Acetylcholinesterase from the invertebrate
Ciona intestinalis is capable of assembling into
asymmetric forms when co-expressed with vertebrate
collagenic tail peptide
Adam Frederick
1
, Igor Tsigelny
2
, Frances Cohenour
1
, Christopher Spiker
1
, Eric Krejci
3
,
Arnaud Chatonnet
4
, Stefan Bourgoin
1
, Greg Richards
1
, Tessa Allen
1
, Mary H. Whitlock
1
and
Leo Pezzementi
1
1 Department of Biology, Birmingham-Southern College, Birmingham, AL, USA
2 Department of Chemistry and Biochemistry, San Diego Supercomputer Center, University of California at San Diego, La Jolla, CA, USA
3 Institut National de la Sante

(Received 14 November 2007, revised 7
January 2008, accepted 15 January 2008)
doi:10.1111/j.1742-4658.2008.06292.x
To learn more about the evolution of the cholinesterases (ChEs), acetylcho-
linesterase (AChE) and butyrylcholinesterase in the vertebrates, we investi-
gated the AChE activity of a deuterostome invertebrate, the urochordate
Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for
the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, mole-
cular biology, and molecular modeling confirms that the enzyme is AChE.
Sequence analysis and molecular modeling also indicate that the cDNA
codes for the AChE
T
subunit, which should be able to produce all three
globular forms of AChE: monomers (G
1
), dimers (G
2
), and tetramers (G
4
),
and assemble into asymmetric forms in association with the collagenic
subunit collagen Q. Using velocity sedimentation on sucrose gradients, we
found that all three of the globular forms are either expressed in cells or
secreted into the medium. In cell extracts, amphiphilic monomers (G
1
a
)
and non-amphiphilic tetramers (G
4
na

methylquinolinium iodide; DTNB, 5-(3-carboxy-4nitro-phenyl)disulfanyl-2-nitro-benzoic acid; GPI, glycophosphatidylinositol; HIS buffer, high
ionic strength buffer; IC
50,
half maximal inhibitory concentration; LBA, long branch attraction;
na
, non-amphiphilic; PPII, polyproline II;
PRAD, proline-rich attachment domain; PRiMA, proline-rich membrane anchor; WAT, tryptophan (W) amphipathic tetramerization domain.
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1309
Gnathostome vertebrates have two evolutionarily
related cholinesterases (ChEs), acetylcholinesterase
(AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE;
EC 3.1.1.8). AChE rapidly hydrolyzes the neurotrans-
mitter acetylcholine at cholinergic synapses. BuChE
appears to act as a scavenger of cholinergic toxins, but
may also play a role in synaptic transmission [1,2].
These two enzymes appear to be the result of a gene
duplication event early in vertebrate evolution [3].
Both enzymes have a 20 A
˚
deep catalytic gorge lined
with aromatic amino acids [4]. AChE has fourteen aro-
matic residues lining the gorge; in BuChE, aliphatic
amino acids replace six of the aromatic moieties. In
particular, smaller non-aromatic residues in BuChE
replace the two phenylalanines of the acyl pocket of
AChE (Phe288 and Phe290 in Torpedo californica
AChE), a subsite of the enzyme that plays an impor-
tant role in substrate specificity. Amino acid position
numbers appearing in parentheses represent the homo-
logous positions in mature AChE from Torpedo cali-

Another difference between vertebrate and inverte-
brate ChEs is that vertebrates possess both globular
and asymmetric forms of the enzymes, but inverte-
brates apparently possess only globular forms. The
globular forms of ChEs are monomers (G
1
), dimers
(G
2
), and tetramers (G
4
) of catalytic subunits. The
asymmetric forms are comprised of one (A
4
), two
(A
8
), or three (A
12
) tetramers attached to a triple-
stranded collagenic tail (collagen Q; ColQ) [12,13].
The asymmetric forms associate with the basal lamina
[14].
Alternative splicing of the AChE gene in the verte-
brates produces a number of carboxyl termini [15],
resulting in the multiple molecular forms. mRNAs
containing the H-terminus (AChE
H
) are translated into
glycophosphatidylinositol-membrane-anchored (GPI)

AChE
T
appears to be rare in invertebrates, where
AChE
H
predominates. AChE
T
has been reported
for AChE1 from the nematodes Caenorhabditis spp.
[20,21] and Meloidogyne spp. [22], where it forms G
1
a
and a G
4
form that may associate with a structural
subunit [20,21]. Meedel reported that C. intestinalis lar-
vae have G
1
,G
2
, and G
4
forms of AChE, implying the
presence of AChE
T
in the invertebrate, but did not
find any asymmetric forms [11].
The cloning, in vitro expression, and characterization
of this putative AChE from C. intestinalis should iden-
tify the nature of the enzyme and provide additional

tinalis compared to the vertebrate enzyme (Fig. 1).
Additionally, the carboxyl terminus of the C. intestinal-
is AChE appears to be coded for by a T exon: six of
the seven aromatic residues of the T. californica AChE
WAT domain are conserved; there is a 74% sequence
similarity with T. californica AChE, and the domain
has ability to form an amphipathic helix, characteristic
of the T sequence. A cysteine that mediates interchain
disulfide bonds is also conserved (Fig. 2A,B). We found
no evidence in the genomic sequence of an upstream
H exon in the C. intestinalis AChE gene.
A second gene for AChE in C. intestinalis has been
proposed [8] (Genbank accession no. AK112482; cioin-
acche2 in ESTHER; AChE2; Table 1) [23]. However,
Table 1. Aromatic amino acids in the catalytic gorge of putative
AChEs from C. intestinalis and AChE from T. californica. Numbering
for C. intestinalis AChE2 starts at first methionine residue in the
sequence. Conserved aromatic residues are shown in bold. Desig-
nations of AChE1 and AChE2 are from the ESTHER database [23]
to distinguish the AChE described in the present study (AChE1)
and another sequence proposed to be an AChE from C. intestinalis
(GenBank accession no. AK112482).
Subsite
C. intestinalis
AChE1
C. intestinalis
AChE2
T. californica
AChE
Peripheral site Ile97 Ser124 Tyr70

T. californica (X03439), Myxine glutinosa (U55003), C. intestinalis (TPA: BK006073), B. floridae (U74381), S. purpuratus (XM_777020; pre-
dicted similar to AChE), Drosophila melanogaster (X05893), Anopheles stephensi (228651), C. elegans (X75332), Meloidogyne incognita
(AF075718), Loligo opalescens (AF065384), and Boophilus microplus (AJ223965).
A. Frederick et al. AChE from C. intestinalis
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1311
the derived amino acid sequence shows only 28% iden-
tity with the AChE from T. californica, and only 30%
homology with the C. intestinalis AChE described in
the present study. Although the three pairs of con-
served cysteine residues involved in intrachain disulfide
bonding in AChEs are found in the sequence, only two
members of the catalytic triad are present: serine and
glutamate. The third residue, histidine, is replaced by a
cysteine. This replacement would probably inactivate
the enzyme; in T. californica and human AChEs,
respectively, H440Q and H447Q mutants lack activity
[24,25]. Additionally, of the fourteen aromatic amino
acids that line the catalytic gorge of vertebrate AChE,
only six are conserved in the sequence (Table 1); how-
ever, the sequence shows the invertebrate acyl pocket
conformation, which provides a seventh aromatic resi-
due in the gorge. Nevertheless, in BuChE, eight of the
residues are conserved [1]. Particularly important is the
absence of the tryptophan of the choline-binding site.
In human AChE, a W86A mutation increases K
m
by
660-fold [26]. Finally, the sequence clearly does not
have a carboxyl terminus coded for by an AChE
T

AChE. Green and yellow residues are hydrophobic. Red, blue, and orange residues are hydrophilic.
AChE from C. intestinalis A. Frederick et al.
1312 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS
sis of ATCh and butyrylthiocholine (BTCh) by enzyme
that was secreted into the medium by the COS-7 cells,
enzyme extracted from the cells, and enzyme extracted
from adult C. intestinalis. Only ATCh is hydrolyzed
appreciably, as indicated by the low values of
V
max
BTCh
⁄ V
max
ATCh
. It proved difficult to determine
accurate kinetic parameters for BTCh hydrolysis given
the low activity that the enzyme showed for the sub-
strate, and it was not possible to detect BTCh hydroly-
sis by extracts of adult organisms; nevertheless, the
kinetic parameters determined are in reasonable agree-
ment. The enzymes also show substrate inhibition
(i.e. lower enzyme activity at high substrate concentra-
tions, and b parameter values of < 1) (Fig. 3;
Table 2). The selective hydrolysis of ATCh is charac-
teristic of AChE.
Pharmacological characterization of the
recombinant ChE from C. intestinalis expressed
in vitro and native enzyme expressed in vivo
confirms that the enzyme is AChE
To determine further the nature of the cholinesterase

a
and G
4
na
because the G
1
form shifts to a higher
sedimentation coefficient in the absence of detergent.
The forms of AChE secreted into media are G
2
a
and
Substrate (M)
10
–6
10
–5
10
–4
10
–3
10
–2
10
–1
10
–7
Cholinesterase activity (mAb·min
–1
)

ATCh
V
max
BTCh
(mAb ⁄ min)
K
m
BTCh
(mM)
K
ss
BTCh
(mM) b
BTCh
V
max
BTCh
⁄ V
max
ATCh
Medium 495 ± 91 188 ± 53 275 ± 78 0.19 4.15 ± 1.38 4.80 ± 3.09 83 ± 45 0.03 0.012 ± 0.006
Cells 1101 ± 27 223 ± 15 100 ± 27 0.32 3.21 ± 1.06 1.57 ± 0.73 171 ± 102 0
a
0.003 ± 0.001
Organism 132 ± 25 100 ± 19 501 ± 244 0.22 0
b
–––0
a
Values of b less than 0.02 are indistinguishable from zero.
b

testinalis AChE catalytic subunits could assemble into
asymmetric forms of AChE in the presence of a colla-
genic tail, we co-transfected COS-7 cells with cDNAs
for the C. intestinalis catalytic subunit and for R. nor-
vegicus ColQ, and analyzed cell extracts on sucrose
gradients. In addition to peaks corresponding to G
1
and G
4
, a peak of enzyme activity appears at approxi-
mately 16S, which is characteristic of the A
12
form of
AChE. Collagenase digestion at 37 °C converts the
putative A
12
form to a lytic G
4
; a shoulder of residual
undigested A
12
is visible (Fig. 6; Table 5). We have not
found genes for ColQ or PRiMA in the C. intestinalis
genome.
Molecular modeling of C. intestinalis AChE also
indicates that the catalytic subunit can assemble
into asymmetric forms
Molecular modeling, in addition to sequence analysis,
also indicates that the catalytic gorge of C. intestinalis
AChE is similar to that of vertebrate AChEs, showing

AChE from C. intestinalis. Data are the mean ± SE of three or
more determinations. Sources of enzyme are the same as in
Table 1.
Source Physostigmine BW284c51 Ethopropazine Iso-OMPA
Medium 5.09 ± 0.66 0.93 ± 0.17 768 ± 203 > 3000
Cells 7.35 ± 0.28 1.91 ± 0.01 650 ± 93 > 3000
Organism 14.1 ± 0.76 1.23 ± 0.76 741 ± 60 > 3000
0 5 10 15 20 25
Fractional AChE activity on gradient
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Sedimentation coefficient
0 5 10 15 20 25
Fractional AChE activity on gradient
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Fig. 5. Velocity sedimentation analysis of the globular molecular

units into a tetramer through interaction with the
PRAD domain of ColQ. The [AChE
T
]–ColQ com-
plex model was built based on the PRAD–WAT
interaction; inter-subunit interactions involving the
catalytic domains were considered secondary [27]. As
a result, the complex has a quasi-four-fold axis of
symmetry (Fig. 7A,B). The four WAT domains of
the tetramer form a-helices and coil around a single
antiparallel PRAD domain, which approximates a
left-handed polyproline II (PPII) helical conforma-
tion. The three tryptophans of the WAT domain ori-
ent inwards to interact with ColQ, and come into
close contact and stack with the prolines of the
PRAD domain (Fig. 7C,D).
Phylogenetic analysis of AChE sequences
supports a classical phylogeny for deuterostome
invertebrates
A phylogenetic analysis of vertebrate and deutero-
stome and protostome invertebrate ChEs places
C. intestinalis AChE intermediate between the echino-
derms and the cepalochordate amphioxus (see supple-
mentary Fig. S3). This placement is consistent with
conventional phylogenetic trees based primarily on
morphological data [28]. Note, however, that the
branch length for C. intestinalis AChE is the longest in
the tree, and the bootstrap value for the branching
between amphioxus and C. intestinalis is one of the
weakest in the tree.

0 5 10 15 20 25
Fractional activity on gradient
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Fig. 6. Velocity sedimentation analysis of globular and asymmetric
forms of AChE produced by cotransfection with cDNAs for C. intes-
tinalis catalytic subunit and rat ColQ. Total HIS cell extracts were
digested with collagenase and analyzed on sucrose gradients as
described in the Experimental procedures. Control (d); collagenase
digestion (s).
Table 5. Sedimentation coefficients of C. intestinalis AChE cata-
lytic subunit co-expressed with ColQ with and without digestion by
collagenase. Data are the mean ± SE of ‡ 7 determinations.
Conditions Sedimentation coefficients
)Collagenase 5.10 ± 0.07 11.48 ± 0.10 16.09 ± 0.14
+Collagenase 5.16 ± 0.12 11.54 ± 0.08 15.65 ± 0.20
a
Molecular form
b
G
1
G
4

and rat collagenic tail, ColQ, results in the assembly
of the A
12
asymmetric form. Sequence analysis and
molecular modeling support both of these conclu-
sions. In some respects, the AChE from C. intestinal-
is more closely resembles the AChE of the
vertebrates than any other invertebrate AChE and
provides information about the evolution of the
ChEs.
The ChE from the invertebrate C. intestinalis is
an AChE that resembles vertebrate AChE
Our kinetic data are consistent with those of Fromson
and Whittaker [9] and Meedel and Whittaker [10],
who investigated ChE activity in extracts of larval
C. intestinalis, and also concluded that the activity is
due to AChE. They found that the hydrolysis of
BTCh was 4.5% of that for ATCh at 25 mm [8], and
that high concentrations of ATCh produced substrate
inhibition [10]. They do not show a hydrolysis curve
for BTCh and, in the present study, we were unable
to detect hydrolysis of BTCh by extracts of adult
C. intestinalis. Our estimates of their values for K
m
(approximately 100 lm) and K
ss
(approximately
100 mm) for ATCh hydrolysis data are comparable to
our own [10].
Our pharmacological results are also consistent with

B
C
D
Fig. 7. Modeled structures of C. intestinalis
AChE [AChE
T
]–ColQ complex. (A, B) The
[AChE
T
]-ColQ complex modeled on the
basis of the [WAT]
4
–PRAD structure, from
the side and bottom respectively. Each cata-
lytic subunit is shown in a different color
(purple, yellow, blue and orange), as is ColQ
(green). (C) Hydrophobic interactions
between WAT and PRAD helices. The view
is down and into the PRAD helix in the
center of the figure. The four WAT helices
are shown colored as in (A) and (B). The
magenta space-filled residues are the Trps
of the WAT domains, which all face inward
and surround the PRAD. (D) Cut away view
showing the Trps (in space-filling format) of
two WAT domains (colored as above) inter-
acting with the PRAD PPII helix. The Trp
side-chains zipper into the grooves of the
PPII helix.
AChE from C. intestinalis A. Frederick et al.

analysis of deuterostome AChEs supports the classical
phylogeny and is similar to the phylogenetic tree for
AChE of various vertebrates and deuterostome inver-
tebrates provided by Vienne and Pontarotti [36]. Note,
however, that the branch length for C. intestinalis
AChE is the longest in the tree; this long branch
length is typical of many C. intestinalis genes and is a
result of rapid evolution in the species [34,37]. This
rapid evolution and the resultant long branch length
gives rise to an artifact called long branch attraction
(LBA), which has a number of effects. Most impor-
tantly in this case, LBA results in the grouping of two
sequences that evolve more rapidly than the others
do: C. intestinalis AChE and a putative AChE from
the echinoderm Strongylocentrotus purpuratus. LBA is
also a problem in metaphylogenies, but can be cor-
rected for more easily, and a consensus is forming
around the revised deuterostome phylogeny, with the
urochordates actually being the sister group to the
vertebrates [28,34–36]. Not only does LBA compro-
mise our AChE phylogeny, but also the bootstrap
value for the branching between amphioxus and C. in-
testinalis AChEs is one of the weakest in the tree,
indicating its uncertainty. If it is assumed that the
urochordates are the closest living relative of the ver-
tebrates, the acyl pocket of C. intestinalis may in fact
be ancestral to that of the vertebrates. What may have
been responsible for the shift in acyl pocket structure
during the transition from invertebrates to vertebrates,
or nonchordates to chordates and vertebrates, remains

na
forms of enzyme were produced. The amphiphilici-
ty of G
1
and G
2
is due to the exposure of the hydropho-
bic T peptide of their carboxyl termini, which interact
with detergent micelles on the gradients; while the
T peptide of G
4
is sequestered away from solvent and
unable to interact with detergent [38]. Extracts of adult
C. intestinalis contained G
1
a
and G
4
na
forms. By con-
trast, it was reported that extracts of the larvae produce
all three globular forms, possibly indicating a develop-
mental difference in AChE assembly between the larvae
and adults [11]. Nevertheless, all three G forms pro-
duced in vivo are also produced in vitro.
Inspection of the T peptide sequence shows that all
of the tryptophans of the WAT domain are conserved
in the C. intestinalis sequence. However, one of the
seven aromatic amino acids, Tyr20, is replaced by
Ser20. In Torpedo marmorata AChE, the mutations

Co-expression of C. intestinalis AChE catalytic sub-
unit with rat ColQ resulted in the production of the
A
12
asymmetric form of AChE. These results confirm
our molecular modeling, which indicated that the
appropriate interactions between the WAT domain of
the catalytic subunit and the PRAD domain of ColQ
were present to assemble the catalytic tetramers of the
asymmetric forms. The A
12
form consists of three such
tetramers attached to the triple-stranded helix of ColQ.
This result is the first demonstration of the assembly
of catalytic subunits of an invertebrate AChE into
asymmetric forms.
The evolution of the T peptide and tetrameric
forms of AChE
However, one question arises: what, if anything,
assembles the C. intestinalis G
4
tetramers in the
absence of ColQ in vivo or in vitro? T peptide
sequences have been identified in vertebrates; deutero-
stome invertebrates, the urochordate C. intestinalis and
the echinoderm S. purpuratus; and in protostome
invertebrates, the mollusk Aplysia californica and vari-
ous nematodes, including Caenorhabditis elegans, sug-
gesting that the peptide is widespread in nature. The
presence of the T peptide in both branches of the ani-

nitro-benzoic acid (DTNB), iso-OMPA, ethopropazine, and
physostigmine were purchased from Sigma (St Louis, MO,
USA). Type-3 collagenase was obtained from Worthington
(Lakewood, NJ, USA). 7-[(diethoxyphosphoryl)oxy]-1-
methylquinolinium iodide (DEPQ) was a gift from Yacov
Ashani. Adult specimens of C. intestinalis were purchased
from The Marine Biological Laboratory (Woods Hole,
MA, USA). We thank Andrew Gannon for help with the
C. intestinalis dissection.
Gene synthesis and analysis
The ci0100132088 gene from the urochordate C. intestinalis
is now identified in the Department of Energy Joint Gen-
ome Institute (DOE JGI) Database (-psf.
org/Cioin2/Cioin2.home.html) as an AChE gene. The
sequence for this gene is embedded in the C. intestinalis
genome sequence (GenBank accession no. AABS01000124)
[7,8]. We spliced out the intronic sequences and translated
the coding exonic sequences in silico. Nucleotide sequence
and derived amino acid sequence data reported are avail-
able in the Third Party Annotation Section of the
DDBJ ⁄ EMBL ⁄ GenBank databases under the accession no.
TPA: BK006073. These sequence data are also available on
the DOE JGI Database. The amino acid sequence for the
protein has also been deposited in the Esther database as
cioin-acche1 ( />what=index) [23]. A BLAST search was conducted at
NCBI with the translated sequence, and it was found to be
similar to many AChE amino acid sequences in that data-
base, showing 72% homology with the AChE of Ciona sav-
ignyi. GenScript Corporation (Piscataway, NJ, USA)
synthesized and subcloned a cDNA for the protein into

EDTA. Extracts were centrifuged at 20 000 g for 20 min and
the supernatants were assayed for AChE activity.
The same HIS buffer was used to extract adult C. intesti-
nalis tissue but, given the low activity in the adult, equal
amounts of tissue and buffer on a weight ⁄ volume basis
were used. The interstitial fluids in C. intestinalis are isos-
motic with seawater. Typically, specimens of C. intestinalis
were dissected to separate the outer tunic from the internal
organs; subsequently, the digestive system was emptied of
its contents. The tunic and remaining viscera were then sep-
arately flash frozen in liquid nitrogen. The viscera, which
contained more enzyme, was used for kinetic and sedimen-
tation velocity experiments; the tunic was used for pharma-
cological experiments. For velocity sedimentation on
sucrose gradients, extracts were made with HIS buffer con-
taining 10 mm NaHPO
4
,pH7,1m NaCl, 1% Triton
X-100, 1 mm EDTA, 0.02 mgÆmL
)1
pepstatin, 0.2 mgÆmL
)1
aprotinin, 1 mgÆmL
)1
bacitracin, and 0.3 mgÆmL
)1
benz-
amidine [48].
Measurement and analysis of AChE activity
and inhibition

max
⁄ [enzyme]) because DEPQ could not be used to
accurately titrate the enzyme, even after overnight expo-
sure. Although Triton X-100 can activate AChE, and dif-
ferent concentrations of the detergent can artifactually
alter enzyme activity, total activities for different prepara-
tions were never compared, only V
max
ratios for BTCh and
ATCh hydrolysis, which were always determined sequen-
tially for the same enzyme preparations. Values of IC
50
for
the inhibitors used were determined by incubating enzymes
with various concentrations of drug for 20 min and then
assaying for enzyme activity in the presence of ATCh.
sigmaplot was then used to fit the data to a three-parame-
ter logistic function, yielding IC
50
. Since we were just look-
ing for classical diagnostic differential inhibition, it was
not necessary to determine k
i
, K
I
,oraK
I
values for the
inhibitors [10,52,53].
Velocity sedimentation on sucrose gradients:

1EA5) as a template. The two amino acid sequences were
aligned with t-coffee software [55], as clustalw mis-
aligned conserved cysteines involved in intra-molecular
disulfide bonding. A two-sequence blast confirmed the
t-coffee results [56]. The structure was minimized for
10 000 iterations of steepest descent in vacuo using the
distance-dependent dielectric constant by the discover
program (Accelrys). Volumes of active site gorges were
calculated with CASTp [57]. For modeling of the C. intesti-
nalis G
4
–ColQ complex, the crystal structures of the
[WAT]
4
–PRAD complex (pdb ID 1VZJ) and the mouse
[AChE
T
]
4
–ColQ complex model (a generous gift from
D. Zhang and J. A. McCammon) were used and modeling
was performed as described previously [27]. After modeling,
the complex underwent 10 000 iterations of steepest descent
minimization.
A. Frederick et al. AChE from C. intestinalis
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1319
Sequence and phylogenetic analysis
For analysis of acyl pocket structures, multiple amino acid
sequences were aligned with clustalw [58]. By contrast to
the pairwise alignment, there was no obvious problem with

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