Báo cáo khoa học: Non-enzymatic developmental functions of acetylcholinesterase – the question of redundancy docx

Non-enzymatic developmental functions of
acetylcholinesterase – the question of redundancy
Glynis Johnson, Chrisna Swart and Samuel W. Moore
Divisions of Paediatric Surgery ⁄ Molecular Biology and Human Genetics, University of Stellenbosch, Tygerberg, South Africa
Acetylcholinesterase (AChE) is defined by its enzy-
matic role in the hydrolysis of the neurotransmitter
acetylcholine (ACh) in the synapse and neuromuscular
junction. It is also expressed in cells and tissues that
lack cholinergic innervation, for example, in the early
embryo [1]. This has suggested that AChE may have
non-classical functions, which may be broadly defined
as any function outside the context of the synapse or
neuromuscular junction. Such functions could be either
cholinergic (enzymatic) or non-cholinergic (presumably
mediated by structural sites). The latter possibility was
supported by the discovery that AChE is homologous
to a number of non-enzymatic cell adhesion and sig-
nalling molecules that are active in neural development
[2]. Evidence for non-cholinergic functions has been
sought, and it has been found that AChE is capable of
promoting cell adhesion and neurite outgrowth [3],
amyloidosis [4] and apoptosis [5] in vitro. Interactions
with a number of proteins and peptides have been
reported; these include laminin-111 [6,7], collagen IV
[6], fibronectin [8], the nicotinic acetylcholine receptor
[9], the prion protein [10] and the amyloid beta-peptide
Keywords
acetylcholinesterase; heparan sulfate;
laminin; neuroligin; perlecan
Correspondence
G. Johnson, Divisions of Paediatric

Surgery ⁄ Molecular Biology and Human
Genetics, Faculty of Health Sciences,
University of Stellenbosch, PO Box 19063,
Tygerberg 7505, South Africa
Fax: +27 21 933 7999
Tel: +27 21 938 9422
E-mail:
(Received 13 June 2008, revised 13 August
2008, accepted 14 August 2008)
doi:10.1111/j.1742-4658.2008.06644.x
Despite in vitro demonstrations of non-enzymatic morphogenetic functions
in acetylcholinesterase (AChE), the AChE knockout phenotype is milder
than might be expected, casting doubt upon the relevance of such functions
in vivo. Functional redundancy is a possible explanation. Using in vitro
findings that AChE is able to bind to laminin-111, together with detailed
information about the interaction sites, as well as an epitope analysis of
adhesion-inhibiting anti-AChE mAbs, we have used molecular docking and
bioinformatics techniques to explore this idea, investigating structurally
similar molecules that have a comparable spatiotemporal expression pat-
tern in the embryonic nervous system. On this basis, molecules with which
AChE could be redundant are the syndecans, glypicans, perlecan, the
receptor tyrosine kinase Mer, and the low-density lipoprotein receptor. It
is also highly likely that AChE may be redundant with the homologous
neuroligins, although there is no evidence that the latter are expressed
before synaptogenesis. AChE was observed to dock with Gas6, the ligand
for Mer, as well as with apolipoprotein E3 (but not apolipoprotein E4),
both at the same site as the laminin interaction. These findings suggest that
AChE may show direct functional redundancy with one or more of these
molecules; it is also possible that it may itself have a unique function in the
stabilization of the basement membrane. As basement membrane molecules

are characterized by multiple molecular interactions, each contributing
cumulatively to the construction and stability of the network, this may
account for AChE’s apparently promiscuous interactions, and also for the
survival of the knockout.
Abbreviations
ACh, acetylcholine; AChE, acetylcholinesterase; ApoE, apolipoprotein E; BChE, butyrylcholinesterase; BM, basement membrane; ECM,
extracellular matrix; FGF, fibroblast growth factor; HSPG, heparan sulfate proteoglycan; LDL, low-density lipoprotein; PRiMA, proline-rich
membrane anchor.
FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS 5129
[11]. A number of structural sites on AChE that medi-
ate these interactions have also been described
[9,11,12]. The addition of anti-AChE mAbs to neural
cells was found to ablate cell adhesion and neurite out-
growth [13,14] and also to induce apoptosis [14], which
suggested that the site on AChE recognized by the
antibodies is necessary for both adhesion and survival.
By contrast, and seemingly contrary to the in vitro
evidence, the AChE knockout mouse survives. It was
found that the related cholinesterase, butyrylcholin-
esterase (BChE), compensates, to some degree, for the
lack of AChE in synapses and neuromuscular junc-
tions [15]. BChE, however, has not generally been
observed to promote non-cholinergic cell adhesion
[14,16], and so is unlikely to replace AChE in this
context. The knockout has severe abnormalities: it is
largely immobile, with deficiencies in muscle structure
and function [17], requires a liquid diet in order to sur-
vive, has significant behavioural abnormalities, and has
nervous system defects, in particular, in the develop-
ment of the eye [18]. The last-named, in particular,

suggests the presence of non-classical functions, and
that AChE is indispensable in this context. However,
another in vivo study, using a catalytically inactive, but
otherwise structurally intact, AChE in the zebrafish
(which does not have BChE) showed little evidence for
non-cholinergic developmental functions [19]. The
zebrafish study did, however, show evidence of choli-
nergic non-classical functions. Neurotransmitters are
known to have morphogenetic activity; ACh in partic-
ular, inhibits cell adhesion and neurite outgrowth [20].
AChE, by hydrolysing and thus removing ACh, is
indirectly able to promote the opposite effect, namely,
the stimulation of cell adhesion and neurite outgrowth.
Nevertheless, the lack of indisputable in vivo evidence
for non-cholinergic functions has led to a questioning
of their relevance [21].
There is therefore a discrepancy between the in vivo
evidence of the knockouts and the documented effects
and interactions in vitro. Presumably, AChE is indeed
capable of producing the effects seen in vitro. A possi-
ble explanation, and one that is able to reconcile
both sides of the debate, is that of functional redun-
dancy. Redundancy appears to be fairly common in
higher organisms, as suggested by the number of
knockouts with no apparent phenotype. It seems to
be more frequent in proteins expressed in develop-
mental, rather than ‘housekeeping’, contexts. This
may be due to the tendency for developmental
proteins to be expressed in precise spatiotemporal
patterns with a relatively smaller margin for error;

redundancy may promote robustness by providing a
backup or fail-safe device.
Analysis of AChE in embryonic development sug-
gests that there are two discrete phases of expression:
the morphogenetic, corresponding to the migration
and differentiation of neural crest cells; and the syn-
aptogenetic, corresponding to synapse formation [1].
The start of differentiation is characterized by an
increase in AChE expression. This involves the assem-
bly of largely intracellular monomeric forms into tetra-
mers, which are initially secreted. Concomitant with
neurite outgrowth is a shift in expression from secreted
to membrane-associated tetramers [22]. The tetramers
are anchored in the membrane by an association
with the proline-rich membrane anchor (PRiMA), a
type I integral membrane protein [23]. The PRiMA
has a full transmembrane domain, as well as a short
31-residue cytoplasmic domain; it is not known, how-
ever, whether the PRiMA interacts with cytoplasmic
molecules or with the cytoskeleton. Cells transfected
with AChE cDNA show high AChE immunoreactivity
on the outer margins of cell bodies and growth cones
[24]. A number of in vitro studies has shown that pro-
viding AChE in the culture medium or as a plate-coat-
ing induces neurite outgrowth [14,25,26]. This suggests
the possibility that AChE may be able to exert a mor-
phogenetic effect from a location exterior to the cell as
well, perhaps corresponding to the secreted tetrameric
forms of early differentiation.
The extracellular matrix (ECM) is a web-like net-

work of proteins and proteoglycans that provides the
cell with both structural support and information
about its environment. The basement membrane (BM)
is the layer of specialized ECM immediately surround-
ing the cell. The most abundant components of the
BM are laminins (laminin-111 in the developing ner-
vous system) and collagen IV, which interact and self-
associate to form a scaffold to which other ECM
components, such as nidogen ⁄ entactin, fibronectin and
perlecan, bind [27]. ECM molecules tend to be large,
and the majority are modular, many with domains
resembling those of other ECM molecules; this results
in a multiplicity of binding sites and interactions, pro-
ducing a strong and resilient molecular web which is
furthermore anchored to the cell by interactions with
cell-surface receptors, in particular, the integrins and
a-dystroglycan [28]. The BM and the ECM in general
provide the milieu through which growth factors dif-
fuse, neural precursors migrate and through which the
developing axons, or neurites, grow. For these pro-
cesses to occur, there must be informative associations
between the cell, via its surface receptors, and the BM.
It is well documented that BM components are
involved in such associations and promote cell migra-
tion and neurite outgrowth [29].
Acetylcholinesterase redundancy G. Johnson et al.
5130 FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS
We have recently identified the epitopes of seven
adhesion-inhibiting anti-AChE mAbs by long synthetic
peptides, and also by a microarray of short overlap-

ping conformationally constrained peptides [12]. These
antibodies showed a common epitope, centred on the
40
PPMGPRRFL and
90
RELSED sequences, which are
linked by a salt bridge between
46
R and
94
E. Docking
of the mouse AChE and laminin structures showed the
major interaction site on AChE to be
90
RELSED, with
contributions from
40
PPM,
46
R and
60
VDATT
(Fig. 1A). The interaction site on laminin was also
conformational, consisting of a number of clusters:
2718
VRKRL,
2738
YY,
2789
YIKRK and

2819
RK in the
alpha1 G4 domain (Fig. 1B).
In this study, we used bioinformatics and in silico
docking to explore the possibility of functional redun-
dancy. We took the premise that, when the mAbs
ablate adhesion and induce apoptosis in neuroblas-
toma cells in vitro, they may be interacting, not only
with AChE, but also with another molecule or mole-
cules that have similar sites. AChE may function as a
backup to these molecules in vivo. We limited our
investigation specifically to the AChE–laminin interac-
tion, for which we have detailed information, and to
those molecules expressed during the migration and
differentiation ⁄ neurite outgrowth stages of neural
development. This would be a preliminary step to
defining ways in which AChE may indeed function
non-cholinergically in vivo.
Results and Discussion
Clues from the laminin site
The site on laminin to which AChE binds overlaps
with the heparin-binding site [30]. This site was previ-
ously identified with the peptide AG73 (which also
binds AChE), which forms part of the site [31]. AChE
competes with heparan sulfate for binding to laminin
[12], suggesting that AChE may be redundant with
heparin-containing molecules.
Many proteoglycans are expressed during neural
development, both in the ECM and on the cell surface
[32]. Although much of our knowledge of proteoglycan

expression patterns and function is sketchy to say the
least, there is accumulating evidence that they play
important roles in development, promoting cell adhe-
sion, cell–cell interactions and growth factor signalling
[33,34]. The protein core may be decorated with hepa-
ran, chondroitin, or less frequently, dermatan sulfate,
alone or in combination. Intermolecular interactions
have been shown to occur both by the sugars and the
protein core [32,33]; variations in sugar composition
and length, together with the diversity of proteins to
which they are attached, provide a multiplicity of
potential binding and signalling structures. Heparan
A
B
Fig. 1. Interacting sites of mouse AChE and the mouse laminin
alpha1 chain. (A) Laminin-binding site on AChE. Detail of the mouse
AChE dimer (1J06.pdb) showing the peripheral anionic site residues
in yellow, with the arrow indicating the direction of the active site
gorge. The laminin-binding residues are shown in magenta. (B)
AChE-binding site on laminin alpha1. Detail of the G4 domain of the
mouse laminin alpha1 G4-5 domain pair (2JD4.pdb). Residues inter-
acting with AChE are shown in cyan.
G. Johnson et al. Acetylcholinesterase redundancy
FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS 5131
sulfate proteoglycans (HSPGs) in the developing
nervous system have been observed to bind to a heter-
ogeneous group of molecules, including proteins
(NCAM, slit proteins, laminin, fibronectin and the
thrombospondins) and growth factors [members of the
fibroblast growth factor (FGF), Wnt, transforming

growth factor b and Hedgehog families and pleiotro-
phin] [33]. Binding may occur exclusively by the
heparan sulfate chains, or there may be contributions
from the protein core as well. HSPGs that show
similar spatiotemporal expression to AChE are the
membrane-associated syndecans, glypicans and
testicans, and the extracellular molecules perlecan,
agrin and collagen XVIII (Table 1).
The syndecans are a family of four transmembrane
receptors that are expressed in a variety of tissues
and appear to have multiple biological functions [35].
Syndecans carry both heparan and chondroitin sul-
fate chains, and their extracellular domains may be
shed as functional molecules into the matrix [36].
Although all four syndecans are expressed in the
developing nervous system, there are differences in
their spatiotemporal distribution, and it is likely that
they have different functions. Syndecans have been
observed to bind various growth factors, as well as
ECM molecules and LDL [37]. Knockouts of synde-
cans show no obvious phenotypes [32]. Syndecan-1
has been shown to bind laminin-111 through interac-
tion of the heparan sulfate with the AG73 site in the
LG4 domain [38].
The glypicans are glycosylphosphatidylinositol-
linked membrane HSPGs that appear to play impor-
tant roles in cell growth and differentiation [39]. Like
the syndecans, individual glypicans also show differ-
ences in their developmental expression patterns, sug-
gesting distinct functions; glypicans also modulate

growth factor signalling through their heparan sulfate
chains. Glypican-2 is exclusive to the nervous system,
and has been shown to bind laminin-111 in vitro [40],
as has glypican-1. There is no documentation of lami-
nin binding by other members of the glypican family.
The testicans are a subgroup of the BM-40 ⁄
SPARC ⁄ osteonectin family of modular proteins. There
is no documented evidence of them binding laminin.
They have an inhibitory effect on neurite outgrowth
[41], which would suggest they are unlikely to show
redundancy with AChE.
Perlecan is a large multidomain HSPG that cross-
links many cell-surface and ECM components. Apart
from its role in the formation of the basement
Table 1. HSPGs in the developing nervous system. The information is taken from [32–34], unless otherwise indicated. Notes
A–F
indicate
the relevant references.
Family HSPG Expression
Ligands
Reference
Growth factors Proteins
Syndecans Syndecan-1 Cell surface FGF family transforming growth
factor b family pleiotrophin
Laminin, fibronectin, tenascin-C
a
, LDL
ba
[39]
b

[55]
Syndecan-2 Cell surface FGF2 Laminin, synbindin
c
, fibronectin
c
[59]
Syndecan-3 Cell surface FGF2, midkine, pleiotrophin Laminin, EGFR
dd
[60]
Syndecan-4 Cell surface FGF family Laminin, synbindin
c
, fibronectin
c
[59]
Glypicans GLP-1 Cell surface FGF family Laminin, slit-1 slit-2
GLP-2 Cell surface FGF2 Laminin, thrombospondin
GLP-3 Cell surface IGFII
e
FGF2
e
[61]
GLP-4 Cell surface FGF family
GLP-5 Cell surface
GLP-6 Cell surface
Testicans Testican-1 Cell surface
Testican-2 Cell surface
Testican-3 Cell surface
Perlecan ECM Laminin, collagen, fibronectin,
tenascin, amyloid precursor
protein

f
f
[45]
Agrin ECM FGF family Laminin, NCAM, tenascin-C,
thrombospondin
Collagen XVIII ECM Laminin, heparan sulfate
Acetylcholinesterase redundancy G. Johnson et al.
5132 FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS
membrane, it is believed to support various biological
functions including cell adhesion, growth-factor bind-
ing and apoptosis [42]. It is expressed from very early
stages of development. Perlecan consists of five
domains including domain I which contains the
heparan sulfate attachment sites, domain II contain-
ing LDL receptor repeats and domain V which is
homologous to the laminin G domains. Perlecan is
known to bind laminin through the AG73 site [43].
Perlecan also binds AChE, interacting with the ColQ
collagen-like tail associated with the asymmetric
AChE isoforms in the neuromuscular junction [44].
The ColQ-containing isoforms are not, however,
expressed during the earlier stages of neural develop-
ment. A recent study [45] observed colocalization of
AChE and perlecan near membrane protrusion sites
in fibroblasts and astrocytes, with results suggesting
the possibility of interactions with amyloid precursor
protein. Colocalization may indicate the presence of
functional redundancy.
Agrin is a multidomain HSPG that is best known
for its role in the clustering of ACh receptors during

synaptogenesis. It binds various molecules, including
laminin-111, by both heparan sulfate-dependent and
-independent means [32,33]. The interaction site on
laminin, however, does not correspond to the heparin-
binding site AG73 in the LG4 domain [46]. Further-
more, agrin, when used as a substrate, inhibits rather
than enhances neurite outgrowth. Both these factors
argue against agrin as functionally redundant with
AChE.
Collagen XVIII, and its cleavage product endostatin,
are components of the BM with structural characteris-
tics of both proteoglycans and collagen. Collagen
XVIII acts as a ligand for neural receptor tyrosine
phosphatases, an interaction that modulates axon
growth [47]. It binds laminin, albeit not at the heparin-
binding site, and also itself binds heparan sulfate on
the cell surface [47]. These factors suggest that collagen
XVIII is an unlikely candidate as an AChE-redundant
molecule.
Clues from the AChE site
Homologous proteins
AChE belongs to the a ⁄ b hydrolase fold family of pro-
teins, which includes the cholinesterases (AChE and
BChE), the cholinesterase-domain proteins (the neuro-
ligins, neurotactin, glutactin, gliotactin, the Dictyosteli-
um crystal protein and thyroglobulin), as well as the
carboxylesterases and lipases [2]. Neurotactin, glutactin
and gliotactin are invertebrate proteins, whereas the
neuroligins are expressed in vertebrates.
The AChE site

90
RELSED falls partly within a
carboxylesterase type b signature 2 (signature sequence
EDCLYLNVWTP; ProSite pattern PS00941). This
signature is strongly conserved throughout the
a ⁄ b hydrolase fold family and occurs in the sequence
surrounding a cysteine involved in a disulfide bond.
This sequence conservation implies that at least part of
the
90
RELSED site is conserved in the cholinesterase-
domain proteins also. Additional conserved residues
are found in the
40
PPMGPRRFL sequence where R46
is conserved, as it forms a salt bridge with E94.
Prolines 40 and 41 are also conserved (Fig. 2).
Although BChE is closely homologous to AChE
(70% identity), it does not promote cell adhesion
[14,16]. It also does not bind laminin in vitro [6], nor do
Fig. 2. Sequence alignment of neuroligins 1–4, AChE and BChE. All sequences are human. Conserved residues are indicated by asterisks,
and conservative replacements by dots. The residues (and their equivalents) forming the laminin-binding site in AChE are shown in bold.
Alignment was carried out using
CLUSTALW.
G. Johnson et al. Acetylcholinesterase redundancy
FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS 5133
the proteins dock. The neuroligins are a group of four
transmembrane proteins, located in the postsynaptic
membrane [48]. They form an adhesion complex with
b-neurexins in the presynaptic membrane, promoting

the formation of the synapse. The extracellular domain
of neuroligin-1 shows 34% homology to AChE, with
clear resemblances in both the
90
RELSED
(
147
QDQSED) and
40
PPMGPRRFL (
88
PPTFERRFQ)
sequences (Fig. 2). It has been proposed that neuroligin-
1 and AChE may be functionally redundant [49], with
both binding to b-neurexin. Although this was not
confirmed in a subsequent study [50], the neurexins
show considerable alternative splicing, and it is possible
that isoforms other than those tested bind. The neuro-
ligins, however, do not appear to be expressed before
synaptogenesis, so would not be capable of redundancy
with AChE at earlier stages of development.
Searches for similar motifs in other proteins
Searches for the
40
PPMGPRRFL sequence (and equiva-
lents with conservative replacements) showed only vari-
ous AChEs and neuroligins from a number of species.
Searches for the
90
RELSED sequence (and equiva-

lents with conservative replacements) in neural mole-
cules yielded the syntaxins, ligatin, proto-oncogene
receptor tyrosine kinase Mer, perlecan and the LDL
receptor. Of these, only Mer, perlecan and LDL recep-
tor are expressed during migration and differentiation.
Searches for the subsidiary
60
VDATT motif also
involved in AChE’s interaction with laminin yielded a
large number of candidates. The subset of developmen-
tally associated neural proteins with both the
90
REL-
SED and
60
VDATT motifs was considerably smaller:
perlecan and Mer. It would appear from the position of
the two motifs in the perlecan sequence that they may
be situated far apart. Unfortunately, the structure of
perlecan has not been solved, so this cannot be verified.
Although the structure of the relevant part of Mer
has also not been solved, it appears from the sequence
that the motifs may be relatively close. Mer belongs to
the Ax1 ⁄ Sky ⁄ Mer family of receptor tyrosine kinases,
and is expressed in both embryonic and mature
nervous tissue [51]. Mer appears to induce both cell
adhesion and flattening, and, in combination with
interleukin-3, promotes differentiation. Unlike many
receptor tyrosine kinases, it does not appear to stimu-
late proliferation [52].

Gas6, the product of the growth arrest-specific
gene 6, is a ligand for Mer, as well as for Ax1 and
Sky. It contains two laminin-like G domains, in which
the receptor-binding site is located. We investigated
docking of Gas6 with AChE. It was observed that
Gas6 docks with AChE in the same position as lami-
nin (Fig. 3A). The AChE
90
RELSED motif lies within
2A of Gas6 residues 296-298 (YLG) and 306-309
(VIRL). This site is essentially identical to that
described for Gas6 binding to Ax1 [53]. The AChE
40
PPMGPRRFL peptide lies within 2A
˚
of Gas6
A
B
Fig. 3. Docking of AChE with Gas6 and apolipoprotein E3. (A)
Detail of the docking of the mouse AChE dimer (1J06.pdb) with
human Gas6 (1H30.pdb). AChE is shown in grey, and Gas6 in cyan.
On AChE, the peripheral anionic site residues are shown in yellow,
and the residues 40–42, 46, 60–64 and 90–95 in black. (B) Detail
of the docking of the mouse AChE dimer (1J06.pdb) with human
apolipoprotein E3 (1LPE.pdb). AChE is coloured grey, and apoE3,
cyan. Peripheral anionic site residues are shown in yellow, and the
residues 40–42, 46, 60–64 and 90–95 in magenta.
Acetylcholinesterase redundancy G. Johnson et al.
5134 FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS
residues 339–345 (GMQDSW) as well as F428 and

D432, and the
60
VDATT motif within 2A
˚
of G298
and R299, as well as 329–332 (DPEG), 350–351 (LR)
and 437–440 (IPR). It would thus appear to be a pos-
sibility that AChE and Mer may be functionally
redundant.
The LDL receptor pentapeptide DGSDE
Low-density lipoprotein domain repeats are found in a
number of molecules, including perlecan. Their func-
tion is not known. In the LDL receptor itself, these
regions have been identified as involved in the binding
of LDL. A sequence that is especially important is the
conserved pentapeptide DGSDE [54]. This sequence is
remarkably similar to the AChE
91
ELSED sequence.
Furthermore, LDL, the ligand that binds to DGSDE,
is also known to bind heparin, and it appears that the
AChE site resembles heparin as both bind to the same
site on laminin. It has been reported [55] that LDL
binds syndecan-1.
Lipoproteins are implicated in neurite outgrowth
and plasticity, as well as in the pathology of Alzhei-
mer’s disease, where the presence of the apoE4 allele is
associated with increased risk and earlier age of onset
of the disease [56]. Apolipoprotein E3 (ApoE3) pro-
motes neurite outgrowth, whereas apoE4 inhibits it

[57]; however, the mechanisms by which this occurs
are unclear. ApoE binds the amyloid beta-peptide and
colocalises with amyloid deposits. Although both iso-
forms have been observed to bind, apoE4 binds with
greater avidity.
We investigated docking of the apoE isoforms
with AChE (1LPE.pdb and 1LE4.pdb; apoE3 and
apoE4, respectively). We found that AChE docked
with apoE3 (Fig. 3B) again via the same site that
binds laminin. ApoE residues lying within 2A
˚
of the
AChE
90
RELSED motif were R142, K143, R145
and K146, while those within 2A
˚
of the
40
PPMGPRR sequence were W34, R38, R145 and
L149. Those within 2A
˚
of the
60
VDATT sequence
were L43, Q48, W118, E131, L133, R134, V135 and
R136. The receptor-binding region of apoE has been
localized between residues 135 and 151 [58], which is
the same region that docks with the AChE site.
Many of these residues are basic. By contrast, apoE4

does not dock with AChE.
Conclusions
Redundancy would explain the apparent inconsistency
between the in vitro findings detailing non-cholinergic
functions and the evidence from the knockout models.
In this study, we have concentrated on the develop-
mental functions attributed to AChE during neural
crest cell migration and differentiation and on the
AChE–laminin-111 interaction.
Candidate molecules on the cell surface are the syn-
decans and glypicans, by virtue of their heparan sulfate
chains. The lack of comprehensive information on the
developmental expression and interactions of HSPGs
makes it difficult to narrow the field of possibilities.
The neuroligins are also strong candidates, based
on their homology with AChE, although there is no
documented evidence that they are expressed before
synaptogenesis. Another cell-surface receptor is the
receptor tyrosine kinase Mer, which has similar peptide
motifs to AChE, as does the LDL receptor. The only
candidate for redundancy in the ECM is perlecan,
by the double virtue of its heparan sulfate chains
and sequence similarity to both AChE and the LDL
receptor.
Extracellular matrix molecules characteristically
demonstrate multiple interactions, by means of various
sites. Many are modular with several types of domains;
a number have laminin G-like domains, thus re-
sembling the laminin site with which AChE interacts
in vitro. These include agrin, pentraxin, slit, serum

amyloid P component, Gas6 and b-neurexin. Docking
results indicate that AChE may bind Gas6, and an
interaction of AChE with b-neurexin has been postu-
lated on the basis of the AChE-neuroligin homology
[49]. AChE has been found to interact with a number
of ECM molecules: laminin, collagen IV and fibro-
nectin, as well as the amyloid beta-peptide, and also
appears to have a number of interaction sites itself.
AChE thus, in its potential for multiple interactions,
resembles ECM molecules. Interestingly, a majority of
the molecules – the amyloid beta-peptide, laminin,
collagen, fibronectin, perlecan, various HSPGs, apoE,
agrin, serum amyloid P component – with which
AChE interacts or may interact, are found in amyloid
deposits.
From an evolutionary perspective, the cholinesterases,
cholinesterase-domain proteins and ACh appear to
have been around for a very long time: AChE and
ACh, in particular, are found in bacteria, algae and
protozoa as well as, as far as is known, throughout the
plant and animal kingdoms. Cholinesterase-domain
cell adhesion molecules have been described not only
in mammals and insects, but also in the slime mould
Dictyostelium, suggesting that the split between
enzymes and non-enzymes may have occurred in the
earliest life-forms. Presumably, the common ancestor
had both enzymatic and adhesive characteristics, and
the cholinesterase-domain protein branch of the family
G. Johnson et al. Acetylcholinesterase redundancy
FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS 5135

specialized in cell adhesion and signalling, losing their
catalytic function. AChE, however, retained and
perfected its enzymatic ability, while apparently at the
same time retaining its adhesive capability. Although
early organisms did not have nervous systems, it is
possible that ACh and AChE may have become
involved in morphogenesis. It is thus possible that
AChE’s morphogenetic functions, both enzymatic and
non-enzymatic, may be more ancient than its synaptic
role. Such presumed antiquity suggests there may have
been a distinct selective advantage in retaining these
functions, that they fulfill a definite role and are
neither trivial nor fortuitous.
The bioinformatic evidence presented here indicates
that AChE might mimic functions of the syndecans,
glypicans, Mer or the LDL receptor on the cell mem-
brane, or of perlecan in the BM. This could result in
functional redundancy in the strict sense of the word,
with one molecule substituting directly for another.
This is supported by the findings with antibodies,
where incubation of cells with antibodies resulted in a
loss of adhesion followed by apoptosis, indicating the
blocking of an essential site. It could also, however, be
something less precise: that AChE, through its ability
(demonstrated and postulated) to interact with a vari-
ety of matrix molecules, simply acts to enhance the
stability of the BM. This would be advantageous, so
presumably would have been retained by natural selec-
tion, and would also account for AChE’s ability to
promote cell adhesion and neurite outgrowth, both of

which depend heavily on a favourable matrix. It is also
entirely possible that AChE may function in both
ways, as a direct backup molecule and as an enhancer
of BM stability. The findings also suggest that AChE,
through its multiple interactions, may play a significant
role in amyloidosis.
Experimental procedures
The identification of similar structures was carried out on
ProSite ( The
sequences of mouse (NP 033729) and human (P22303)
AChE were used. Other sequences used were mouse laminin
(NP 032506), human neuroligin 1 (NP 055747), mouse neu-
roligin 1 (NP 619607), human receptor tyrosine kinase Mer
(NP 006334), human perlecan (P98160), mouse perlecan
(Q05793) and human LDL receptor (NP 000518).
Docking was performed by hex 4.5. This program uses
rigid-body docking, and spherical polar Fourier correla-
tions to accelerate docking. Structures were downloaded
from the Protein Data Bank ( />mouse AChE dimer (1J06.pdb), mouse laminin alpha1 G4-5
domain pair (2JD4.pdb), C-terminal LG domain pair of
human Gas6 (1H30.pdb) and the LDL receptor binding
domain of human apolipoprotein E3 (1LPE.pdb) and
human apolipoprotein E4 (1LE4.pdb).
Acknowledgements
We thank the National Research Foundation, the
Medical Research Council of South Africa, and the
Harry Crossley Foundation of the University of Stel-
lenbosch for financial support.
References
1 Layer PG & Willbold E (1995) Novel functions of

cholinesterases in development, physiology and disease.
Prog Histochem Cytochem 29, 1–95.
2 Holmquist M (2000) Alpha ⁄ beta-hydrolase fold
enzymes: structures, functions and mechanisms. Curr
Protein Peptide Sci 1, 209–235.
3 Paraoanu LE & Layer PG (2008) Acetylcholinesterase
in cell adhesion, neurite outgrowth and network forma-
tion. FEBS J 275, 618–625.
4 Inestrosa NC, Dinamarca MC & Alvarez A (2008)
Amyloid–cholinesterase interactions: implications for
Alzheimer’s disease. FEBS J 275, 625–632.
5 Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH,
Guo LH, Alemany M, Zhang LY & Shi YF (2002)
Induction of acetylcholinesterase expression during
apoptosis in various cell types. Cell Death Differ 9,
790–800.
6 Johnson G & Moore SW (2003) Human acetylcholines-
terase binds to mouse laminin-1 and collagen IV by an
electrostatic mechanism at the peripheral anionic site.
Neurosci Lett 337, 37–40.
7 Paraoanu LE & Layer PG (2004) Mouse acetylcholines-
terase interacts in yeast with the extracellular matrix
component laminin-1 beta. FEBS Lett 576, 161–164.
8 Giordano C, Poiana G, Augusti-Tocco G & Biagioni S
(2007) Acetylcholinesterase modulates neurite out-
growth on fibronectin. Biochem Biophys Res Commun
356, 398–404.
9 Greenfield SA, Day T, Mann EO & Bermudez I (2004)
A novel peptide modulates alpha7 nicotinic receptor
responses: implications for a possible trophic:toxic

mechanism within the brain. J Neurochem 90, 325–331.
10 Pera M, Roman S, Ratia M, Camps P, Munoz-Torrero
D, Colombo L, Manzoni C, Salmona M, Badia A &
Clos MV (2006) Acetylcholinesterase triggers the aggre-
gation of PrP 106–126. Biochem Biophys Res Commun
346, 89–94.
11 De Ferrari GV, Canales MA, Shin I, Weiner LM,
Silman I & Inestrosa NC (2001) A structural motif of
acetylcholinesterase that promotes amyloid beta-peptide
fibril formation. Biochemistry 40, 10447–10457.
Acetylcholinesterase redundancy G. Johnson et al.
5136 FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS
12 Johnson G, Swart C & Moore SW (2008) Interaction of
acetylcholinesterase with the G4 domain of the laminin
alpha1 chain. Biochem J 411, 507–514.
13 Sharma KV & Bigbee JW (1998) Acetylcholinesterase
antibody treatment results in neurite detachment and
reduced outgrowth from cultured neurons: further
evidence for a cell adhesive role for neuronal acetyl-
cholinesterase. J Neurosci Res 53, 454–464.
14 Johnson G & Moore SW (2000) Cholinesterases
modulate cell adhesion in human neuroblastoma
cells in vitro. Int J Dev Neurosci 18, 781–790.
15 Xie W, Stribley JA, Chatonnet A, Wilder PJ, Rizzino A,
McComb RD, Taylor P, Hinrichs SH & Lockridge O
(2000) Postnatal developmental delay and supersensi-
tivity to organophosphate in gene-targeted mice lacking
acetylcholinesterase. J Pharmacol Exp Ther 292, 896–
902.
16 Mack A & Robitzki A (2000) The key role of butyry-

lcholinesterase during neurogenesis and neural disor-
ders: and antisense-5¢-butyrylcholinesterase-DNA study.
Prog Neurobiol 60, 607–628.
17 Vignaud A, Fougerousse F, Mouisel E, Bertrand C,
Bonafas B, Molgo J, Ferry A & Chatonnet A (2008)
Genetic ablation of acetylcholinesterase alters muscle
function in mice. Chem Biol Interact doi: 10.1016/
j.cbi.2008.04.035.
18 Bytyqi AH, Lockridge O, Duysen E, Wang Y, Wolfrum
U & Layer PG (2004) Impaired formation of the inner
retina in an AChE knockout mouse results in degenera-
tion of all photoreceptors. Eur J Neurosci 20, 2953–
2962.
19 Behra M, Cousin X, Bertrand C, Vonesch JL, Biell-
mann D, Chatonnet A & Strahle U (2002) Acetylcholin-
esterase is required for neuroanl and muscular
development in the zebrafish embryo. Nat Neurosci 5,
111–118.
20 Biagioni S, Tata AM, De Jaco A & Augusti-Tocco G
(2000) Acetylcholine synthesis and neuron differentia-
tion. Int J Dev Biol 44, 689–697.
21 Cousin X, Strahle U & Chatonnet A (2005) Are there
non-catalytic functions of acetylcholinesterases? Lessons
from mutant animal models. Bioessays 27, 189–200.
22 Ferrand C, Clarons D, Delteil C & Weber M.J (1986)
Cellular localization of the molecular forms of acetyl-
cholinesterase in primary cultures of rat sympathetic
neurons and analysis of the secreted enzyme. J Neuro-
chem 46, 349–358.
23 Perrier AL, Massoulie J & Krejci E (2002) PriMA: the

membrane anchor of acetylcholinesterase in the brain.
Neuron 33, 275–285.
24 Karpel R, Sternfeld M, Ginzberg D, Guhl E, Graess-
mann A & Soreq H (1996) Overexpression of alterna-
tive human acetylcholinesterase forms modulates
process extensions in cultured glioma cells. J Neurochem
66, 114–123.
25 Bigbee JW, Sharma KV, Chan EL & Bogler O (2000)
Evidence for the direct role of acetylcholinesterase in
neurite outgrowth in primary dorsal root ganglion
nerves. Brain Res 861, 354–362.
26 Olivera S, Rodriguez-Ithurralde D & Henley JM (2003)
Acetylcholinesterase promotes neurite elongation, syn-
apse formation and surface expression of AMPA recep-
tors in hippocampal neurones. Mol Cell Neurosci 23,
96–106.
27 Timpl R & Brown JC (1996) Supramolecular assembly
of basement membranes. Bioessays 18, 123–132.
28 Talts JF, Andac Z, Gohring W, Brancaccio A & Timpl
R (1999) Binding of the G domains of laminin alpha1
and alpha2 chains and perlecan to heparin, sulfatides,
alpha-dystroglycan and several extracellular matrix
proteins. EMBO J 18, 863–870.
29 Perris RM & Perissinotto D (2000) Role of the extracel-
lular matrix during neural crest cell migration. Mech
Dev 95
, 3–21.
30 Harrison D, Hussain SA, Combs AC, Ervasti JM,
Yurchenco PD & Hohenester E (2007) Crystal structure
and cell surface anchorage sites of laminin alpha1 G4-5.

J Biol Chem 282, 11573–11581.
31 Hoffman MP, Engbring JA, Nielsen PK, Vargas J,
Steinberg Z, Karmand AJ, Nomizu M, Yamada Y &
Kleinman HK (2001) Cell type-specific differences in
glycosaminoglycans modulate the biological activity of
a heparin-binding peptide (RKRLQVQLSIRT) from
the G domain of the laminin alpha1 chain. J Biol Chem
276, 22077–22085.
32 Hartman U & Maurer P (2001) Proteoglycans in the
nervous system – the quest for functional roles in vivo.
Matrix Biol 20, 23–35.
33 Bandtlow CE & Zimmerman DR (2000) Proteoglycans
in the developing brain: new conceptual insights for old
proteins. Physiol Rev 80, 1267–1290.
34 Selleck SB (2000) Proteoglycans and pattern formation:
sugar biochemistry meets developmental genetics.
Trends Genet 16, 206–212.
35 Tkachenko E, Rhodes JM & Simons M (2005) Synde-
cans: new kids on the signaling block. Circ Res 96, 488–
500.
36 Kim CW, Goldberger OA, Gallo RL & Bernfield M
(1994) Members of the syndecan family of heparan sul-
fate proteoglycans are expressed in distinct cell-, tissue-,
and development-specific patterns. Mol Biol Cell 5,
797–805.
37 Bernfield M & Sanderson RD (1990) Syndecan, a devel-
opmentally regulated cell surface proteoglycan that
binds extracellular matrix and growth factors. Phil
Trans R Soc Lond B Biol Sci 327, 171–186.
38 Hoffman MP, Nomizu M, Roque E, Lee S, Jung DW,

Yamada Y & Kleinman HK (1998) Laminin-1 and
laminin-2 G-domain synthetic peptides bind syndecan-1
and are involved in acinar formation of a human
G. Johnson et al. Acetylcholinesterase redundancy
FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS 5137
submandibular gland cell line. J Biol Chem 273, 28633–
28641.
39 Bernfield M, Gotte M, Park PW, Reizer O, Fitzgerald
ML, Lincecum J & Zako M (1999) Functions of cell
surface heparan sulfate proteoglycans. Annu Rev Bio-
chem 68, 729–777.
40 Stipp CS, Litwack ED & Lander AD (1994) Cerebro-
glycan: an integral membrane heparan sulfate proteo-
glycan that is unique to the developing nervous system
and expressed specifically during neuronal differentia-
tion. J Cell Biol 124, 149–160.
41 Schnepp A, Komp Lindgren P, Hulsmann H, Krogel S,
Paulsson M & Hartmann U (2005) Mouse testican-2.
Expression, glycosylation and effects on neurite out-
growth. J Biol Chem 280, 11274–11280.
42 Farach-Carson MC & Carson DD (2007) Perlecan – a
multifunctional extracellular proteoglycan scaffold.
Glycobiology 17, 897–905.
43 Brown JC, Sasaki T, Gohring W, Yamada Y &
Timpl R (1997) The C-terminal domain V of perlecan
promotes beta1 integrin-mediated cell adhesion,
binds heparin, nidogen and fibulin-2 and can be modi-
fied by glycosaminoglycans. Eur J Biochem 250, 39–46.
44 Kimbell LM, Ohno K, Engel AG & Rotundo RL
(2004) C-terminal and heparin-binding domains of

collagenic tail subunit are both essential for anchoring
acetylcholinesterase at the synapse. J Biol Chem 279,
10997–11005.
45 Anderson AA, Ushakov DS, Ferenczi MA, Mori R,
Martin P & Saffell JL (2007) Morphoregulation by
acetylcholinesterase in fibroblasts and astrocytes.
J Cell Physiol 215, 82–100.
46 Denzer AJ, Schulthess T, Fauser C, Schumacher B,
Kammerer RA, Engel J & Ruegg MA (1998) Electron
microscopic structure of agrin and mapping of its bind-
ing site in laminin-1. EMBO J 17, 335–343.
47 Marneros AG & Olsen BR (2005) Physiological role of
collagen XVIII and endostatin. FASEB J 19, 716–728.
48 Chubykin AA, Liu X, Comoletti D, Tsigelny L, Taylor
P & Sudhof TC (2005) Dissection of synapse induction
by neuroligins: effect of a neuroligin mutation associ-
ated with autism. J Biol Chem 280, 22365–22374.
49 Grifman M, Galyam N, Seidman S & Soreq H (1998)
Functional redundancy of acetylcholinesterase and
enuroligin in mammalian neuritogenesis. Proc Natl
Acad Sci USA 95, 13935–13940.
50 Comoletti D, Flynn R, Jennings LL, Chubylin A, Mat-
sumura T, Hasegawa H, Sudhof TC & Taylor P (2003)
Characterisation of the interaction of a recombinant
soluble neuroligin-1 with neurexin-1 beta. J Biol Chem
278, 50497–50505.
51 Graham DK, Bowman GW, Dawson TL, Stanford
WL, Earp HS & Snodgrass HR (1995) Cloning and
developmental expression analysis of the murine c-mer
tyrosine kinase. Oncogene 10, 2349–2359.

52 Guttridge TD, Luft JC, Dawson TL, Koslowska E,
Mahajan NP, Varnum B & Earp HS (2002) Mer
receptor tyrosine kinase ignaling: prevention of apop-
tosis and alteration of cytoskeletal architecture without
stimulation of proliferation. J Biol Chem 277, 24057–
24066.
53 Sasaki T, Knysev PG, Cheburkin Y, Gohring W, Tisi
D, Ulrich A, Timpl R & Hohenester E (2002) Crystal
structure of a C-terminal fragment of growth arrest-spe-
cific protein Gas6. Receptor tyrosine kinase activation
by laminin G-like domains. J Biol Chem 277, 44164–
44170.
54 Noonan DM, Fulle A, Valente P, Cai S, Horigan E,
Sasaki M, Yamada Y & Hassell JR (1991) The
complete sequence of perlecan, a basement membrane
heparan sulfate proteoglycan, reveals extensive
similarity with laminin A chain, low density lipoprotein-
receptor, and the neural cell adhesion molecule. J Biol
Chem 266, 22939–22947.
55 Fuki IV, Kuhn KM, Lomasov IR, Rothman VL, Tus-
zynski GP, Iozzo RV, Swenson TL, Fisher EA & Willi-
mans KJ (1997) The syndecan family of proteoglycans:
novel receptors mediating internalisation of atherogenic
lipoproteins in vitro. J Clin Invest 100, 1611–1622.
56 Strittmatter WJ, Saunders AM, Schmechel D, Pencak-
Vance M, Enghild J, Salveson GS & Roses AD (1993)
Apolipoprotein E: high avidity binding to beta-amyloid
and increased frequency of type 4 allele in late-onset
familial Alzheimer’s disease. Proc Natl Acad Sci USA
90, 1977–1981.

57 Beffert U, Stolt PC & Herz J (2002) Functions of lipo-
protein receptors in neurons. J Lipid Res 45, 403–409.
58 Datta G, Chaddha M, Garber DW, Chung B-H, Tytler
EM, Dashti WA, Bradley SH, Gianturco SH & Anan-
tharomaiah GM (2000) The receptor binding domain of
apolipoprotein E, linked to a model class A amphi-
pathic helix, enhances internalisation and degradation
of LDL by fibroblasts. Biochemistry 39, 213–220.
59 Ethell IM, Hagihara K, Miura Y, Irie F & Yamaguchi
Y (2000) Synbindin, a novel syndecan-2-binding protein
in neuronal dendritic spines. J Cell Biol 151, 53–68.
60 Hienola A, Tumova S, Kulesskiy E & Rauvala M
(2006) N-syndecan deficiency impairs neural migration
in brain. J Cell Biol 174, 569–590.
61 Song HH, Shi W, Xiang YY & Filmus J (2005) The
loss of glypican-3 induces changes in Wnt signaling.
J Biol Chem 280, 2116–2125.
Acetylcholinesterase redundancy G. Johnson et al.
5138 FEBS Journal 275 (2008) 5129–5138 ª 2008 The Authors Journal compilation ª 2008 FEBS