Respective roles of the catalytic domains and C-terminal
tail peptides in the oligomerization and secretory
trafficking of human acetylcholinesterase and
butyrylcholinesterase
Dong Liang
1,2
, Jean-Philippe Blouet
1
, Fernanda Borrega
1
, Suzanne Bon
1
and Jean Massoulie
´
1
1 Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supe
´
rieure, Paris, France
2 Key Laboratory of Brain Functional Genomics, MOE&STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal
University, China
In vertebrates, butyrylcholinesterase (BChE
T
) and the
T splice variant of acetylcholinesterase (AChE
T
)
consist of a catalytic domain of approximately 500
residues, followed by C-terminal tail (t) peptides [1,2].
These peptides of 41 and 40 residues, respectively, con-
tain seven strictly conserved aromatic residues, includ-
ing three evenly spaced tryptophans, and a cysteine

) possess
a characteristic C-terminal tail (t) peptide. This t peptide allows their
assembly into tetramers associated with the anchoring proteins ColQ and
PRiMA. Although the t peptides of all vertebrate cholinesterases are
remarkably similar and, in particular, contain seven strictly conserved
aromatic residues, these enzymes differ in some of their oligomerization
properties. To explore these differences, we studied human AChE (Aa) and
BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged
(Ab and Ba). We found that secretion was increased by deletion of the
t peptides, and that it was more efficient with a than with b. The patterns
of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicat-
ing a predominant influence of the catalytic domains. However, addition of
a cysteine within the aromatic-rich segment of the t peptides modified the
oligomeric patterns: with a cysteine at position 19, the proportion of tetra-
mers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser
extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms,
from monomers to hexamers. These results indicate that both the catalytic
domains and the C-terminal t peptides contribute to the capacity of cho-
linesterases to form and secrete various oligomers. Sequence comparisons
show that the differences between the t peptides of AChE and BChE are
remarkably conserved among all vertebrates, suggesting that they reflect
distinct functional adaptations.
Abbreviations
AchE
T
, T splice variant of acetylcholinesterase; BChE, butyrylcholinesterase; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide;
Nbs
2
, 5,5¢-dithiobis(2-nitrobenzoic acid); PRAD, proline-rich attachment domains; t, tail.
94 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works

tetramerization and association with PRAD-containing
proteins, because addition of a t peptide at the C-ter-
minus of green fluorescent protein or alkaline phos-
phatase allowed the formation of tetramers associated
with an N-terminal fragment of ColQ [17]. However,
the catalytic domains are also involved in quaternary
interactions that certainly participate in the formation
and stability of these oligomers. In particular, the tet-
ramers are formed of two pairs of subunits, in which
a
7,8
and a
10
helices from each subunit form a four
helix bundle, with a hydrophobic contact zone [16,18].
The respective contributions of the catalytic domains
and the t peptides in oligomers has not been evaluated.
The formation of AChE
T
tetramers associated with
PRAD-containing proteins is physiologically important
because it ensures their functional localization by ColQ
in the basal lamina at neuromuscular junctions [19], as
well as by PRiMA in cell membranes, particularly
in the brain [20]. Similarly, the formation of BChE
T
tetramers conditions the secretion of this enzyme and
its stability in the bloodstream.
Injection of AChE or BChE offers a very efficient
protection against poisoning by anti-cholinesterase

we associated the catalytic domain of each enzyme
with the t peptide of the other. For convenience, the
large catalytic domains are designated by capital letters
(A and B), whereas the small t peptides are designated
by lower case letters (a and b), so that the wild-type
AChE and BChE are Aa and Bb, and the chimeras are
Ab and Ba. Comparisons of wild-type enzymes and
chimeras, as well as of various mutants, show that
both domains contribute critically to the oligomeriza-
tion and to the efficiency of secretion.
Results
Exchange of t peptides between human AChE
and BChE
The T variants of human AChE and human BChE are
composed of a catalytic domain of approximately 500
residues, followed by small C-terminal t peptides of 40
and 41 residues, respectively. In the present study, the
catalytic domains are indicated by capital letters ( A and
B) and the C-terminal peptides by lower case letters (a
and b), so that the wild-type enzymes are abbreviated as
Aa and Bb. The C-terminal t peptides of human AChE
T
(a) and BChE
T
(b) are highly homologous, with 24 iden-
tical residues (60%), including the seven aromatic resi-
dues and the cysteine located at )4 from the C-terminus,
being strictly conserved among all vertebrate cholines-
terases (Fig. 1A). However, they present significant dif-
ferences, particularly between the residues immediately

residual activity, plotted as a function of the amount
of DEPQ, were identical for A, Aa and Ab, with
acetylthiocholine as substrate, as well as for B, Ba
and Bb, using either acetylthiocholine or butyrylthio-
choline as substrates. Because of excess substrate inhi-
bition, AChE presented a maximal rate for
approximately 2 mm acetylthiocholine. The rates of
hydrolysis of acetylthiocholine and butyrylthiocholine
(at 6 mm) by BChE were approximately 14% and
24% of the rate of hydrolysis of acetylthiocholine
(at 2 mm) by AChE.
Influence of the C-terminal t peptides on activity,
secretion and oligomerization
As expected, the truncated mutants A and B, without
t peptides, produced only monomers, sedimenting
around 4S (not shown). The levels of cellular activity
were lower for these mutants than for the wild-types but
secretion was increased (Fig. 2A,B), in agreement with
our previous conclusions that t peptides induce a partial
misfolding of the polypeptides, as well as an intracellular
degradation of a fraction of active subunits [3].
Cells expressing wild-type human AChE (Aa)
secreted approximately 15% of their content per hour
and produced mostly monomers and dimers, with a
small proportion of tetramers (less than 10% of the
A
B
Fig. 1. Structures of AChE and BChE t peptides. (A) Sequences of the C-terminal t peptides of human AChE and BChE. These peptides
(a and b) are encoded by 3¢ exons from the cholinesterase genes; in the present study, we numbered from their first residue. The seven
aromatic residues, which are conserved in all vertebrate cholinesterases, are shown in blue; acidic residues are shown in red and basic resi-

Ab SSVGL
Ab N19C
Ab N19C N18S
Ab N19C MD22VH
Ab N19C N18S MD22VH
-
B-
Ba
Ba S19C
-
Bb
Bb SSVGL
Bb A12C
Bb H15C
Bb N19C
Bb D23C
Bb N26C
Bb S37C
Bb N19C N18S
Bb N19C MD22VH
Bb N19C N18S MD22VH
-
Cellular activity
A
B
Secreted activity
0
1
2
3

activities. A and B represent AChE and BChE from which the t peptides were deleted; Aa and Bb represent the wild-type enzymes with
their t peptides, Ab and Ba represent chimeras in which the t peptides were exchanged; mutations in the t peptides are indicated. For each
mutant, the cellular and secreted activities are shown by bars to the left and the right. AChE and BChE activities were determined by the
Ellman assay with acetylthiocholine and butyrylthiocholine as substrates, respectively: AChE activities are indicated as grey bars and BChE
activities as hatched bars. AChE and BChE activities were normalized to the wild-type enzymes (Aa and Bb, respectively). (B) Ratio
of secreted to cellular activity. Note that the double mutation M22V ⁄ D23H is abbreviated as MD22VH.
D. Liang et al. Oligomerization and secretion of AChE and BChE
FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 97
the fact that mutants lacking some of the N-glycosyl-
ation sites, which were provided by O. Lockridge [36],
did not produce a higher proportion of tetramers
(not shown).
For the chimeras Ab and Ba, the rates of secretion
were approximately 5% and 15% of the cellular con-
tent per hour, respectively, and therefore appeared to
be mainly determined by the t peptides. By contrast,
Fig. 3B shows that the sedimentation profiles were
very similar for Aa and Ab, and for Ba and Bb,
except that the BChE species sedimented faster than
their AChE counterparts, in agreement with the
higher mass of BChE subunits [37]. This indicates a
predominant influence of the catalytic domain on
oligomerization.
Role of the C-terminal cysteine
Mutation of the cysteine located at )4 from the C-ter-
minus to a serine in the a or b peptides suppressed the
formation of Aa or Bb dimers, but not the production
of a small proportion of tetramers (not shown). These
mutations increased the ratio of secreted to cellular
activity in both cases (Fig. 2). However, in the case of

studies on Torpedo AChE [38].
Role of cysteines in oligomerization – effects of
introducing an additional cysteine
In a previous study, we found that mutating residue
19 in the t peptide of Torpedo AChE considerably
increased the production and secretion of tetramers
Cell extract
Medium
G
1
G
2
G
4
G
1
G
2
G
4
G
1
G
2
G
4
G
1
G
2

Aa
Bb
Ba
Ab
Aa
AChE activity (arbitrary units)BChE activity (arbitrary units)
Sedimentation coefficients
55101015 15
Wild-type t peptides
AB
With added cysteine
19C
Bb
Ba
Ab
19C
19C
19C
Fig. 3. Sedimentation profiles indicating the proportions of oligo-
meric forms produced by AChE, BChE, chimeras and mutants. (A)
Left panels: Aa, Ab, Ba, Bb. (B) Right panels: mutants containing a
cysteine at position 19 (S19C in peptide a, N19C in peptide b). The
profiles corresponding to cell extracts are shown with filled sym-
bols (d, AChE;
, BChE) and a continuous line, and those corre-
sponding to the medium with empty symbols (s, AChE; h, BChE)
and a dashed line. The peaks corresponding to tetramers, dimers
and monomers are indicated as G
4
,G

. As observed in the pre-
ceding section, the ratio of secreted to cellular
activity again appeared to depend essentially on the
t peptides: it was much higher for Aa
19C
and Ba
19C
than for Ab
19C
and Bb
19C
. The 19C mutations
enhanced the difference between the two peptides
because the secreted ⁄ cellular ratio was increased with
peptide a
19C
compared to a and decreased with pep-
tide b
19C
compared to b.
By contrast to the oligomeric patterns obtained
without a cysteine at position 19, we observed a
much stronger similarity between enzymes possessing
the same C-terminal peptide (a
19C
or b
19C
) than
between those possessing the same catalytic domain
(Fig. 3B). Thus, mutation S19C in a

tides possessing a cysteine at position 19 had a stron-
ger effect on the secretability of cholinesterases than
wild-type t peptides, and exerted a dominant influence
on oligomerization.
Effects of introducing cysteines at different
positions in BChE
Our previous study of Torpedo AChE showed that the
pattern of oligomerization depended critically on
the position at which a cysteine was introduced in the
t peptide [4]. Because the presence of a cysteine
induced tetramerization at position 19 of peptide a,
but not at position 19 of peptide b, we explored the
effects of cysteines at other positions in BChE. We
mutated residues that, similar to N19, are located
within the aromatic-rich segment of peptide b, but are
oriented in the opposite sector of the a helix, produc-
ing mutants A12C, H15C, D23C and N26C (Fig. 1B).
We also added a second cysteine near the C-terminus
(S37C), changing the C-terminal peptide from SCVGL
to CCVGL.
These mutations had little effect on the cellular or
secreted activities compared to wild-type BChE, except
that the secreted ⁄ cellular ratio presented a minimum
with a cysteine at position 19, and was notably
increased in the mutant possessing two C-terminal
cysteines (S37C). As shown in Fig. 4, the sedimenta-
tion profiles of cellular enzyme varied mostly in the
proportions of monomers and dimers, whereas tetra-
mers remained low. The ratio of dimers to monomers
was markedly increased with cysteines in the N-termi-

that mutation W8R increased both cellular activity and
secretion, in agreement with the notion that aromatic
residues induce degradation of AChE through an endo-
plasmic reticulum associated degradation process [3].
In all these mutants, the cellular extracts contained
only a trace of tetramers, as observed for Ab and
D. Liang et al. Oligomerization and secretion of AChE and BChE
FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 99
Ab
19C
(Fig. 5). The sedimentation profiles of the
secreted enzyme were similar to those obtained with
Ab
19C
, except that the proportion of tetramers (G
4
)
was somewhat increased with N18S. The M22V muta-
tion mostly increased the 13.5S species, and the D23H
mutation did not increase G
4
by itself, but their combi-
nation, M22V ⁄ D23H, induced a significant increase in
the proportion of secreted tetramers.
Hoping to obtain a higher yield of secreted tetra-
mers, we then combined the N18S and M22D ⁄ D23H
mutations in Ab
19C
. The combination of mutations
N18S, N19C, M22D and D23H, abbreviated as S, pro-

Ab oligomers (Fig. 6A). The major oligomers were iso-
lated from gradient fractions. By comparison with the
standard proteins b-galactosidase and alkaline phospha-
tase, we obtained Stokes radii values, as indicated in
Fig. 6B. We then determined the mass of these oligomers,
assuming that it is proportional to the product of the sed-
imentation coefficient and Stokes radius, as expected for
proteins of similar density. The values thus obtained indi-
cated a globular structure because the mass was in fact
proportional to S
3 ⁄ 2
. This relationship allowed us to
determined the mass of the minor components, sediment-
ing at 8.5S and 12.3S (Fig. 6A). Figure 6C shows that
Cell extract
Medium
G
1
G
2
G
4
G
1
G
2
G
4
G
1

N19C
Bb
D23C
Bb
N26C
Bb
S37C
Fig. 4. Sedimentation profiles of mutants of human BChE (Bb)
with cysteines at positions 12, 15, 19, 23 and 26. The profiles
obtained for Bb
19C
, also shown in Fig. 3, are repeated here for
comparison with the other mutants. The symbols are as in Fig. 3.
Tetramers, dimers and monomers are indicated as G
4
,G
2
and G
1
,
respectively. The mutations replacing various residues by cysteines
in the C-terminal peptide are indicated.
Oligomerization and secretion of AChE and BChE D. Liang et al.
100 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works
the masses of the six observed oligomers represent multi-
ples of the smaller one, demonstrating that they represent
monomers (G
1
), dimers (G
2

studies showed that oligomers of AChE
T
subunits pos-
sessed the same turnover rate per site, but this did not
exclude a possible influence of the nature of C-terminal
peptides. To examine this question, we titrated the
active sites of truncated, wild-type and chimeric cho-
linesterases with the irreversible inhibitor DEPQ, and
compared their activities with the substrates acetylthio-
choline and butyrylthiocholine. We found that the cat-
alytic rate per active site only depends on the catalytic
domain: it was identical for truncated enzymes (A or
B) and with enzymes possessing either a or b C-termi-
nal peptides, in agreement with previous studies [40]
showing that the variants AChE
T
, AChE
R
and a trun-
cated mutant possessed the same K
m
value and excess
substrate inhibition. These results also show that the
catalytic activity is not influenced by the oligomeric
state of the enzymes, and thus justifies quantitative
comparisons between the activities of the various
mutants investigated in the present study.
Effect of the C-terminal t peptides on folding
and secretion
The t peptides of cholinesterases form amphiphilic

G
1
G
2
G
4
G
6
G
1
G
2
G
4
G
1
G
2
G
4
G
3
G
6
G
1
G
2
G
4

G
1
G
2
G
6
Bb N18S
N19C
Bb N18S N19C
M22V D23H
Bb N19C
M22V D23H
AChE activity (arbitrary units)
BChE activity (arbitrary units)
51015
Ab N19C
Fig. 5. Effect of mutations suppressing differences between a and
b, on the distribution of oligomeric forms. (A) Left panels and top
right panel: Ab
19C
. (B) Lower right panels: Bb
19C
. Sedimentation
patterns are shown as in Fig. 3. The sedimentation profiles of the
Ab
19C
mutant (top right panel) are repeated for comparison with
those obtained with additional mutations, which suppressed some
of the differences with peptide a. Note that the effects of muta-
tions M22V and D23H are not additive.

formed oligomers, including tetramers. Because these
tetramers were obtained without co-expression with a
PRAD-containing protein, they most probably repre-
sent homotetramers, in which the four t peptides may
form a coiled coil complex with all aromatic residues
oriented inwards, but without a central PRAD. This
hypothesis is supported by the fact that, although the
presence of a PRAD only induces the assembly of tet-
ramers, expression of some mutants without a PRAD
produces tetramers together with other oligomers,
including molecular forms sedimenting as trimers, pen-
tamers and hexamers. The odd-numbered complexes
are not likely to represent heteromeric associations
containing other proteins because they only occur with
some Ab mutants with an added cysteine, and their
masses correspond exactly to those expected for multi-
ples of AChE subunits. Because the formation of these
unusual oligomers appears to depend strictly on the
presence of an additional cysteine, they are probably
stabilized by a network of inter-catenary disulfide
bonds, linking all subunits together.
The Ab
19C
chimera formed all oligomeric forms
from monomers to hexamers, illustrating the versatility
of oligomeric associations based on the t peptide, in
association with the catalytic domain. It should be
noted that hexamers have been observed in transfected
COS cells expressing wild-type rat AChE, and
appeared as a transient mode of association, which

4.3 S
6.5 S
10.5 S
13.5 S
8.5 S
12.3 S
Masses of oligomers (kDa)
Numbers of subunits
G6
G5
G4
G3
G2
G1
AB C
Fig. 6. Determination of the Stokes radius and mass of AChE
B
oligomers. (A) Oligomers were isolated from sucrose gradients of medium
from cells expressing the Ab
19C-29D-30H
mutant. The profile of cellular activity was identical to that shown in Fig. 3B for the Ab
19C
mutant.
(B) Elution of oligomers in gel filtration chromatography. The elution parameters were defined as K
e
=(V
e
– V
o
) ⁄ (V

).
Oligomerization and secretion of AChE and BChE D. Liang et al.
102 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works
contrast, Bb
19C
only formed the classical monomers,
dimers and tetramers, possibly because of steric
constraints due to the catalytic domain or to its associ-
ated N-glycans.
Although the nature and proportions of oligomers
depended on the presence of the t peptides and their
cysteines, the catalytic domains also influenced the
oligomerization patterns. The cellular and secreted
oligomers formed by Aa and Ab were very similar, as
well as those formed by Ba and Bb, suggesting a pre-
dominant influence of the catalytic domains on oligo-
merization. This may be due in part to the difference
in N-glycosylation of AChE and BChE, which carry
four and nine N-glycan chains, respectively [35]; we
therefore compared the oligomeric patterns of
wild-type BChE and mutants in which part of the
N-glycosylation sites were mutated [36], but observed
no difference (not shown). The relative influence of the
C-terminal t peptide appeared to be strongly increased
when a cysteine was added at position 19 because the
patterns obtained for Aa and Ba were almost the same,
except for a shift in the sedimentation coefficients,
which are higher for BChE than for AChE.
By contrast to Aa and Ab, oligomers of Ba and Bb
represented a significant proportion of cellular activity,

Bb
19C
, the linkage between the two domains might well
play a crucial role in the quaternary associations of
the cholinesterase subunits. The first three residues of
peptides a and b are indeed different, but their replace-
ment in Ab
19C
(GNI to DTL) had little effect on either
secretion or oligomerization. It is also noteworthy that
the effects of the combined mutations M22D ⁄ V23H
could not be simply accounted for by the effects of the
separate mutations M22V and D23V. This suggests
that the secretory trafficking of molecules containing
peptides a and b depends on global properties of the
peptides rather than on individual residues.
AChE and BChE are expressed differentially during
embryogenesis [42–44]. They appear to play distinct
roles, which may be based on their catalytic activity,
but also on protein–protein interactions [45], because
their catalytic domain is homologous to adhesion pro-
teins such as neuroligin [46,47]. For example, AChE
may be involved in neurite extension during brain
development [40,48,49]. Both catalytic and noncatalytic
functions clearly require appropriate oligomeric orga-
nization and localization and therefore depend on the
C-terminal t peptides, which may be directly involved
in distinct interactions with partner proteins.
The two cholinesterases present a complex relation-
ship with the development of Alzheimer’s disease, which

FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 103
of these sister enzymes. The t peptides of higher mam-
mals, including rat, mouse, rabbit, horse, bovine, dog,
cat and primates, are identical and share 22 common
residues with the t peptide of chicken AChE (however,
there are more differences with marsupials) (Fig. 7).
The t peptides of BChE show more variation between
mammalian species: there are a few differences between
man and Maccaca mulatta, and the K variant of
human BChE, which occurs with high frequency in
European and American populations, consists of the
replacement of A6 by a threonine [58], but it does not
affect the assembly of tetramers [59]. Quite surpris-
ingly, the human t peptide shares 34 common residues
with that of chicken and only 13 of these residues are
common to both AChEs and BChEs (ten if one
considers marsupials), including the seven conserved
aromatic residues and the C-terminal cysteine. Thus,
the differences between the t peptides of AChE and
BChE are conserved in vertebrates, suggesting that
they are functionally significant. It is possible that,
although the physiological localization of AChE in
cholinergic tissues depends on its association with its
anchoring proteins ColQ and PRiMA, the major func-
tion of BChE rather depends on other interactions,
particularly on the formation of soluble tetramers
circulating in the bloodstream.
Production of recombinant AChE or BChE
tetramers
We have shown that mutation S19C in the C-terminal

evolution of vertebrates probably reflects their subtly
distinct functions, associated with the respective roles
of AChE and BChE in synaptic and nonsynaptic
contexts.
Experimental procedures
Mutations and constructs
The coding sequences of human AChE
T
(T variant, Aa)
and BChE
T
(Bb), inserted in the pGS vector, were gener-
ously provided by O. Lockridge. The residues of the t pep-
tides are numbered from the first residue following the
catalytic domain. To exchange the C-terminal t peptides,
BsiWI restriction sites were introduced at the junction
between regions encoding the catalytic domains and the
t peptides. Fragments encoding the t peptides were cut
between these sites and a downstream SacII site in the
vector, purified in agarose gels, and religated with the
appropriate complementary fragment. The nucleotides sepa-
rating the coding sequences of the catalytic domains and
C-terminal peptides were then removed by site-directed
mutagenesis with the method of Kunkel et al. [60]. Other
mutations were performed with this method.
Cell cultures and transfection
Transient transfection of COS cells was performed by the
DEAE-dextran method as described previously [61], usually
AChE T Chicken
GPPEDAEREWRLEFHRWSSYMGRWRTQFEHYSRQQPCATL

) followed by centrifugation for 15 min at 10 000 g
at 4 °C. The culture medium containing the secreted
enzyme was also centrifuged at 10 000 g for 10 min to
remove cell debris before analysis.
Enzyme assays
Enzyme activity was quantified by the colorimetric
method of Ellman et al. [62]. The reaction medium con-
tained 0.5 mm acetylthiocholine or butyrylthiocholine,
0.5 mm 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs
2
) and
0.05 m Na
+
phosphate (pH 7). The reaction was moni-
tored at 414 nm in a Multiskan RC automatic plate
reader (Labsystems, Helsinki, Finland); the optical density
was recorded at 20 s intervals over a period of 10 min.
Alkaline phosphatase and b-galactosidase from Escherichia
coli were assayed with the chromogenic substrates
p-nitrophenyl phosphate and o-nitrophenyl galactoside,
respectively. For titration of active sites, the culture
media were adjusted to pH 7 with 1 m Tris–HCl (pH
9.5) and 50 lL samples were treated with 100 lLofa
series of dilutions of DEPQ in 10 mm Na
+
phosphate
buffer (pH 7.4) overnight at room temperature; the
remaining AChE activity was then determined with the
Ellman colorimetric method, by addition of 150 lLof
50 mm Na

total volume with potassium ferricyanide; the standards
used were b-galactosidase (6.9 nm, 464 kDa) and alkaline
phosphatase (3.3 nm, 87 kDa).
Prediction of secondary structure
The secondary structure of mutated t peptides was pre-
dicted according to Rost [63], using the predictprotein
website ().
Acknowledgements
We thank Dr Oksana Lockridge for the generous gift of
vectors expressing wild-type and mutated human AChE
and BChE, and Dr Yaacov Ashani for DEPQ. This
work was supported by the US Defense Threat Reduc-
tion Agency under contract W91ZLK-06-C-0020, by the
Centre National de la Recherche Scientifique, and by
the Association Franc¸ aise contre les Myopathies.
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