Chris and Xavier, Institut Pasteur, 12/1995
Summary
The acetylcholinesterase (AchE) from the venom of the Central Asian cobra Naja naja oxiana is highly insensitive towards the inhibitor fasciculin. Starting from partial amino-acid sequence data, its complete sequence was determined by cDNA sequencing. The modification by site-directed mutagenesis of some peripheral site residues yielded an enzyme form which is about four oders of magnitude more sensitive to fasciculin than wild type Naja naja AChE.
General background
Acetylcholinesterase (AChE) is the most prominent member of the protein family of cholinesterases. Its main function is signal termination at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The primary structure of AChE from several animal species is known; the enzyme isolated from the electric organ of Torpedo californica has been crystallized, and the crystal structure has been resolved. Its main characteristic is its active site, which is buried in the middle of the globular protein at the bottom of a deep gorge that is lined by an array of aromatic residues. Efficient catalysis is brought about by a catalytic triad similar to the one known from serine proteases, and the substrate specificity is achieved by a tryptophan residue (W84) which had earlier been predicted to be near the active site ("anionic subsite") by affinity-labelling studies (Weise at al, 1990). The knowledge of the three-dimensional structure has fuelled a series of mutational studies the outcome of which has been to establish the importance of the various aromatic residues.
For further information on AChE have a look at the ESTHER data base.
Snake venom AChE
Another rich source of AChE is the venom of Elapidae snakes which, in
addition to the toxic components directed at the neurotransmitter receptor,
contains a set of anabolic enzymes that probably serve to deliver the toxins
to their target molecules. AChE has been purified from the venom of the
Central Asian cobra Naja naja oxiana by affinity chromatography
(Raba et al., 1979), and its catalytic properties have been described in
some detail. Altogether, they are rather similar to those of the Torpedo
enzyme, with the notable exception of substrate inhibition, which is markedly
decreased in cobra AChE. In the Torpedo enzyme, substrate inhibition seems
to be mediated by a region that is located on the rim of the gorge and
forms an additional binding site for AChE; this region seems to be different
in cobra AChE (Kreienkamp et al., 1991). A systematic study into the properties
of AChEs from different Elapidae venoms was performed recently and
the AChE from the venom of Bungarus fasciatus has been cloned and
sequenced (Cousin, X. et al, to be published) at the Institut Pasteur in
Paris.
In contrast to other AChEs, snake venom AChE is monomeric (Cousin,
X., Créminon, C., Grassi, J., Meflah, K., Cornu, G., Saliou, B.,
Bon, S., Massoulié, J., and Bon, C., FEBS Lett. 387:196-200,
1996) ; monomeric forms find increasingly wide use as a model systems in
biochemical and biophysical experiments on AChE (e.g. Porschke et al.,
Biophys.J. 70, 1603-1608, 1996).
Particularly interesting for the studies performed here is the observation that the sensitivity of snake venom AChE from different snake species towards fasciculin, a peptidic toxin purified from the venom of the mamba Dendroaspis, varies greatly following the species (Frobert, Y., Créminon, C., Cousin, X., Rémy, M. H., Chatel, J. M., Bon, S., Bon, C. and Grassi, J. Biochim. Biophys. Acta 1339: 253-267, 1997). In particular, AChE from the venom of the cobra Naja sp. is almost insensitive to this peptidic inhibitor (with an IC50 value >> 10-5 M).
Preparatory protein-chemical work
Partial sequence data including the N terminus and the surroundings of the active serine residue of Naja naja AChE were obtained in 1990 (Weise, 1990). Since then, we have continued the protein-chemical analysis; up to now, peptides covering more than 70% of the total sequence have been analysed by combining Edman sequencing and peptide mass spectrometry. In the course of these studies, we have also obtained some information on disulfide bridges and the sites of post-translational modification (N-glycosylation) which may be helpful for the understanding of structural data. The cobra AChE sequence is highly similar to the Torpedo sequence (about 65% sequence identity, which is very promising for homology modelling studies), but sufficiently different to expect interesting structural differences.
Results
This work was presented at the 6th cholinesterase meeting (March 1998, San Diego, USA) and at the Xth International Symposium on Cholinergic Mechanisms (September 1998, Arcachon, France). It is to be published in a biochemical journal.
Part 1: Cloning of Naja naja oxiana AchE
During a stay of Chris Weise at the Institut
Pasteur, Paris (Unité des Venins, Prof.
Cassian Bon) in autumn 1995, sponsored with an EMBO short term fellowship),
PCR cloning was tried directly on DNA reverse-transcribed from total RNA
of the Naja naja venom gland, using a set of specific oligonucleotides
made in the Paris laboratory from the previous cloning work on AChE from
the venom of Bungarus fasciatus. In this way the complete sequence of Naja
naja AchE was established. The peptidic sequence AChE (538 amino-acid residues)
is 93 % identical to that of Bungarus AchE
In an earlier paper (Cousin, X. et al., J.Biol.Chem. 271, 15 099-15 108, 1996), it had been shown that the low sensitivity to fasciculin of AChE from the venom of the krait Bungarus fasciatus (bfAChE) can be attributed to the presence of a methionine residue in position 70 and to a lysine residue in position 285, in place of a tyrosine and an acidic residue, respectively.
Naja AchE shares the features explaining the lower sensitivity of bfAChE
to fasciculin (M70, K285), and additional differences are observed mainly
in the peripheral site region, rendering that portion of the molecule more
positively charged in Naja naja AChE.
Chris and Xavier, Institut Pasteur, 12/1995
Part 2: Site-directed mutagenesis - Identification of residues
involved in insensitivity towards fasciculin
This part of the work was carried out in autumn 1997 at INRA Montpellier
(Unité de Différenciation Cellulaire et Croissance) in collaboration
with Xavier Cousin.
The modification by site-directed mutagenesis of these peripheral site residues to their counterparts in Torpedo or Bungarus AChE greatly increases the sensitivity of Naja naja AChE to fasciculin. We generated a triple mutant which, at low ionic strength, is about four oders of magnitude more sensitive to fasciculin than wild type Naja naja AChE.
This shows that point mutations affecting charges in the peripheral site region have a pronounced effect on AChE sensitivity towards fasciculin and clearly demonstrates the importance of electrostatic interactions in the interplay between AChE and this peptide inhibitor, although the complex was described as mainly stabilized by hydrophobic interactions.
Moreover for this mutant, the inhibition by gallamine is strongly enhanced,
whereas the inhibition by propidium and bis-quaternary compounds is almost
not affected. This shows that the area around the mutated residues forms
a subsite essential for the binding of gallamine.