with high affinity to a specific
and saturable binding site in a cytosolic fraction produced from mouse liver [14.15]. This high-affinity
cantharidin binding site was subsequently
identified in several other tissues including:
heart. kidney, lung, pancreas, skin. spleen, brain, blood and stomach [15]. Recently, Li and Casida [ 161 purified the cantharidin-binding protein (CBP) to homogeneity,
identifying it as protein phosphatase
type 2A (i.e. the amino acid sequences
of seven peptides from CBP were identical with deduced
amino acid sequences for PP2A,. CPB had phosphorylase a phosphatase
activity which was inhibited by both
okadaic acid and cantharidic
acid, and okadaic acid
inhibited the binding of [‘Hlcantharidin
to CBP).
The present report characterizes
the inhibitory activity of cantharidin
on the activity of the purified catalytic
subunits of serine/threonine
protein phosphatases
types
1 (PPl ), 2A (PP2A) and 2B (PP2B) and in whole cell
homogenates
of PC1 2 cells. These studies indicate that,
like okadaic acid. cantharidin
is a strong inhibitor
of
PPI and PP2A and a weak inhibitor of PP2B.
F51.
The toxic properties of cantharidin
are well characterized. Dermal contact produces blisters characterized
by a distinctive vesiculobullous
eruption [7,8]. Oral ingestion leads to severe irritation
and ulceration
of the
gastrointestinal
and urinary tract epithelial linings [3,9],
and intraperitineal
injection causes severe congestion
and edema of the liver, with a LD,, = 1 mg/kg [lO,ll].
The cellular mechanisms
underlying
the toxicity of
cantharidin
have been under investigation
for several
Publmhed by Elsevier Science PuhlisherJ B I!
2. MATERIALS
AND
METHODS
Phosphorylase
kmase (EC 2.7.1.38) and protein kinase A (3’.5’cyclic AMP-dependent
protein k1nase) from rabbit muscle. histone
(type 2AS). PP2B (calcmeunn)
and calmodulin
(phosphodiesterase
3’:5’-cyclic nucleotide activator) from bovine brain. andp-nitrophenyl
phosphate
(PNPP) were obtained from Sigma Company and utilized
without further punfication.
Cantharidin
was purchased from Sigma
283
Volume 330. number
3
FEBSLETTERS
September
1993
chetmcal company and exhIbIted >98% purity by CC/MS analysis.
Okadalc acid was generously provided by Dr. R. Dickey.
2.2. Detrrrmnatmn ojprotem phosphatase uc’trvrt?,
The catalytic subunIt of PPI and PPZA were purified to apparent
homogeneity.
demonstrating
a single band upon SDS-PAGE and silver stainmg. as described by Honkanen et al. [I 7-l 91. Inhibition assays
conducted with purified PPI and PP2A contained
125 pM PPI or 25
pM PP2A having phosphohistone
phosphatase
activity of 265 + 15
and 1250 + 62 nmol/mm/mg
of protein, respectively. Phosphohistone,
specific activity 4.5 x 1O6dpmlnmol
of incorporated
phosphate,
was
prepared by the phosphorylation
of lustone (type 2AS from Sigma)
with CAMP-dependent
protein kinase (protein kinase A) m the presence of [y-“P]ATP
(150 ,uM ATP) as described m detail previously
[18]. Protein phosphatase
activity against phosphohistone
was determmed by the liberation of “P usmg either the purified catalytic subunits of the mchcated protein phosphatases
(Figs. 2 and 3) or a dilute
whole cell homogenate
of PC12 cells (Fig. 4). The dephosphorylation
of substrate was c 10% of the total, and reactions were linear with
respect to enzyme concentration
and time [I 81. “P-phosphate
liberated
by the enzymes was extracted as a phosphomolybdate
complex and
measured according
to the methods of Kilhlea et al [20]. Inhibition
of phosphatase
activity was determined
bq the addition of toxins to
the assay mixture IO mm before Initiation of the reaction with the
addition of substrate
[IS]. Okadaic acid and cantharidin
were &ssolved m dlmethyl formamide
(DMF), which was added in equal
amounts to controls. PP2B activity was measured using PNPP as a
substrate m the presence of calmodulin
and Ca”. as described previously [18].
Previous experiments
have indicated that for compounds
that behave as ‘tightly binding inhibitors’ the determination
of K, by fitting
dose-mhlbitlon
relatlonshlps
to the theoretical
function by an ordinary nonlinear least-squares
methods does not provide reliable confidence hmits [21]. However, as demonstrated
for okadaic acid, the
dose-inhibitlon
of ‘tightly bmdmg Inhibitors’ is well described by the
followmg equation, derived with steady-state
assumptions,
s = [(I?,-1,-K,)
+ d(E,-1,-K,)’
+ 4 E;KJ
/2E,
(1)
Cantharidin
Fig.
1 Structure
of cantharidin.
at a >500-fold higher concentration
than that which
inhibits PPl (Fig. 2). Under identical assay conditions
okadaic acid inhibits the activity of PP2A (IC,,-0.04 nM)
at a > 1,OOO-fold lower concentration
than cantharidin
(Fig. 3).
To determine whether cantharidin
inhibits the activity of the native forms of PPl and PPZA at a comparable concentration
to that observed with the respective
purified catalytic subunits. we examined the effects of
cantharidin
in whole cell homogenates
of PC12 cells.
Fig. 4 shows the inhibitory
dose-response
of cantharidin and okadaic acid in a dilute whole cell homogenate of PC12 cells (60 ,ug protein/ml).
Both toxins inhibited the divalent cation-independent
protein phosphatase activity (predominately
PPl and PP2A) in the
whole-cell homogenate,
with 0.1 ,uM okadaic acid and
100pM cantharidin
causing 100% inhibition. Nonlinear
regression analyses of the dose-inhibition
curves obtained with both okadaic acid and cantharidin
indicate
that the divalent cation-independent
phosphatase
activity contained
in the PC12 cell homogenate
best fits a
where s stands for the fractional activity v,/v,, K, 1s the dissociation
constant, and E, and Z, are the total concentration
of the enzyme and
inhibitor.
respectlvelq
[21] For the set of observations
(1,. .u) and
(I’,. ~‘1. rearrangement
of equation 1 gives.
-.
I( 1-u)E,
+ ( 1-.Y)K, = YI, and I’( 1-I’)/?,
-PPx?
+ ( 1-.C)K, = ?.I,’
\
K, values were estimated as means of the solutions obtained by solving
the simultaneous
equations for all possible sets of observations
[?I].
This procedure
is the direct-linear
plot method for estimation of enzyme-kinetic
constants
[22] as modified by Takai et al. [21]. Theoretlcal hmlts of this application
have been discussed previously [21,23].
‘.
0
3. RESULTS
The dephosphorylation
of phosphohistone
by the purified catalytic subunits of both PPl and PPZA is inhibited by cantharidin,
which also inhibits
the activity
of PP2B at higher concentrations
(Fig. 2, Table I).
Cantharidin
inhibits
the activity of PPl and PP2A
in a dose-dependent
manner, inhibiting
PP2A (IC,,, =
0.16 5 0.06 PM; mean +_S.D., n = 4) at a 10.6- fold
lower concentration
than PPl(IC,,
= 1.70 2 0.5 PM;
mean + S.D.. rz = 4); PPZB is inhibited by cantharidin
284
Y
x
7
-Log
h
[Cantharldin;
5
1
3
(M)]
Fig. 2. Inhibition of protem phosphatases
by cantharidm.
The catalytic subunits of PPI and PP2A were assayed using “P-labeled lustone
as a substrate and PPZB using PNPP as a substrate as described in
Section 2. The data is expressed as % of control, with control activity
against phospholustone
bemg 265 + IS and I.250 + 62 nmol/min/mg
protein for PPI and PP2A. respectively. Cantharidm
was mlxed with
the enzymes for IO min at 23°C prior to the mitlation of the reactlon
with the addition of substrate. The data represents the mean ? S.D.
of all data obtained from at least 4 separate experiments employing
two different preparatlona
of each enzyme tested.
FEBSLETTERS
Volume 330, number 3
September 1993
i
12
11
10
9
8
-Log [Toxin;
7
6
5
4
-Log [Toxin;
(M)]
Fig. 3. Comparison of the inhibttory effect of okadaic acid and cantharidin on the activity of the purified catalytic subunit of PPZA from
muscle. Assays were conducted
as described
in Figure 2 using
phosphohistone
as a substrate and with enzymes having comparable
nntial activity. Both toxms were mixed with the enzyme 10 min at 23°C
prior to the addition
of substrate.
Each point represents
the
mean 2 SD. (n = 4).
(M)]
Fig. 4. Effect of okadatc acid and canthartdm on protein phosphatase
activity contamed in a homogenate
of PC12 cells. Cells from a single
-50% cont’ment IOO-mm dish were rinsed with 4°C phosphate-buffered
saline, scraped and somcated in 1 ml of Trts-buffer (20 mM Tris-HCl.
1 mM EDTA and 2 mM dithiothreitol.
pH 7.4). The homogenate
was
then diluted below the tttration end point (the concentratton
after
u hich further dilution no longer affects the IC,,, [l&19]. -6Opg protein/
ml) and assayed for phosphohtstone
phosphatase
acttvtty as described
in Fig. 2 Each pomt represents the mean f S D. of three experiments
conducted
in triplicate.
two-site model, which is consistent with that predicted
for an assay containing
a mixture of both PPl and
PP2A [l&19].
tautomycin,
a dialkylmaleic
anhydride
produced by a
strain of Streptonzyces spiroverticillutus [29-311.
Like okadaic acid and the cyanobacterial
toxins (micorcystins and nodularin),
the inhibitory
effect of cantharidin
differs substantially
among the cantharidinsensitive phosphatases.
The purified catalytic subunit of
PP2A from muscle is inhibited by cantharidin
at a concentration
that is 5-lo-times
lower than that required
to inhibit the activity of purified PPl (Table I, Fig. 2).
With okadaic acid, PP2A is inhibited at a lo-lOO-fold
lower concentration
than PPl [18,24,25], while both microcystin-LR
[19,26] and nodularin
[18] are 460-fold
more potent inhibitors
of PP2A than PPl. However,
due the extremely high apparent affinity of microcystinLR and nodularin
for both PPl and PP2A, the difference in the relative potency of these compounds
can
only be observed in very dilute or high volume assays
employing radiolabeled
substrates with very high spe-
4. DISCUSSION
This study shows that cantharidin,
a natural toxicant
of several species of blister beetles [ 1,3] is a potent inhibitor of serine/threonine
protein
phosphatases
types
l(PP1) and 2A (PP2A) and a weak inhibitor
of the
Ca’+/calmodulin-dependent
phosphatase,
PP2B. Thus,
cantharidin
may be added to a growing list of natural
toxins that strongly inhibit the activity of both PPl and
PP2A which include: okadaic acid, a polyether carboxylic acid produced
by several
species of marine
dinoflagelates
[24,25]; the microcystins,
a family of cyclic heptapeptides
produced by certain strains of cyanobacteria
[ 19,26,27]; nodularin,
a cyclic pentapeptide
produced by Nodzduriu spumigena [18]; calyculin A, a
Spiro ketal obtained
from a marine sponge [28] and
Table I
Inhibition
of PPl and PP2A by cantharidin
PPl
PP2A
Inhtbitor
Canthartdin
The dissociation
constants
confidence (in parentheses);
Substrate
Phosphohistone
K, 01M)
12
0.194
(0.037)
84
K W)
1.11
(0.27)
II
35
PPllPP2A
5.72
(K,) estimated by the dose-inhibition
analysis are listed for PPl and PP2A. The K, values are expressed in,uM with 95%
n denotes the total number of determinattons
utthzed in deriving K, esttmates. and the PPl/PP2A is the ratio of K, values
for PPl to that for PPZA using phosphohistone
as a substrate.
285
Volume 330, number 3
FERS LETTERS
cific activity [18,19]. In contrast, calyculin A inhibits the
activity of both PPI and PPZA at a similar concentration [28], and tautomycin,
which is a relatively unstable
compound
[31], has been reported to inhibit PPl at a
slightly lower concentration
than PPZA [29,30].
Because of the inhibitory
effects it has on PPl
and PP2A, okadaic acid has proven to be a valuable
research tool for determining
the physiological
and
serine/threonine
pathological
roles
of
protein
phosphatases
[24,33]. Cantharidin,
which is readily
available and economical,
may also be useful as a research tool. In dilute whole cell homogenates,
PPl and
PP2A make up the majority of the divalent cation independent protein phosphatase
activity contained in most
mammalian
tissues [32,33]. Like okadaic
acid, cantharidin
completely
inhibits the divalent cation-independent
protein phosphatase
activity contained
in a
crude PC12 cell homogenate,
with essentially complete
inhibition
occurring at a concentration
~100 ,uM (Fig.
4). Therefore, cantharidin
also appears to be an effective inhibitor
of the ‘native forms’ of PPl and PP2A.
Furthermore.
like okadaic acid, the dose-inhibition
relationship
produced with cantharidin
is biphasic, with
characteristics
similar to that predicted from a solution
containing
both PPl and PP2A [18,19]. Nonetheless,
additional experiments addressing the specificity, stability and metabolism
of cantharidin
will be required to
fully assess the value of this toxin as a research tool for
the detection and analysis of protein phosphatases.
Acknotvledgmenrs:
Dr. A. Bencsath. Mass Spectrometry
Center. University of South Alabama, for the GC/MS-analysts
of cantharidin.
Dr.
R.W. Drckey, Fishery Research Branch. Food and Drug Admmrstration, for provtding the okadatc acid used m these studies. and T -S.N
Ku for data entry used in statistical and kinetic analysis Thus mvesttgation was supported
by an Intramural
Research Grant award from
the College of Medicine. University of South Alabama.
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