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Tài liệu Báo cáo khoa học: Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide doc

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
´
et de la Recherche Me
´
dicale U686, Universite
´
Paris Descartes, Biologie des Jonctions Neuromusculaires,
Paris, France
4 Institut National de la Recherche Agronomique, Montpellier, France
Keywords
acetylcholinesterase; asymmetric forms;
butyrylcholinesterase; Ciona intestinalis;
evolution
Correspondence
L. Pezzementi, Department of Biology,
Birmingham-Southern College, Box 549022,
Birmingham, AL 35254, USA
Fax: +1 205 226 3078
Tel: +1 205 226 4806
E-mail: lpezzeme@bsc.edu
Website: http://faculty.bsc.edu/lpezzeme/
Database
The nucleotide sequence and derived amino
acid sequence data reported for the AChE
from Ciona intestinalis are available in the
Third Party Annotation Section of the
DDBJ ⁄ EMBL ⁄ GenBank databases under the
accession no. TPA: BK006073. The align-
ment used to determine the phylogenetic
tree for vertebrate and invertebrate cholines-
terases presented here is deposited at the


EMBL-ALIGN database as ALIGN_001208
(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
) are found. Amphiphilic dimers (G
2
a
)
and non-amphiphilic tetramers (G
4
na
) are secreted into the medium.
Co-expression of the catalytic subunit with Rattus norvegicus collagen Q
produces the asymmetric A
12
form of the enzyme. Collagenase digestion of
the A
12
AChE produces a lytic G
4
form. Notably, only globular forms are
present in vivo. This is the first demonstration that an invertebrate AChE is
capable of assembling into asymmetric forms. We also performed a phylo-
genetic analysis of the sequence. We discuss the relevance of our results
with respect to the evolution of the ChEs in general, in deuterostome inver-
tebrates, and in chordates including vertebrates.
Abbreviations
a
, amphiphilic; AChE, acetylcholinesterase; AChE
H,
splice variant H; AChE
T,
splice variant T; ATCh, acetylthiocholine; BTCh,
butyrylthiocholine; BuChE, butyrylcholinesterase; ChE, cholinesterase; ColQ, collagen Q; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-
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-
fornica; residues of the T peptides of AChE
T
from
different species are numbered from 1 to 48 to facili-
tate comparisons. As a result, BuChE can accommo-
date larger and more diverse substrates and inhibitors
compared to AChE [5]. By contrast to the dichoto-
mous acyl pocket situation of ChEs in the vertebrates,
invertebrates have a wider diversity in the structure of
this subsite. In approximately 90% of invertebrate
ChEs, the acyl pocket is formed in a fundamentally
different way [6]. Instead of Phe288 and Phe290 form-
ing the pocket, it is formed by phenylalanines at posi-
tions homologous to Phe290 and Val400. For example,
in ChE2 from the cephalochordate amphioxus, which
is very specific for the substrate acetylthiocholine
(ATCh), the acyl pocket is composed of Phe312
(Phe290) and Phe422 (Val400) [6]. One of the excep-
tions to this invertebrate pattern is found in the
sequence for a putative AChE from the urochordate
Ciona intestinalis [7,8], a deuterostome invertebrate
that is a close relative to the vertebrates. In this
enzyme, phenylalanines homologous to those of the
acyl pocket of vertebrates appear to form the acyl
pocket [6]. Previously, based on substrate and inhibitor
specificity, it was reported that C. intestinalis possesses
an AChE in vivo [9–11]. However, that work was con-
ducted before the techniques of molecular biology were
available, precluding the definitive identification of the
enzyme.
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)
G
2
forms of AChE. By contrast, transcripts containing
the alternatively spliced T-terminus (AChE
T
) are
capable of forming all globular forms: amphiphilic
monomers (G
1
a
), amphiphilic dimers (G
2
a
), and non-
amphiphilic tetramers (G
4
na
), but not GPI-membrane-
anchored G
2
a
. More importantly, AChE
T
, via its
tryptophan (W) amphipathic tetramerization domain
(WAT) sequence [16], can associate with the proline-
rich attachment domain (PRAD) of the collagenic sub-
unit ColQ to form asymmetric enzyme [17,18] or with
the proline-rich membrane anchor (PRiMA) protein
[19].
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
information about the evolution of the ChEs, including
the origins of the acyl pocket, the T exon, and the
asymmetric forms of ChE in the vertebrates.
Results
The sequence of the ChE from C. intestinalis
suggests that the enzyme is an AChE
T
The sequence for C. intestinalis AChE contains 618
amino acids (see supplementary Fig. S1). The mem-
bers of the catalytic triad of AChE are found as Ser
229, Glu 356, and His 471. The three pairs of con-
served cysteine residues involved in intrachain disul-
fide bonding are also found as Cys 94–Cys 121, Cys
293–Cys 297, and Cys 431–Cys 562. Another cysteine
(Cys 616) near the carboxyl terminus of the sequence
probably mediates interchain disulfide bonding. Of
AChE from C. intestinalis A. Frederick et al.
1310 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS
the fourteen aromatic amino acids that line the cata-
lytic gorge of vertebrate AChE, 13 are conserved in
the C. intestinalis AChE (AChE1; Table 1). The
sequence shows 41% identity with the AChE from
T. californica.
The formation of the acyl pocket of C. intestinalis
AChE may more closely resemble that of vertebrate
AChE rather than invertebrate AChE (Fig. 1; see
molecular modeling below). However, the acyl pockets
of C. intestinalis and T. californica are clearly not iden-
tical because, as is the case for other invertebrates, there
is a deletion in the region of the acyl pocket of C. intes-
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
Tyr152 Phe176 Tyr121
Trp311 Ala336 Trp279
Choline binding site
and hydrophobic
site
Trp111 –
a
Trp84
Tyr161 Tyr185 Tyr130
Tyr359 Phe387 Phe330
Phe360 Ile388 Phe331
Acyl pocket Phe317 Tyr342 Phe288
Phe319 Glu346 Phe290
Val430 Phe450 Val400
Wall of gorge Trp145 Trp169 Trp114
Trp262 Tyr289 Trp233
Tyr363 Val391 Tyr334
Trp463 Cys484 Trp432
Tyr473 Ser498 Tyr442
a
There is a deletion in the CLUSTALW alignment in this region of the
sequence for AChE2.
Fig. 1. Amino acid residues surrounding the acyl pocket of some vertebrate and invertebrate acetylcholinesterases. This figure illustrates
the differences between the construction of the acyl pocket in vertebrate and invertebrate AChEs. The line separates the vertebrate and
invertebrate AChEs. The numbers at the top of the figure correspond to the amino acids in T. californica. In the vertebrates, the acyl pocket
is composed of Phe288 and Phe290. In the invertebrates, the acyl pocket phenylalanines homologous to the Phe290 and Val400 positions
form the acyl pocket (
CLUSTALW aligned the amino acid sequences [58]). The alignment of the sequence for C. intestinalis AChE was slightly
adjusted manually (the QE sequence) to emphasize the similarity with vertebrate AChEs. The GenBank accession nos are: Homo sapiens
(M55040), Bos taurus (BC123898), Mus musculus (X56518), R. norvegicus (S50879), Gallus gallus (U03472), Bungarus fasciatus (U54591),
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
exon,
as only one of the seven aromatic residues is preserved.
It is highly unlikely that this protein is an active AChE
because it is missing a member of the catalytic triad
and the main aromatic residue for binding of substrate.
Additionally, the protein would not be expected to pro-
duce all three globular forms because it does not con-
tain a WAT domain. However, it could represent a
GPI-anchored protein because it has a putative signal
sequence, and a putative hydrophobic C-terminus and
cleavage site (not shown). What, if any, role the protein
may play in the organism has not yet been determined;
although it shows highest homology with ChEs and not
other ChE-like adhesion molecules.
Kinetic characterization of recombinant ChE from
C. intestinalis expressed in vitro and native
enzyme expressed in vivo indicates the enzyme
is AChE
To determine the nature of the cholinesterase activity
of the recombinant C. intestinalis enzyme and to com-
pare it with the native AChE, we assayed the hydroly-
Fig. 2. Amino acid sequences of T peptides found in vertebrates and deuterostome and protostome invertebrates. Top, alignment of
T amino acid sequences: Vertebrates, H. sapiens (M55040), M. musculus (X56518), T. californica (X03439); deuterostome invertebrates,
C. intestinalis (TPA: BK006073), S. purpuratus (XM_775310); protosome invertebrates, Apysia californica (AASC01147222.1), C. elegans
(X75332). An alternatively spliced exon codes for the T peptide and numbering starts at the first amino acid of the peptide. The six
conserved aromatic amino acids of the WAT domain are indicated by
; the one nonconserved aromatic residue by h. The S. purpuratus
sequence is associated with a putative AChE; the A. californica sequence has not been associated with AChE. Sequences aligned with
CLUSTALW. Bottom: helical wheel representation of the WAT domain organized as an amphipathic a-helix [62]. The conserved aromatic,
hydrophobic (green diamonds) cluster at the top of the wheel. The arrow points to the nonconserved Tyr in the WAT domain of C. intestinalis
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
activity of the recombinant enzyme, we determined half
maximal inhibitory concentration (IC
50
) values of the
enzymes for the inhibitors (3aS-cis)-1,2,3,3a,8,8a-hexa-
hydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol methyl-
carbamate (physostigmine), which inhibits all
cholinesterases; [4-[5-[4-(dimethyl-prop-2-enyl-ammo-
nio)phenyl]-3-oxo-pentyl]phenyl]-dimethyl-prop-2-enyl-
azanium dibromide (BW284c51), which inhibits AChE
preferentially; and 10-(2-diethylaminopropyl) phenothi-
azine hydrochloride (ethopropazine) and N-[bis(pro-
pan-2-ylamino)phosphoryloxy-(propan-2-ylamino)phos-
phoryl]propan-2-amine (iso-OMPA), which inhibit
BuChE at low concentrations. Physostigmine and
BW284c51 inhibit the enzymes at lm concentrations;
by contrast, much higher concentrations of ethoprop-
azine and iso-OMPA are required for inhibition
(Fig. 4; Table 3). This pattern is characteristic of
AChE.
COS-7 cells transfected with cDNA for AChE
of C. intestinalis produce all three globular
molecular forms of AChE
To determine the molecular forms of AChE produced
in vitro by COS-7 cells transfected with the catalytic
subunit for C. intestinalis AChE, we performed vel-
ocity sedimentation on sucrose gradients in the pres-
ence and absence of Triton X-100. Cell extracts have
G
1
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
)
0
200
400
600
800
1000
1200
Fig. 3. Representative experiment showing concentration depen-
dencies for ATCh and BTCh hydrolysis by an extract of COS-7 mon-
key cells expressing recombinant C. intestinalis AChE cDNA.
Transfected COS-7 cells producing C. intestinalis AChE were
extracted in HIS buffer and assayed with ATCh (d) or BTCh (s)as
described in the Experimental procedures.
Table 2. Kinetic parameters for recombinant and native AChE from C. intestinalis. Data are the mean ± SE of four or more determinations.
Sources of enzyme: medium, enzyme secreted into the medium, usually 12 mL; cells, enzyme extracted with 5 mL of HIS buffer from the
COS-7 cells as described in the Experimental procedures; and organism, enzyme extracted from adult C. intestinalis, as described in the
Experimental procedures.
Source
V
max
ATCh
(mAb ⁄ min)
K
m
ATCh
(lM)
K
ss
ATCh
(mM) b
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
High concentrations of endogenous reducing compounds in the adult tissue
increased the background in the Ellman’s assay and, despite correction, obscured whatever low levels of BTCh hydrolysis there may have
been; kinetic parameters for BTCh hydrolysis could not be obtained.
A. Frederick et al. AChE from C. intestinalis
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1313
G
4
na
because the sedimentation coefficient of the
G
2
form also increases in the absence of detergent. For
extracts of adult C. intestinalis,G
1
a
and G
4
na
are seen
on the gradients. In both extracts and media, the
sedimentation coefficient of the G
4
form remains
unchanged (Fig. 5; Table 4).
COS-7 cells co-transfected with cDNAs for the
catalytic subunit of C. intestinalis and ColQ from
the rat produce the A
12
form of AChE
To determine whether the catalytic subunits of C. in-
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
Fractional AChE activity
1.00
0.75
0.50
0.25
0.00
Inhibitor (
M)
10
–8
10
–7
10
–6
10
–5
10
–4
10
–3
Fig. 4. Representative experiment showing concentration depen-
dencies for inhibition of ATCh hydrolysis by recombinant AChE
from C. intestinalis. Media from transfected COS-7 cells secreting
C. intestinalis AChE was collected and incubated with inhibitors for
20 min prior to being assayed for activity. The inhibitors used were
BW284c51 (d), physostigmine (s), ethopropazine (.), and iso-
OMPA (,).
Table 3. IC
50
values (lM) for inhibition of recombinant and native
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
forms of C. intestinalis AChE produced in vitro and in vivo. Medium
from COS-7 cells transfected with cDNA for the catalytic subunit
for C. intestinalis AChE, total HIS (d); extracts of the transfected
cells (h) and total HIS extracts of adult C. intestinalis tissue (
)
were analyzed on sucrose gradients in the presence (top) and
absence (bottom) of Triton X-100 as described in the Experimental
procedures.
AChE from C. intestinalis A. Frederick et al.
1314 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS
an AChE-like acyl pocket, a hydrophobic patch
(including the choline binding site), and an oxyanion
hole (see supplementary Fig. S2). The distance between
the acyl pocket phenylalanines, Phe317 and Phe319, is
3.7 A
˚
, the same as for T. californica AChE. However,
the volume of the catalytic gorge for C. intestinalis
AChE is 780 A
˚
3
, whereas the volume of the gorge for
T. californica AChE is 986 A
˚
3
.
Molecular modeling of monomeric C. intestinalis
AChE catalytic subunits with the PRAD domain
of ColQ indicates that the WAT domain of the
C. intestinalis AChE is capable of organizing the sub-
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.
Discussion
We have expressed in vitro a synthetic recombinant
ChE from the urochordate C. intestinalis. Based on
Table 4. Sedimentation coefficients of recombinant and native forms of AChE from C. intestinalis. Data are the mean ± SE of ‡ 8 determi-
nations for recombinant enzyme and three and four determinations for enzyme extracted from adult C. intestinalis in the presence and
absence of Triton X-100, respectively. Sources of enzyme are the same as in Table 1.
Conditions
Sedimentation coefficients
Extract Medium Organism
+Triton X-100 4.66 ± 0.17 10.78 ± 0.11 6.58 ± 0.14 10.61 ± 0.16 4.97 ± 0.13 11.15 ± 0.15
)Triton X-100 5.67 ± 0.15 10.88 ± 0.15 7.06 ± 0.24 10.80 ± 0.24 6.35 ± 0.25 11.01 ± 0.11
Molecular form G
1
a
G
4
na
G
2
a
G
4
na
G
1
a
G
4
na
Sedimentation coefficient
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
A
12
a
Estimated from residual activity.
b
Amphiphilic or non-amphiphilic
forms are not designated because the appropriate velocity sedi-
mentation experiments on sucrose gradients in the presence and
absence of Triton X-100 were not performed. The forms are
assumed to be G
1
a
and G
4
na
.
A. Frederick et al. AChE from C. intestinalis
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1315
substrate and inhibitor specificity, the enzyme is
AChE. The AChE is AChE
T
because transfected
COS-7 cells produce G
1
,G
2
, and G
4
forms. Co-
expression of C. intestinalis AChE catalytic subunit
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
previous studies of C. intestinalis demonstrating that
physostigmine and BW284c51 were effective inhibitors
of the activity, but that iso-OMPA was not [9,10].
The only IC
50
that can be obtained from these data is
for BW284c51 (approximately 1 lm) [9], which is vir-
tually identical to that found in the present study.
Not only does the congruence of the kinetic and phar-
macologic data indicate that C. intestinalis possesses
AChE, but it also argues that the cDNA expressed
in vitro in the present study corresponds to the gene
expressed in vivo.
Sequence analysis of important residues in the cata-
lytic gorge also supports the assertion that the enzyme
is AChE. Only one of the 14 aromatic amino acids
that line the catalytic gorge of T. californica and most
other vertebrate AChEs is missing in C. intestinalis
AChE, Tyr70, a member of the peripheral site, which
is replaced by Ile97. The K
ss
of C. intestinalis AChE
for ATCh is rather high and this substitution could
contribute to this value [29].
More interesting is the nature of the acyl pocket. In
all vertebrate AChEs, the acyl pocket is comprised of
two phenylalanines close to one another in the pri-
mary sequence. By contrast, for approximately 90%
of invertebrate AChEs, the acyl pocket is composed
A
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.
1316 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS
of two phenylalanines far apart in the primary
sequence, corresponding to Phe290 and Val400 in
T. californica AChE. The only known exception in
this subset of invertebrate AChEs is C. intestinalis
AChE, where the acyl pocket phenylalanines are
homologous to those of the vertebrates, suggesting
that the C. intestinalis acyl pocket is ancestral to that
of the vertebrates. This conclusion is confounded by
the acyl pocket conformations of the two acetyl-
cholinesterases from the cephalochordate amphioxus
(Branchiostoma floridae); the cephalochordates have
long been considered to be the sister group of the ver-
tebrates. ChE2 from this organism shows the typical
invertebrate acyl pocket structure, whereas ChE1
apparently has a novel acyl pocket, unlike the typical
vertebrate or invertebrate conformations [6,30,31]. The
echinoderms, urochordates (tunicates, C. intestinalis),
cephalochordates (amphioxus, B. floridae), and verte-
brates are members of the deuterostome branch of the
animal kingdom, with the echinoderms generally con-
sidered as the most basal of the groups, the urochor-
dates intermediate, and the cephalochordates closest
to the vertebrates [28,32,33]. However, recent data
from metaphylogenies and phylogenomics have chal-
lenged this view, with Blair and Hedges [34], Delsuc
et al. [35], and Vienne and Pontarotti [36] proposing
that the urochordates are actually the closest living
relatives of the vertebrates, with the cephalochordates
intermediate to the echinoderms. Our phylogenetic
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
a matter of speculation.
The AChE from C. intestinalis is AChE
T
and is
able to assemble into asymmetric forms
organized by vertebrate ColQ
Analysis of the carboxyl terminus sequence indicates
that the C. intestinalis AChE is AChE
T
, which should
be capable of forming the three globular forms: G
1
a
,
G
2
a
, and G
4
na
. When the catalytic subunit of AChE
from C. intestinalis was expressed in vitro,G
1
a
,G
2
a
, and
G
4
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
Y20A and Y20P decrease the amphipathic nature of
the T peptide a-helix and abolish the assembly of
secreted tetramers when catalytic subunits are co-
expressed in the presence or absence of a truncated,
soluble version of ColQ [39]. In the [WAT]
4
–PRAD
model of Dvir et al. [18] (PDB ID code 1VZJ), there is
an edge-on p-p interaction between the edge of Phe14
in WAT strand A and the face of Tyr20 in chain D.
This interaction is not observed for the other Phe14-
Tyr20 combinations. The Y20A and Y20P mutations
would disrupt this interaction and apparently destabi-
lize the tetramer. Clearly, this is not the case for the
AChE tetramer of C. intestinalis, which forms tetra-
mers in the absence and presence of ColQ. The WAT–
PRAD interaction of our tetrameric molecular model
is in good agreement with the corresponding structure
of Dvir et al. [18], and indicates that the side-chain of
Ser20 in strand D is oriented towards and in close
proximity to the edge of Phe14 in strand A. One
A. Frederick et al. AChE from C. intestinalis
FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1317
possibility is that the tetramer is stabilized by the for-
mation of a weak C–HÆO hydrogen bond between the
hydroxyl oxygen of Ser20 and a slightly polar C–H
group of the aromatic ring of Phe14. Such bonds were
first proposed in 1982 for phenylalanines in proteins
by Thomas et al. [40], and have received considerable
attention in recent years [41–44].
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-
mal kingdom indicates that it may be as old as and
conserved for ‡ 900 million years because it would
have had to evolve prior to the protostome-deutero-
stome split [34]. Interestingly, all of the phyla that
have the T sequence also have G
4
AChE, and use ace-
tylcholine as a neurotransmitter at their neuromuscular
junctions, suggesting both are a prerequisite for effi-
cient synaptic transmission at the junctions. Given the
recent research on the interaction between WAT
domains of AChE catalytic subunits and the PRAD
domains of ColQ and PRiMA [38,39,45,46], the fact
that G
4
AChE interacts with a noncatalytic subunit in
nematodes [19,20], the recent finding of small PRAD-
containing polypeptides associated with soluble
tetramers of vertebrate BuChE [47], and the apparent
ubiquity of the T domain, we propose that PRAD-
containing proteins mediate tetramerization of AChE
throughout evolution, with ColQ and PRiMA of the
vertebrates comprising just two of the many examples
of such proteins.
Experimental procedures
Materials
DMEM, fetal bovine serum, and OptiMEM medium were
purchased from Invitrogen (Carlsbad, CA, USA). FuGene
was obtained from Roche (Indianapolis, IN, USA). ATCh,
BTCh, BW284c51, 5-(3-carboxy-4nitro-phenyl)disulfanyl-2-
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 (http://genome.jgi-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 (http://bioweb.ensam.inra.fr/ESTHER/general?
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
pcDNA3.1 (Invitrogen) after linker sequences containing
EcoRI and XbaI restriction sites were added to the 5¢- and
3¢-ends of the cDNA, respectively, for ligation of the cDNA
into the expression plasmid. Double-strand DNA sequenc-
ing confirmed the sequence. The recombinant plasmid was
then used to transform competent Escherichia coli (XL1-
Blue; Stratagene, La Jolla, CA, USA). Qiagen maxi-preps
(Qiagen, Valancia, CA, USA) were used to obtain plasmid
DNA for transfections.
In vitro expression and extraction of enzymes
COS-7 monkey cells (American Type Culture Collection,
Manassas, VA, USA) were grown in DMEM containing
AChE from C. intestinalis A. Frederick et al.
1318 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS
10% fetal bovine serum. Cells were plated at a density of
2.5 · 10
6
cells ⁄ 75 cm
2
culture flask, incubated overnight,
and transferred to OptiMEM medium. FuGene was then
used to transfect the cells with 7.8 lg of DNA. For co-trans-
fection experiments, 7.8 lg of DNA for the catalytic subunit
and for R. norvegicus ColQ (GenBank accession no.
BC107386) was used. The cells were then incubated for 48 h
at 30 °C, before the medium was harvested and the cells were
extracted in 1–5 mL of high ionic strength (HIS) buffer:
10 mm NaHPO
4
,pH7,1m NaCl, 1% Triton X-100, 1 mm
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
Acetylcholinesterase activity was measured according to
the method of Ellman et al. [49] as modified by Doctor
et al. [50] in 100 mm NaHPO
4
, pH 7, 0.3 mm DTNB, and
167 mm NaCl; the final concentration of Triton X-100 was
0.17% for assays performed with cell extracts or media,
and 0.085% for extracts of adults. ATCh and BTCh were
used as substrates at various concentrations; for pharmaco-
logical analyses and assays of sucrose gradients, the con-
centration of ATCh was 1 mm. The kinetic parameters K
m
,
K
ss
, b, and V
max
, were determined as described by Radic
´
et al. [51] and Kaplan et al. [29]; the parameter, b, indi-
cates the relative catalytic efficiency of the two-substrate
bound complex compared to the single-substrate form. If
b < 1, the enzyme shows substrate inhibition; if b >1,
the enzyme shows substrate activation, and if b =1,
Michaelis–Menten kinetics is observed. sigmaplot (Systat
Software, San Jose, CA, USA) was used to fit the kinetic
data. It was not possible to determine the turnover number
k
cat
(V
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:
collagenase digestion
The molecular forms of AChE were analyzed by velocity
sedimentation in 5–25% isokinetic sucrose gradients
prepared in HIS buffer containing 1 mgÆmL
)1
BSA.
Sedimentation was in a Beckman SW 41 rotor at
30 000–37 000 r.p.m. for times satisfying the equation
[(r.p.m.)
2
· t (h)]=2.5 · 10
10
, as described previously [53].
Apparent sedimentation coefficients were calculated relative
to the sedimentation of catalase (11.3S). Data were plotted
as fractional activity of total AChE activity on the gradient
as a function of sedimentation coefficient. For collagenase
digestion, HIS extracts were adjusted to 10 mm CaCl
2
and
incubated at 37 °C for 1 h with or without 200 lgÆmL
)1
collagenase as described previously [54].
Molecular modeling
Molecular modeling was performed on an Indigo O2 com-
puter (Silicon Graphics, Sunnyvale, CA, USA) using the
discover and insight ii programs (Accelrys, San Diego,
CA, USA). The 3D structure of C. intestinalis monomeric
AChE was built using the Homology module of insight ii
and the crystal structure of Torpedo AChE (pdb index
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
the multiple alignment. For phylogenetic analysis, a multi-
ple sequence alignment and phylogenetic tree based on
the Neighbour-joining method were generated with
clustalx [59,60]. Bootstrap values for 1000 replicates are
indicated [61]. This alignment is deposited at the EMBL-
ALIGN database as ALIGN_001208.
Acknowledgements
This research was supported by a National Institutes
of Health Academic Research Award (no. 1 R15
GM072510-01) to LP. Additional support was pro-
vided by Birmingham-Southern College.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Alignment of C. intestinalis and T. californica
AChE sequences.
Fig. S2. Molecular model of the catalytic gorge of
C. intestinalis AChE. Some of the key residues com-
prising the gorge are shown.
Fig. S3. Phylogenetic tree inferred from the alignment
of amino acid sequences of vertebrate and invertebrate
ChEs.
This material is available as part of the online article
from http://www.blackwell-synergy.com
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
AChE from C. intestinalis A. Frederick et al.
1322 FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS

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