Tài liệu Báo cáo khoa học: A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken

A synthetic weak neurotoxin binds with low affinity to Torpedo

and chicken a7 nicotinic acetylcholine receptors

Siew Lay Poh1,2, Gilles Mourier1

, Robert Thai1

, Arunmozhiarasi Armugam2

, Jordi Molgo´

3

, Denis Servent1

,

Kandiah Jeyaseelan2 and Andre´ Me´ nez1

1

CEA, Saclay, Gif-sur-Yvette, France; 2

National University of Singapore, Singapore; 3

UPR, CNRS, Gif-sur-Yvette, France

Weak neurotoxins from snake venom are small proteins

with five disulfide bonds, which have been shown to be poor

binders of nicotinic acetylcholine receptors.We report on the

cloning and sequencing of four cDNAs encoding weak

neurotoxins from Naja sputatrix venom glands. The protein

encoded by one of them, Wntx-5, has been synthesized by

solid-phase synthesis and characterized. The physicochemi￾cal properties of the synthetic toxin (sWntx-5) agree with

those anticipated for the natural toxin. We show that this

toxin interacts with relatively low affinity (Kd ¼ 180 nM)

with the muscular-type acetylcholine receptor of the electric

organ of T. marmorata, and with an even weaker affinity

(90 lM) with the neuronal a7 receptor of chicken. Electro￾physiological recordings using isolated mouse hemidia￾phragm and frog cutaneous pectoris nerve–muscle

preparations revealed no blocking activity of sWntx-5 at lM

concentrations. Our data confirm previous observations that

natural weak neurotoxins from cobras have poor affinity for

nicotinic acetylcholine receptors.

Keywords: snake neurotoxins; nicotinic acetylcholine

receptors.

During the past three decades, the most
obvious venom

toxins have been uncovered either because they are present

in large amounts and/or because they have been directly

associated with the search for an important target. At

present, two additional approaches may be considered to

discover new toxin functions. One of them is a proteomic￾type approach, which aims at isolating all components of

the
toxinome [1,2]. The second approach involves investi￾gation of the vast number of venom components that have

already been isolated, and sometimes chemically character￾ized, but whose biological activity still remains mysterious.

These functionally unknown components are often classi￾fied as miscellaneous types of toxins, even though they

usually belong to well-identified structural families [3]. This

is the case of the so-called
weak neurotoxins (Wntxs) found

in elapid snakes and isolated for the first time 26 years ago

from the venom of Naja melanoleuca [4]. Since then, more

such toxins have been isolated [5–19].

The Wntxs possess 62–68 amino acids and belong to the

structural family of
three-fingered folded toxins, which

includes the cardiotoxins, muscarinic toxins, acetylcholin￾esterase inhibitors and the a-neurotoxins that block mus￾cular and/or neuronal nicotinic acetylcholine receptors

(AChRs) [20–22]. The fold adopted by all these toxins is

characterized by three adjacent loops rich in b-pleated sheet,

tethered by four conserved disulphides. A fifth loop is

sometimes observed in the second loop of the a/j-neuro￾toxins and j-neurotoxins [22], where it specifically contri￾butes to the binding of the toxins to the neuronal AChR

[23–26]. Wntxs also possess a fifth disulfide bond, but this is

located in the first loop [16,27,28].

Using Wntxs isolated from venom, it was shown that

these molecules interact with AChRs but with low affinities

[10,29]. Many efforts have been made to obtain pure Wntxs.

However, it cannot be completely ruled out that their low

activity may be due to the presence of minor but highly

potent contaminants, as was previously observed in the case

of j-bungarotoxin [30]. We identified four cDNAs enco￾ding Wntxs in venom glands of the cobra N. sputatrix

(previously known as Naja naja sputatrix [31]) and selec￾ted one of them. Then, we synthesized the correspond￾ing Wntx (Wntx-5) by chemical means, characterized its

physicochemical properties and investigated its biological

properties. We show that sWntx-5 is a weak binder of

muscular-type AChR from Torpedo marmorata’s electric

organ and an even weaker binder of the a7 neuronal-type

receptor from chicken. Our data generally agree with a

report published recently [29]. Moreover, the low AChR

binding activity of Wntx-5 can be accounted for by the

presence of a few residues that are also found in potent

three-fingered snake neurotoxins [22,32,33].

MATERIALS AND METHODS

Materials

Bacterial strains used, JM109 [34] and Epicurean
coli

SURE
cells, were from Stratagene (USA). Oligonucleo￾tides were synthesized at the National University of Singa￾pore. Molecular biology reagents were from Amersham

International Inc. (UK), Promega, New England Biolabs,

Correspondence to A. Me´nez, De´partement d’ Inge´nierie et d’Etudes

des Prote´ines, CEA, Saclay, 91191 Gif-sur-Yvette Cedex, France.

E-mail: [email protected]

Abbreviations: Wntx, weak neurotoxin; AchR, nicotinic

acetylcholine receptor; TCEP, tris(2-carboxyethyl)-phosphine

hydrochloride; Bgtx, bungarotoxin; Ea, erabutoxin a.

Note: The cDNA sequences reported in this paper have the

GenBank accession numbers AF026891, AF026892, AF098923 and

AF098923.

(Received 18 February 2002, revised 17 June 2002,

accepted 12 July 2002)

Eur. J. Biochem. 269, 4247–4256 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03113.x