Chapter 15: Structure and functions of acetylcholinesterase and butyrylcholinesterase

A.C. Cuello (Editor) Progress in Brain Research, Vol. 98 0 1993 Elsevier Science Publishers B.V. All rights reserved.

139 CHAPTER 15

Structure and functions of acetylcholinesterase and butyrylcholinesterase Jean MassouliCl, Joel Sussman2, Suzanne Bonl and Israel Silman3 ‘Luboratoire de Neurobiologie, CNRS URA 295, Ecole Normale Supirieure, 46 rue d’Ulm, 75005 Paris, France; Departments of ’Structural Biology and 3Neurobiology, Weizmann Institute of Science, Rehovot, Israel

Introduction Vertebrates possess two cholinesterases, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1 A). Cholinesterases catalyze a very simple reaction, hydrolysis of the ester bond of acetylcholine. The role of AChE in cholinergic transmission, although admittedly secondary to that of the presynaptic machinery responsible for the synthesis and release of acetylcholine, and of the postsynaptic receptors, is crucial for synaptic function. The role of ChE is not clear: this enzyme is, in fact, dispensable, since its absence in humans does not correlate with any physiological abnormality (Kattamis et al., 1967). Despite the fact that they do not, at first sight, appear as glamorous as receptors, cholinesterases display a number of fascinating features, and pose important questions concerning their structure and functions. AChE is one of the fastest enzymes known (Quinn, 1987; Quinn et al., 1992) and possesses an unusual molecular structure (Sussman et al., 1991). Both AChE and BChE display a repertoire of molecular forms which differ in their quaternary structure and may be anchored in different ways to synaptic structures (MassouliC and Bon, 1982). Cholinesterases are thought to exert non-cholinergic functions, e.g. in morphogenesis, during early embryonic development, in the modulation of neuronal activity and in the elimination of various toxic compounds, which may explain their presence outside the context of cholinergic transmission. They are also expressed abnormally in some tumors and in other pathological states (Soreq et al., 1991; Zakut et al., 1990, 1992). In this chapter, we focus on the following aspects: the atomic structure of cholinesterases and their catalytic mechanism; the structure and biosynthesis of their molecular forms; the possibility that these enzymes participate in cellular interactions in addition to their catalytic activity. A

more complete discussion of the structure and function of the cholinesterases can be found in a recent review article (Massoulib et al., 1992).

Three-dimensional structure of acetylcholinesterase The crystallization of the GPI-anchored G, form of AChE from Torpedo califomica, subsequent to solubilization by PIPLC (Sussman et al,, 1988), was followed by the X-ray crystallographic determination of its three-dimensional structure (Sussman et al., 1991). AChE belongs to the class of a@ proteins (Richardson, 1985), and consists of a large central mixed P-sheet surrounded by 15 a-helices (Fig. 1). Solution of the structure made it possible to visualize, for the first time, at atomic resolution, a protein binding pocket for the neurotransmitter, acetylcholine (ACh). It was found that the active site consists of a catalytic triad (S200-H440E327), located near the bottom of a deep and narrow gorge, about 20 8, deep, lined with the rings of 14 aromatic amino acid residues, which account for approx. 40% of its surface area. Despite the complexity of this array of aromatic rings, it was proposed, on the basis of modelling which involved docking of the ACh molecule in an all-trans conformation, that the quaternary group of the choline moiety makes close contact with the indole ring of W84. This is in agreement with the recent affinity-labelling study of Weise et al. (1990), which identified W84 as being part of the putative “anionic” (choline) binding site. Recently, by soaking suitable competitive inhibitors into the native AChE crystals, it has been possible to obtain direct structural evidence concerning the quaternary binding site. In the complex of AChE with the quaternary inhibitor, edrophonium, it can be seen that the quaternary group indeed nestles in close contact with W84, as sugges-

140

ted both by modelling (Sussman et al., 1991) and by affnity labelling (Weise et al., 1990) (Fig. 2). The metahydroxyl group of edrophonium appears to establish hydrogen bonds with both S2000r and with H440NE2,providing a structural basis for the fact that this isomer is a much better inhibitor of AChE than the corresponding para and ortho isomers of the parent anilinium compound (Hobbiger, 1952). Another complex, tacrine, the heterocyclic compound currently on clinical trial for the management of Alzheimer’s disease (Gauthier and Gauthier, 1991) was also studied. Tacrine is known to be a powerful competitive inhibitor of AChE (Heilbronn, 1961), and the Xray data show that it, too, makes close contact with W84, lying in a plane parallel to the plane of the indole ring. This

confirms an earlier prediction, based on the observation of a charge-transfer complex of Elecirophorus AChE with the structurally similar N-methylacridinium ion (Shinitzky et al., 1973). In both complexes, the ring of an additional aromatic residue, F330, moves significantly to make an aromatic-aromatic interaction with the bound inhibitor. Kieffer et al. (1986) showed earlier that the peptide, GSXF, in Electrophorus AChE, was labelled by the photoaffinity label, DDF. This peptide corresponds to GSFF (residues 328-331) in Torpedo AChE. The ring structure of an additional aromatic residue, the indole of W279, at the top of the gorge, undergoes a conformational change in both complexes, even though it is located at least 8 8, from the bound ligand.

Fig. 1. Three-dimensional structure of GZaAChE from Torpedo. The structure is represented in a ribbon diagram showing p strands (green) and a helices (red). The active site gorge is located above the central p sheet and the arrow marks the location of the active-site serine. SertOO.

141

’*-OH EDR

Fig. 2. Close-up view, in the vicinity of the active site, of the complex of edrophonium (EDR) with Torpedo AChE.

Site-directedmutagenesis of amino acids in the active-site gorge The solution of the three-dimensional structure of AChE raised many questions about the role of various amino acids within the active-site gorge. The technique of site-directed mutagenesis, expressing the mutated sequence in COS cells, as described above, permitted a direct experimental approach to these questions. One question concerned the fact that the three-dimensional structure suggested that a glutamic acid residue, E327, participates in a catalytic triad with S200 and H440. This was unexpected since the acidic residue participating in the catalytic triad of various serine hydrolases had previously always been an aspartic residue. A conserved aspartate residue, D326, is adjacent to E327, although the X-ray structure indicates that it points away from the active site. Site-directed mutagenesis indeed showed that mutagenesis of D326 to an asparagine had little or no effect on the activity of the enzyme, whereas mutation of E327, even to an aspartate, abolished enzymic activity almost completely (Duval et al., 1992~). The role of the various aromatic residues in the activesite gorge is of especial interest. Comparison of the sequences of AChE from several sources with those of known BChE sequences revealed that six of the fourteen conserved aromatic residues which line the active site

gorge in AChE are absent in BChE. Human BChE bears great similarity to Torpedo AChE; it displays 53% sequence homology, and the first 534 residues, which include all those believed to be involved in catalytic activity, may be aligned completely. This permitted modelling of human BChE on the basis of the three-dimensional structure of Torpedo AChE. In the model so obtained, it could be seen that two of the six missing aromatic residues, F288 and F290, which are replaced by L and V, respectively, in BChE, may prevent entrance of butyrylcholine into the acyl-binding pocket in wild-type Torpedo AChE. Their mutation to L and V, by site-directed mutagenesis, produced a double mutant which hydrolysed butyrylthiocholine almost as well as acetylthiocholine (Harel et al., 1992). The mutant enzyme was also inhibited well by the bulky, BChE-selective organophosphate inhibitor, isoOMPA (Austin and Berry, 1953). The tryptophan residue, W279, at the entrance to the gorge, is another aromatic residue present in AChE, but lacking in BChE. Modelling designated it as part of the “peripheral” anionic site of AChE which is absent in BChE, where in the human enzyme, it is replaced by Ala. The mutant, W279A, of Torpedo AChE, showed strongly reduced inhibition by the “peripheral” site-specific ligand, propidium, relative to the wild-typc enzyme, whereas its inhibition by the catalytic site inhibitor, edrophonium, was hardly affected (Harel et al., 1992).

Molecular forms: asymmetric forms; amphiphilic and non-amphiphilic globular forms The major molecular forms of vertebrate cholinesterases are schematically illustrated in Fig. 3 (MassouliC et al., 1992). Asymmetric or collagen-tailed forms are characterized by the presence of a collagen-like tail, associated with one (A4), two (A,) or three (Alz) catalytic subunit tetramers. These forms display an unusually large Stokes’ radius and aggregate at low ionic strength in the presence of polyanionic compounds such as glycosaminoglycans (Bon et al., 1978). They lose both these properties upon digestion by collagenase. Globular forms, which are devoid of a collagen-like tail, are heterogeneous. We have defined amphiphilic and nonamphiphilic globular forms on the basis of their capacity to associate with micelles of non-denaturing detergents (e.g. Triton X-100, Brij-96). There are several types of arnphiphilic forms: (a) glycophosphatidylinositol (GP1)anchored dimers (Type I amphiphilic dimers) exist in the nervous system of insects (Drosophila), in Torpedo electric organ and muscle and on the surface of mammalian erythrocytes and lymphocytes (Silman and Futerman, 1987); (b) Type I1 amphiphilic forms occur in other

142

Fig. 3. Genomic structure the AChE gene in Torpedo, differential splicing of mRNA and quaternary structure of the major oligomeric forms of cholinestermes in vertebrates. Alternative splicing generates mRNAs encoding H and T subunits, which differ in their C-terminal peptides. H subunits produce glycophosphatidylinositol (GP1)-anchored dimers (Type I amphiphilic dimers). T subunits produce amphiphilic monomers and dimers (Type I]), as well as tetrmers and hetero-oligorners which incorporate structural collagenic (Q) or hydrophobic (P) subunit$. Note that in Torpedo, collagen-tailed forms also contain 100 kDa structural subunits of unknown function (Lee and Taylor, 1982) (after Massoulit5 et d.,1993).

Torpedo tissues and can be distinguished from the Type I forms because they are insensitive to GPI-specific phospholipases (PI-PLC) and do not aggregate in the absence of detergent (Bon et al., 1988a,b); such forms are abundant in the brain and muscles of birds and mammals (Bon et al., 1991); (c) Hydrophobic-tailed tetramers are, like the collagen-tailed forms, hetero-oligomeric forms which are anchored in plasma membranes by a hydrophobic 20 kDa subunit (Gennari et al., 1987; Inestrosa et al., 1987); they are the major species of AChE in mammalian brain.

H and T subunits of Torpedo AChE; expression in transfected cells The Torpedo electric organ provides a valuable experimental tool for biochemical analysis of cholinergic elements. It contains two main types of AChE molecules, asymmetric or collagen-tailed forms (A forms) and glycolipid-anchored dimers (GPI-GZa form), which appear to be associated respectively with the extracellular basal lamina and with pre- and post-synaptic membranes.

The catalytic subunits of the two types of molecular forms display the same catalytic activity per active site. Their N-terminal sequences are identical, but their Cterminal sequences are distinct. The two types of subunit are encoded by the same gene. The coding sequence is contained in two common exons, followed by alternatively spliced exons which specify the two types of catalytic subunit, H and T. The corresponding sequences were determined from cDNA clones, and transiently expressed in COS cells (Duval et al., 1992b). When the cells were cultured at 37°C. we did not obtain any AChE activity resulting from the expression of the Torpedo enzyme, above the low endogenous AChE background. Active Torpedo AChE was, however, produced at lower temperatures, e.g. 27°C. Even in these conditions, however, a comparison of AChE activity and of the amount of AChE protein, as determined from Western blots showed that only a small fraction was active. We characterized the corresponding molecular forms. The H subunits generate glycolipid-anchored dimers, as in vivo, demonstrating that the C-terminal peptide is processed as a GPI-cleavagdattachment site, even in the foreign environment of the COS cell.

for the rest of the coding sequence. In vertebrate tissues, globular forms are much more widely expressed than asymmetric Forms, which are produced only in differentiated nervous tissue and in muscle. The fact that they are assembled from Q and T subunits in COS cells indicates tHat this association does not require a specific biosynthetic capacity that would be restricted to differentiated cells, Therefore, it is likely that the production of A forms, a.g. in muscle cells, is controlled by the expression of the structural Q subunits.

'Che T subunits generate mostly non-amphiphilic tetramers (G4"), as well as amphiphilic monomers (G13 and dimers (GZa), which resemble the Type I1 forms characterized in vlvo. Truncated T subunits, from which most of the distinct C-terminal peptide (Tc) had been deleted by mutagenesis, produced only non-amphiphilic monomers, demonstrating that this 40-amino acid peptide is responsible for the hydrophobic character of the Type IT amphiphitic forms. We found that attachment of a chemically synthesized Tc peptide was sufficient to confer the capacity to bind detergent micelles to nnn-amphiphilic tetramers, indicating that this property does not require posttranslational modification of the peptide, such as the addition of lipidic groups, although such modification has been repotted (W.R. Randall, personal communication), and might reinforce the anchoring of the enzyme to membranes.

Organisation of the collagenic Q subunit in asymmetric AChE molecules The primary structure of the Q subunit indicates the existence of three main domains in the mature protein: an Nterminal non-collagenic domain (QN), a collagenic domain, and a C-terminal non-collagenic domain (Qc). The size of the collagenic domain corresponds to a triple helix of about 50A, in good agreement with the lerrgth of the tail as observed in electron micrographs of isolated collagentailed molecules (Krejci et a]., 1991a). This domain is flanked by two pairs of cysteine residues, which may form disulfide bonds between each pair of strands, thus stabilizing the triple helical collagenic structure. Antibodies directed against the Qc domain were found to bind intact asymmetric forms, but not collagenase-digested molecules from which a part of the rail had been cleaved. This suggested that the QN domain is associated with the catalytic T subunits, while the Qc domain is located at the distal end of the tail (Duval et al., 1992a). We confirmed the capacity of the QN domain to bind catalytic T subunits by engineering a chimeric protein in which the QN domain is fused to the GPI attachment signal peptide (Hc) of the H subunits (Fig. 4A). When this QN/HC protein was co-expressed with Torpedo T subunits, we ob-

Biosynthesis of asymmetric forms in transfected cells We have recently cloned a cDNA encoding a collagenic subunit of Torpedo asymmetric AChE (Krejci et al., 1991a). The production of asymmetric forms was observed when these collagenic subunits, Q, were expressed together with the T subunit in COS cells. This was not the case, however, with H subunits or with truncated T subunits. The Tc peptide seems, therefore, to be necessary for the association of the T and Q subunits, in agreement with the fact that it contains a cysteine residue (Cys-575) which is involved in inter-subunit disulfide bonds between two subunits or between T and Q subunits (Roberts et a]., 1991). It is noteworthy that hybrid asymmetric forms could be generated by co-expression of Torpedo Q aubunits with rat T subunits (Legay et al., 1993), probably because the Tc peptide is highly conserved between Torpeda and mammals, with 75% identity, as compared to 56% identity

@

COLLAGENIC SUBUNIT

SIGNAL PEmE

W

N-TE?RMINAL. DOMNN(QM Pro-rich region

32

SIGNAL QN DOMAIN PEPTlDE

COLLAGENlC DOMAIN

C-'TERMINAL DOMAIN @s-fi&h region

Pro-rich ngion

--- +

PP

Ln

-4

AChE HC PEPTIDE

1-1-

Fig. 4. A . Schematic representation of the primary structure of the collagenic subunit, Q, and of the chimeric protein, QN/HC.

144

Brij-96

16.1s

9

rat AChET

10

20

30

Fractions

Fig. 4 B. Sedimentation analysis in sucrose gradients, in the presence of the detergent Brij-96, of AChE molecular forms produced by expressing rat AChE T subunits in transfected COS cells. ( A ) (Lower curve) T subunits generate amphiphilic : G ( of Type 11, amphiphilic and monomers and dimers ) non-amphiphilic tetramers (G4a and Gqna ), as well as a nonamphiphilic 13s form of unknown structure (Legay et al., 1992); ( B ) GPI-anchored tetramers which are sensitive to PI-PLC are produced when rat T subunits are co-expressed with a chimeric anchoring subunit. QN/Hc. consisting of the non-collagenic N terminal domain of the Q subunit and the GPI cleavage/attachment signal of the H subunit.

tained GPI-anchored AChE tetramers. These molecules were largely exposed at the cellular surface, from which they could be solubilized by PI-PLC (Duval et al., 1992a). Figure 4B demonstrates that the QN/HC chimeric protein also associates with rat subunits, forming GPI-anchored tetramers in COS cells. This experiment shows that the different domains of the catalytic and structural subunits, which are involved in membrane localization (the GPI attachment signal, H,) and in the association of oligomers (QN and T,), can function as independent units and illustrates the complementarity between the Torpedo QN and rat T, domains.

Non-classical functions of cholinesterases As mentioned above, various observations suggest that the physiological function of the cholinesterases is not restricted exclusively to hydrolysis of acetylcholine at cholinergic synapses. Non-synaptic functions may also depend on their catalytic activity: for example, BChE has been suggested to act as a scavenger for various compounds, such as solanum alkaloids (Neville et al., 1990) and heroin (Lockridge et al., 1980). It has also been suggested that cholinesterases participate in a primitive muscarinic cellular signalling system during early stages of embryogenesis (Drews and Mengis, 1990). It is also possible, however, that cholinesterases may control cellular activities through structural interactions which do not involve their catalytic activity. It has been shown, for example, that the application of AChE modifies the excitability of dopaminergic neurons i n the rat substantia nigra, even after irreversible inhibition of the enzyme by an organophosphorus compound (Greenfield, 1991). In the early chick embryo, AChE and BChE are expressed in spatially and temporally distinct patterns: BChE expression is correlated with cellular proliferation and morphogenetic movements, whereas AChE expression is one of the first signs of neuronal or muscular differentiation (Layer, 1991). In vitro experiments, in which embryonic retinal cells reorganize into retinospheroids, showed that various selective inhibitors of each enzyme produced different morphogenetic effects, suggesting that these effects are not mediated by enzymatic activity (Layer et al., unpublished). The hypothesis that cholinesterases participate in structural interactions has been strengthened recently by the analysis of their homology with other proteins (Krejci et al., 1991b). Proteins containing a cholinesterase-like domain include not only various esterases which possess the elements of a catalytic triad, Glu/Asp-His-Ser, like the cholinesterases, but also structural proteins which lack the active-site serine (mammalian thyroglobulin, Drosophila glutactin and Drosophilu neurotactin). Neurotactin is particularly interesting because the expression of this transmembrane protein confers heterophilic adhesive properties on transfected Schneider cells (Barthalay et al., 1990). This adhesive character must belong to its cholinesterase-like domain, which constitutes the exttacellular region of the protein. Thus, cholinesterase-like domains seem to have been used in evolution as scaffolds for organizing a catalytic site or for structural interactions. It is possible that cholinesterases exert both functions. This idea is supported by the finding that some cholinesterase forms carry a specific glycanic epitope, recognized by monoclonal antibodies such as HNK-I (Bon et al., 1987), which has been

I45 considered as a hallmark of adhesion glycoproteins (Keilhauer et al., 1985).

Concluding remarks As will be apparent from the above presentation, there has been dramatic progress in recent years in several directions relating to the structure and function of the cholinesterases. This is especially true with respect to our understanding of their modes of anchoring, with respect t o characterization of the genes encoding the cholinesterases and with respect to the way in which the various molecular forms are produced. The recent solution of the three-dimensional structure also opens up pathways to gaining a detailed understanding of the mechanism of action. However, a number of interesting questions remain open. Firstly, how does the novel structure of the enzymatic subunit explain the exceptional catalytic efficiency of AChE? What is the role of the peripheral site? Concerning the polymorphism of cholinesterases, it still remains to be established whether the full repertoire of splicing variants has been described and whether additional modes of anchoring exist. Moreover, the mechanisms of folding and assembly by which the different forms of the enzyme are generated as homo- and hetero-oligomers remain almost completely unknown. Perhaps the most intriguing of the open questions relate to the putative non-cholinergic function(s) of the cholinesterases. Thus we are still lacking any biological role for BChE; but even for AChE, its temporal and spatial expression during embryogenesis, in particular, challenge us to establish a biological function distinct from its well-defined role at the cholinergic synapse.

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