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: gjo@sun.ac.za
(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 ª2008 The Authors Journal compilation ª2008 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 ª2008 The Authors Journal compilation ª2008 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 ª2008 The Authors Journal compilation ª2008 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 band 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 bfamily 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 ª2008 The Authors Journal compilation ª2008 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⁄bhydrolase 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⁄bhydrolase 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 ª2008 The Authors Journal compilation ª2008 FEBS 5133