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Enzyme analysis of Atheris snake venom

Authors:
Report and Alstrwts Xl3
T/w n~woro.uic* ~~I~o.~~J~Io~~~~~I.s~~.~ ‘4. laitr~i~~c!~.lo.\.rrr cd cvwroshr
bird lo
thfirrcl Iri+c!flinir,r prolc~irr crc’cvp1or.c in
Torpedo nmmor:tta
chrric
orgcru. I.
K ri3;tj.‘. J. <ira/yna Faurc.’ F. CiubenSek’ and C. Bon’ (‘Department ol
Biochemistry and Molecular Biology, J. St&n Institute. Jamova 39. IO00 Ljubljana. Sloven& and ‘1JnitC dcs
Venins. Inslitut Pasteur. -_.
-6 Rue du Dr Roux. F7.5724 Paris C’edex 15. France).
This study investigated the binding of radtoiodinatcd ammodytoxin C’ tAlxC), a monomeric phospholipasc A:
(PLA:) neurotoxin from C’&,rcr
W~J~OM~IW.~. and
of crotoxin, a dimcric PLA: neurotoxin from
(hmh cluri.wu.~
/twi/ic.rr.v.
to presynaptic membranes from the electric organ of
Torpcvh ~~wwwruu~.
In both cases. two dilTercnt
f&lics of specilic hinding sites were idcntilicd and charactcrirsd. The high-aflinity binding sites for both toxins
are proteins. In contrast. the low-allinity binding sites were not alrccted by proteinascs. heat or low pH. suggesting
the involvement 01’ certain lipid structures in this t)/pc of specilic binding. By ilHinity-lalxlling. ‘“I-AtxC’ was
shown to be associated predominemly with mcmhranc proleins of apparent mol. \CIS of 70.000 and 2tMt!O and
weakly with several proteins of apparent mol. wt ranging bctwccn 39.000 and
57.000.
““I-Crotoxin
was
mainly
bound to a 48.000 mol. WI memhranc protein. All PI..A?s tcs~cd. except /I-hungarotoxin. inhihitcd the low-allinity
spccitic binding of AlxC. whereas only ncurotoxic PL.A:s ;rnd surprisingly. myotoxic PLA~an;d~guc ammod~tin
I.. were able to prevent AtiC’ and crotoxin high-allinity binding and their cross-linking. The inhihition prolilcs
of high-allinity hindine and cross-linking wcrc quite diITcrcnt for ‘“I-crotoxin
and
for
“‘I-AtxC’. AtxC and
crotoxin did not inhibit each other. indicating that they do not shitre the same high-aflinity binding sites on
Torpdo
memhrancs. In contrait with croloxin. the isolated basic suhunit C’B of crotoxin W:IS able IO inhihi1
completely the high-allinity hinding of “‘I-AIx<‘. Thcreforc. the acidic subunit CA of crotoxin dots not simply
act
;IS
:I chaperone li)r the CB subunit.
IWI
it allo confcrb ;I distinct binding spccilicity
IO
~hc croIoGn.
This work
aas wpportcd
hy the I~.uro~;m Molr~ular
Biology
0r~;mitaIion 6-month fcllo\vhhip to I.K.
li~ro~/jt (!f r/rc lrrrrrrilrg
,spic/cr
Cupiennius S&I I
Crcuich).
L. K uhn-Nentwig and W. F;cntwig (Zoologic;tl Institute.
University of Bcrne I%altzerslrassc 3. 3012 Bcrnc. Swit7erlimd ).
The tropical hunting spider C’qGrurirsr
.ur/vi
is C;IS~ to breed in captivity. lives for a hout I
IO 2
years and produces
a venom which cdn be milked
at
qttnmities of
approx.
IO-IS /tl
every
3 wcckz
from
adult animnl\. Venom
protlucIion depends on age. gender and degree of htmpr. and proh;lbly on reproductive status. Inscwts
:tnd
other
arthropods as the main prey group are sensitive
IO
the venom
itI
astonishingly diltirent IcvcI>. Diptsrnns arc’
among the
most
sensitive
laxa:
crichcts. moths :III~ rome r(xachch arc olu, very scnsitivc. Sonic hcc1lc.c. ro;lches
and mits iirc eutrcniely inscnsitivc 10 lhc vcnoni. ii II lcttctl specish ranging over 4 log units. Our hypolhcsis
H'il!.
thr
this dill&n1 reaction to spider
WIOI~I
is caused h! physiological pn)pcrIics of the hacmol\-mph of the prq
item. C’upicrrrth strki inject5 only s1111111 qu;mtitich 01’ \cnom into a given prey 1~111 is ahlc
ICI cwltrol
the amount
of venom injecIcd. According IO tl~c si/c. ;tcttvity tlr dettinsi\s p”tcnti;ll of the prey iIcm the spider rclcascs
suhscqucntly more
VCIIOIII
quantity.
mc
\~‘noni contmiis zcveral do/en pcptidcs. of which I3
(mol. WI 3I)OO 13.(l(Iol wcrc found
IO
have lo\ic propcrtiex. The
most common toxin.
C’STX-I.
cowrs ;IIWIII
hull
of the tot;ll toxicity and consists of 74 amino ;tcids. C’STX-2 has I3 aniino acids less, the lirst 01 amino acids
hcing complctcly identical to (‘STX-I. Since the IoGiIy of(‘Sl’S-2 is only I;20 ofthat ofCSTX-I il H:IS axsumcd
thaw
the ‘Iail of 13 amino ;icitls and the corr~spontlin$ Isrtiq
l
truc’turc
C;IIISC
tl~c toxicity of this pcpridc. Among
thcsc I3 amino ;Icid$ ilrc scvcn Igsinc. thcrciore Ihis ;miino acid i> cxtrcmcly importmit lirr ~hc toxicity of ~hc
crude venom. The toxic utl‘cct of CSTX-I 1s cauzctl h> ~III inhibition of high-threshold C‘it“ channclh (L-IF~~)
of glut;m~;ltsr~illic synapses (Crux. J.. Lac~rda Hcir;to. p. S. and L.cao. R. M.. unpublished). The crude
~c’nom nl
C. .d,i ill\0
conliiins orhcr non-toxic pcptidss (tmlinc~\r ii function). a vcrl active h! altironid:i~ (spre:ltlinp litctor)
hut no protsasc. amino acids. hist;miinc. free pol\:;tmin~s. other II)M’ mol. u I suhsIitnccs or ions lpossihle
syncrgislic ftmcrions).
/%:,t~rrc irno/,~:ri.\ I)!’ Atherih d/u& rt710/~1. I). M&3.’ A. I-‘:lch’ and H.-W. Herrm;tnn’ (Ventruni dcr
Rechtsmcdirin. Umvcrsity of Frankl’urt. Frankfurt. Cicrmany: mid -Kiilncr A~~uitritm~ am ZOO. C’olognc.
Germany).
The
venom
of the African green hush viper
(Arhri.~ .squtmi.qw)
with l’ound IO exhibit a
xtroug co~~gl~l;mt
;tctivity.
whc’n lihrinogcn \Vas used as subsIrate. cmtsing incoagulahlc blood in a paticn1. Several uthcr venom s;mlplcs
(from
il
*inglc specimen) from .4.
sqmuuigcv
;md
I‘roIn :I. c
Ir/orcdri.v . A. thtri.vi. A. Iri.tpklrrs. .4. r~irshi
and .>I.
.wp*rc~i/itrri.r
wcrc tested for the following enrymc aclivities: t.-;miino acid :)xid;lsc. c;tscin ;md
BAE.E-hldrolysib. kallikrein. pliozphodicsIcr;lse. phospholip;t*c A:. lihrinogcn co;~gulasc. h;teml)rrhagic and
myotosic ;tsIivity. Surprisingly, all venoms tcs~cd posscsscd r~markahly high ccqulant activity on hhrinogcn.
Although
symptoms in hum;m
snakchitc
LIWS rcp~wtcd
in the IiIcruturc *cm to hc rather mild (with the e\ccption
of .q. sc/r/tnrri,gc~ hitch. where
a I;lIal asc
has besn de.scrihed). thchc snakes should hc considered IO be cap;lble
of
causing
pott!ntially severe envenoming. Biochemical stuCicx
OH
the coagulant enqmc and its inhibition by
conimcrci;il ilnli~c’nom~
will hc reported.
... Some victims develop acute renal failure and hypertension [17,18]. However, studies on characterization of venom enzymes to explain these sequelae, are scarce and are incomplete [19]. In fact, even superficial reports on the components of whole venoms are equally rare. ...
Full-text available
Article
Secretory phospholipase A₂ (sPLA₂) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A₂ precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a "shotgun cloning" strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA₂ proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.
Article
Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity. Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.
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