Glynis Johnson

Human acetylcholinesterase binds to mouse laminin-1 and human collagen iv by an electrostatic mechanism at the peripheral anionic site.

Acetylcholinesterase (EC 3.1.1.7; AChE) is known to induce neurite outgrowth and differentiation, but its ligands are as yet unknown. Laminin-1 and collagen IV were investigated as potential ligands for AChE. We observed specific saturable binding of biotinylated human AChE to mouse laminin and human collagen, with K(d) values of 4.9482 nM (SE 0.3145 nM) and 1.1617 nM (SE 0.1921 nM) respectively. Peripheral anionic site inhibitors (fasciculin, BW284c51, propidium and gallamine) also significantly reduced binding with fasciculin being the most effective. Significant reductions in AChE-laminin and AChE-collagen interactions were produced by a monoclonal anti-AChE antibody known to react with the peripheral anionic site, and a partial reduction with an antibody that partially recognises the site. Self-association of AChE was also observed (K(d)=16.3235 nM; SE 5.8120 nM); increasing markedly at low pH, but not significantly affected by either inhibitors or antibodies, suggesting a non-specific aggregation phenomenon. Binding to laminin and collagen was significantly reduced by increasing ionic strength and decreasing pH, indicating a dominant role for electrostatic interactions, and suggesting that the site may be different from the hydrophobic site identified for the AChE-amyloid interaction.

Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene

While acetylcholinesterase (EC 3.1.1.7) has a clearly defined role in neurotransmission, the functions of its sister enzyme butyrylcholinesterase (EC 3.1.1.8) are more obscure. Numerous mutations, many inactivating, are observed in the human butyrylcholinesterase gene, and the butyrylcholinesterase knockout mouse has an essentially normal phenotype, suggesting that the enzyme may be redundant. Yet the gene has survived for many millions of years since the duplication of an ancestral acetylcholinesterase early in vertebrate evolution. In this paper, we ask the questions: why has butyrylcholinesterase been retained, and why are inactivating mutations apparently tolerated? Butyrylcholinesterase has diverged both structurally and in terms of tissue and cellular expression patterns from acetylcholinesterase. Butyrylcholinesterase-like activity and enzymes have arisen a number of times in the animal kingdom, suggesting the usefulness of such enzymes. Analysis of the published literature suggests that butyrylcholinesterase has specific roles in detoxification as well as in neurotransmission, both in the brain, where it appears to control certain areas and functions, and in the neuromuscular junction, where its function appears to complement that of acetylcholinesterase. An analysis of the mutations in human butyrylcholinesterase and their relation to the enzymes structure is shown. In conclusion, it appears that the structure of butyrylcholinesterases catalytic apparatus is a compromise between the apparently conflicting selective demands of a more generalised detoxifier and the necessity for maintaining high catalytic efficiency. It is also possible that the tolerance of mutation in human butyrylcholinesterase is a consequence of the detoxification function. Butyrylcholinesterase appears to be a good example of a gene that has survived by subfunctionalisation.

Cholinesterases modulate cell adhesion in human neuroblastoma cells in vitro.

Cholinesterases are expressed non‐synaptically during embryonic development, neoplasia and neurodegeneration. We have investigated the effects of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and, conversely, anti‐AChE and ‐BChE antibodies and inhibitors on cell adhesion and neurite outgrowth in human neuroblastoma cells. Analysis of cholinesterase levels and isoforms in undifferentiated and differentiated cells indicated a significant rise in AChE levels on differentiation. This increase was related to both cell‐associated and secreted enzyme, and was predominantly the G4 isoform. BChE levels and isoforms, on the other hand, showed no significant variation. Coating the tissue culture plate with AChE stimulated neurite outgrowth, while BChE had an anti‐adhesive effect. Cell adhesion was affected by the BChE inhibitor, ethopropazine, and the AChE peripheral site inhibitor, BW284c51, but not by eserine which binds to the active site. This indicates that the adhesion function is non‐cholinergic, a finding supported by the lack of effect of AE‐2, a monoclonal antibody that inhibits AChE, on cell adhesion. Four out of a panel of nine anti‐AChE antibodies inhibited adhesion to varying degrees. Of these antibodies, two are catalytic, with epitopes associated with the peripheral anionic site of AChE, and the remaining two have epitopes overlapping this site. Neither of the two anti‐BChE antibodies used had any effect on adhesion. These results indicate the importance of AChE in neuroblastoma cell adhesion and neurite outgrowth, and suggest that the peripheral anionic site may be involved in these processes.

Cholinesterase-like catalytic antibodies: reaction with substrates and inhibitors

We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.

Interaction of acetylcholinesterase with the G4 domain of the laminin α1-chain

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChEs PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminins heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.

Catalytic antibodies with acetylcholinesterase activity

We describe three catalytic cholinesterase-like catalytic antibodies (Ab1), as well as anti-idiotypic (Ab2) and idiotypic (Ab3) antibodies, to one of the Ab1s. The Ab1s were raised against the human erythrocyte acetylcholinesterase (AChE), and are unusual in that they both recognise and resemble acetylcholinesterase in their catalytic activity. No contamination of the antibody preparations with either acetylcholinesterase or butyrylcholinesterase (BChE) was found. None of the Ab2s showed catalytic activity, whereas four Ab3s did (an incidence of 1.26% of all Ab3s). Although there is considerable resemblance between Ab1s and Ab3s, there are significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites, and were inhibited by the cholinesterase active site inhibitors iso-OMPA and pyridostigmine, suggesting the similarity of the sites to those of cholinesterases. The Ab3s resemble the Ab1s in their ability to hydrolyse both acetyl and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K(m) values (for acetyl, but not for butyrylthiocholine) and increased turnover numbers (K(cat)), rate enhancements (K(cat)/K(uncat)) and K(cat)/K(m) ratios, for both substrates, although these values by no means approach those of the natural enzymes. The Ab1s appear to have structures resembling the anionic sites of cholinesterases, as shown by their reaction with the anionic site inhibitors (edrophonium and tetramethylammonium). No such reactions were observed in the Ab3s. None of the antibodies show evidence of the sites resembling the peripheral anionic site (PAS) of acetylcholinesterase. All the antibodies recognise, to varying degrees, the peripheral anionic site of acetylcholinesterase. This was shown by their ability to inhibit acetylcholinesterase, to compete with peripheral site inhibitors, and to block acetylcholinesterase-mediated cell adhesion, a property of this site. The results indicate idiotypic mimicry of a catalytic antibodys active site, and suggest that the development of the catalytic activity in the anti-acetylcholinesterase antibodies may be related to the structural features of the peripheral anionic site of acetylcholinesterase.

The carboxylesterase/cholinesterase gene family in invertebrate deuterostomes

Carboxylesterase/cholinesterase family members are responsible for controlling the nerve impulse, detoxification and various developmental functions, and are a major target of pesticides and chemical warfare agents. Comparative structural analysis of these enzymes is thus important. The invertebrate deuterostomes (phyla Echinodermata and Hemichordata and subphyla Urochordata and Cephalochordata) lie in the transition zone between invertebrates and vertebrates, and are thus of interest to the study of evolution. Here we have investigated the carboxylesterase/cholinesterase gene family in the sequenced genomes of Strongylocentrotus purpuratus (Echinodermata), Saccoglossus kowalevskii (Hemichordata), Ciona intestinalis (Urochordata) and Branchiostoma floridae (Cephalochordata), using sequence analysis of the catalytic apparatus and oligomerisation domains, and phylogenetic analysis. All four genomes show blurring of structural boundaries between cholinesterases and carboxylesterases, with many intermediate enzymes. Non-enzymatic proteins are well represented. The Saccoglossus and Branchiostoma genomes show evidence of extensive gene duplication and retention. There is also evidence of domain shuffling, resulting in multidomain proteins consisting either of multiple carboxylesterase domains, or of carboxylesterase/cholinesterase domains linked to other domains, including RING finger, chitin-binding, immunoglobulin, fibronectin type 3, CUB, cysteine-rich-Frizzled, caspase activation and 7tm-1, amongst others. Such gene duplication and domain shuffling in the carboxylesterase/cholinesterase family appears to be unique to the invertebrate deuterostomes, and we hypothesise that these factors may have contributed to the evolution of the morphological complexity, particularly of the nervous system and neural crest, of the vertebrates.

Idiotypic mimicry of a catalytic antibody active site

We have previously described three catalytic antibodies (Ab1s) raised against human erythrocyte acetylcholinesterase (AChE). These antibodies both recognise and resemble AChE in their reaction with substrates and appear with a relatively high frequency. We do not know, however, why catalytic activity should have developed in response to a ground state antigen. This question has implication for autoimmune disorders, which are frequently characterised by the presence of catalytic antibodies, many of which have cytotoxic effects. In this study, we raised anti-idiotypic (Ab2) and anti-anti-idiotypic (Ab3) antibodies to a catalytic Ab1 and examined their properties. None of the Ab2s showed catalytic activity, whereas four of the Ab3s did, an incidence of 1.26%. No contamination of antibody preparations with either AChE or butyrylcholinesterase (BChE) was found. Immunisation of mice with AChE, as well as AChE complexed with various inhibitors, resulted in a significant increase in catalytic immunoglobulins in the serum, compared with non-immunised mice and mice immunised with the Ab1. There appears to be considerable resemblance between Ab1s and Ab3s, but there are also significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites and were inhibited by the cholinesterase active site inhibitors tetraisopropyl pyrophosphoramide (iso-OMPA) and pyridostigmine. The Ab3s resembled the Ab1s in their ability to hydrolyse both acetylthiocholine (ATCh) and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K(M) values (for ATCh but not BTCh) and increased turnover numbers (K(cat)), rate enhancements (K(cat)/K(uncat)) and K(cat)/K(M) ratios. The Ab3s also had reduced affinities for cholinesterase anionic site inhibitors (edrophonium, tetramethylammonium and BW284c51) and no affinity at all for the AChE peripheral anionic site (PAS) inhibitor fasciculin. All the antibodies recognise, to some degree, the PAS of AChE, shown by their ability to inhibit AChE, to compete with peripheral site inhibitors and to block AChE-mediated cell adhesion, a property of the site. These results indicate idiotypic mimicry of the catalytic antibodys active site, suggesting that the catalytic activity is due to affinity maturation of immunoglobulin genes in response to a specific antigen, namely, the PAS of AChE. Further studies are required to determine the structural features of this ground state antigen responsible for the development of catalytic activity.

Non‐enzymatic developmental functions of acetylcholinesterase – the question of redundancy

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 pattern 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.

Association of the HNK-1 epitope with the detergent-soluble G4 isoform of acetylcholinesterase from human neuroblastoma cells

The HNK‐1 carbohydrate epitope is expressed in neural and natural killer cells and is a mediator of cell adhesion. It is well documented that acetylcholinesterase has a secondary function in cell adhesion and differentiation. The presence of HNK‐1 on isoforms of Torpedo and Electrophorus acetylcholinesterase, as well as isoforms from the bovine central nervous system has been described. In this paper, we have investigated the association of the epitope with acetylcholinesterase from human neuroblastoma cells. Acetylcholinesterase was extracted, with or without detergent, purified on immunoaffinity columns and the isoforms separated by sucrose density gradient sedimentation. Secreted acetylcholinesterase, from spent serum‐free culture medium, was similarly treated. The presence of the HNK‐1 epitope was determined by ELISA using the anti‐HNK‐1 and Elec 39 monoclonal antibodies. The epitope was found to be associated with the detergent‐soluble G4 isoform, but not with the hydrophilic G1 nor the secreted hydrophilic G4 isoforms. Likewise, no HNK‐1 was observed associated with human erythrocyte acetylcholinesterase. These results indicate that acetylcholinesterase‐G4, anchored in the extracellular membrane, is capable of mediating cell–substrate adhesion through HNK‐1.