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 [
Trang 1acetylcholinesterase – 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.
Trang 2[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 apossi-ble 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]
Trang 3We 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
40PPMGPRRFL and90RELSED sequences, which are
linked by a salt bridge between46R and94E Docking
of the mouse AChE and laminin structures showed the
major interaction site on AChE to be90RELSED, with
contributions from 40PPM, 46R and 60VDATT
(Fig 1A) The interaction site on laminin was also
conformational, consisting of a number of clusters:
2718VRKRL, 2738YY, 2789YIKRK and 2819RK 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 informainterac-tion, 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.
Trang 4sulfate 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.
Ligands
Reference
Syndecans Syndecan-1 Cell surface FGF family transforming growth
factor b family pleiotrophin
Laminin, fibronectin, tenascin-C a , LDL b a [39] b [55]
Syndecan-3 Cell surface FGF2, midkine, pleiotrophin Laminin, EGFR d d [60] Syndecan-4 Cell surface FGF family Laminin, synbindin c , fibronectin c [59]
GLP-3 Cell surface IGFII e
FGF2
e [61]
GLP-4 Cell surface FGF family
Testicans Testican-1 Cell surface
Testican-2 Cell surface
Testican-3 Cell surface
tenascin, amyloid precursor protein f
f [45]
thrombospondin
Trang 5membrane, 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 90RELSED 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 90RELSED site is conserved in the cholinesterase-domain proteins also Additional conserved residues are found in the 40PPMGPRRFL 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
Trang 6the 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 90RELSED
(147QDQSED) and 40PPMGPRRFL (88PPTFERRFQ)
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 the40PPMGPRRFL sequence (and
equiva-lents with conservative replacements) showed only
vari-ous AChEs and neuroligins from a number of species
Searches for the 90RELSED 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 60VDATT 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 60VDATT 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 90RELSED 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
40PPMGPRRFL 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.
Trang 7residues 339–345 (GMQDSW) as well as F428 and
D432, and the 60VDATT 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 91ELSED 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 90RELSED motif were R142, K143, R145
and K146, while those within 2A˚ of the
40PPMGPRR sequence were W34, R38, R145 and
L149 Those within 2A˚ of the 60VDATT 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
Trang 8specialized 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 (http://www.expasy.ch/tools/scanprosite) 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 (http://www.rcsb.org/pdb/):
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
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