Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase Dong Liang 1,2 , Jean-Philippe Blouet 1 , Fernanda Borrega 1 , Suzanne Bon 1 and Jean Massoulie ´ 1 1 Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supe ´ rieure, Paris, France 2 Key Laboratory of Brain Functional Genomics, MOE&STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, China In vertebrates, butyrylcholinesterase (BChE T ) and the T splice variant of acetylcholinesterase (AChE T ) consist of a catalytic domain of approximately 500 residues, followed by C-terminal tail (t) peptides [1,2]. These peptides of 41 and 40 residues, respectively, con- tain seven strictly conserved aromatic residues, includ- ing three evenly spaced tryptophans, and a cysteine located at position )4 from the C-terminus. The t peptide plays an important role in the biosynthesis of cholinesterases, particularly their folding and export. For example, it has been shown that it induces the misfolding of a significant fraction of newly synthe- sized AChE polypeptides, and that this effect depends on hydrophobicity since it was maintained when the aromatic residues were replaced by leucines. The t pep- tide also reduces export, as indicated by the fact that Keywords acetylcholinesterase; butyrylcholinesterase; cysteines; oligomers; secretion Correspondence J. Massoulie ´ , Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supe ´ rieure, Paris, France Fax: +33 1 44 32 38 87 Tel: +33 1 44 32 38 91 E-mail: jean.massoulie@biologie.ens.fr (Received 13 August 2008, revised 25 September 2008, accepted 24 October 2008) doi:10.1111/j.1742-4658.2008.06756.x Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinester- ase that is predominant in mammalian brain and muscles (AChE T ) possess a characteristic C-terminal tail (t) peptide. This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA. Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties. To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba). We found that secretion was increased by deletion of the t peptides, and that it was more efficient with a than with b. The patterns of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicat- ing a predominant influence of the catalytic domains. However, addition of a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetra- mers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers. These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cho- linesterases to form and secrete various oligomers. Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations. Abbreviations AchE T , T splice variant of acetylcholinesterase; BChE, butyrylcholinesterase; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide; Nbs 2 , 5,5¢-dithiobis(2-nitrobenzoic acid); PRAD, proline-rich attachment domains; t, tail. 94 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works the ratio of secreted to cellular AChE was strongly increased when it was deleted; this effect was sup- pressed by mutation of the aromatic residues to leucines [3–6]. However, the major function of the t peptides is that they allow the assembly of tetramers of AChE T [7] and of BChE T [8] and their association with the structural proteins ColQ and PRiMA [9,10]. These heteromeric structures are based on a tight association between four t peptides, also named tryptophan (W) amphi- philic tetramerization domains, and the poly-proline motifs, or proline-rich attachment domains (PRADs) of ColQ and PRiMA [11–13]. In addition, the BChE tetramers that circulate in the blood plasma have recently been shown to incorporate a similar proline- rich peptide derived from the protein lamellipodin [14]. Crystallographic analyses of a complex of synthetic t and PRAD peptides showed that four a-helical t pep- tides form a coiled-coil around the PRAD, which is arranged in a poly-proline II helix [15]. The assembly of cholinesterase homo-tetramers or PRAD-associated tetramers is entirely conditioned by the presence of a t peptide because truncated AChE subunits lacking the t peptide only produce secreted monomers [16]. This peptide constitutes an autono- mous interaction module, necessary and sufficient for tetramerization and association with PRAD-containing proteins, because addition of a t peptide at the C-ter- minus of green fluorescent protein or alkaline phos- phatase allowed the formation of tetramers associated with an N-terminal fragment of ColQ [17]. However, the catalytic domains are also involved in quaternary interactions that certainly participate in the formation and stability of these oligomers. In particular, the tet- ramers are formed of two pairs of subunits, in which a 7,8 and a 10 helices from each subunit form a four helix bundle, with a hydrophobic contact zone [16,18]. The respective contributions of the catalytic domains and the t peptides in oligomers has not been evaluated. The formation of AChE T tetramers associated with PRAD-containing proteins is physiologically important because it ensures their functional localization by ColQ in the basal lamina at neuromuscular junctions [19], as well as by PRiMA in cell membranes, particularly in the brain [20]. Similarly, the formation of BChE T tetramers conditions the secretion of this enzyme and its stability in the bloodstream. Injection of AChE or BChE offers a very efficient protection against poisoning by anti-cholinesterase agents, such as organophosphorous pesticides, but monomers and dimers are much more rapidly elimi- nated than tetramers after injection in the circulation [21–27]. Although the half life of recombinant enzymes, even monomers, in the bloodstream can be considerably increased by derivatization with polyeth- ylene glycol [28–33], it may be interesting to produce these enzymes as recombinant proteins in a stable tet- rameric form, which also present a greater thermal sta- bility than monomers or dimers [34]. Mutants lacking the cysteine located at )4 from the C-terminus do not form stable dimers, but can form tetramers, particularly in the presence of a PRAD-con- taining protein. It is likely that transient dimerization occurs as a first step in the assembly of tetramers, either associated or not with a PRAD. We recently found that addition of a second cysteine at an appro- priate position in the t peptide of Torpedo AChE greatly increased the formation and secretion of homo- tetramers [4]. We therefore explored the possibility that mutations in the t peptides of human AChE T and BChE T might induce their assembly into tetramers. Because these two enzymes differ in their capacity to form oligomers, we investigated the respective roles of the catalytic domain and of the t peptides. For this purpose, we constructed chimeric proteins, in which we associated the catalytic domain of each enzyme with the t peptide of the other. For convenience, the large catalytic domains are designated by capital letters (A and B), whereas the small t peptides are designated by lower case letters (a and b), so that the wild-type AChE and BChE are Aa and Bb, and the chimeras are Ab and Ba. Comparisons of wild-type enzymes and chimeras, as well as of various mutants, show that both domains contribute critically to the oligomeriza- tion and to the efficiency of secretion. Results Exchange of t peptides between human AChE and BChE The T variants of human AChE and human BChE are composed of a catalytic domain of approximately 500 residues, followed by small C-terminal t peptides of 40 and 41 residues, respectively. In the present study, the catalytic domains are indicated by capital letters ( A and B) and the C-terminal peptides by lower case letters (a and b), so that the wild-type enzymes are abbreviated as Aa and Bb. The C-terminal t peptides of human AChE T (a) and BChE T (b) are highly homologous, with 24 iden- tical residues (60%), including the seven aromatic resi- dues and the cysteine located at )4 from the C-terminus, being strictly conserved among all vertebrate cholines- terases (Fig. 1A). However, they present significant dif- ferences, particularly between the residues immediately following the catalytic domain. Some of the differences D. Liang et al. Oligomerization and secretion of AChE and BChE FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 95 between the peptides a and b might be important for the processing and the activity of AChE and BChE, notably those involving charged residues, the presence in peptide b of an additional tryptophan (W8) and the presence of six instead of five residues between the aromatic-rich region and the cysteine. Both peptides are predicted to form amphiphilic a helices, in which the aromatic resi- dues are clustered in a sector of approximately 140° (Fig. 1B). To analyze the oligomerization properties due to the t peptides of human AChE and BChE, we constructed chimeras Ab and Ba in which we exchanged these pep- tides; we also deleted the C-terminal peptides, produc- ing the truncated enzymes A and B. The different constructs were expressed in transiently transfected COS cells, and we analyzed the cellular and secreted cholinesterase activities (Fig. 2), as well as the oligo- meric patterns, revealed by sedimentation profiles in sucrose gradients (Fig. 3). The C-terminal t peptides do not influence the catalytic activity of AChE and BChE We examined the possible influence of the C-terminal peptides on the AChE and BChE activities by comparing the catalytic rates per active site. The active sites were titrated with the irreversible inhibitor 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) (see Experimental procedures). The slopes of residual activity, plotted as a function of the amount of DEPQ, were identical for A, Aa and Ab, with acetylthiocholine as substrate, as well as for B, Ba and Bb, using either acetylthiocholine or butyrylthio- choline as substrates. Because of excess substrate inhi- bition, AChE presented a maximal rate for approximately 2 mm acetylthiocholine. The rates of hydrolysis of acetylthiocholine and butyrylthiocholine (at 6 mm) by BChE were approximately 14% and 24% of the rate of hydrolysis of acetylthiocholine (at 2 mm) by AChE. Influence of the C-terminal t peptides on activity, secretion and oligomerization As expected, the truncated mutants A and B, without t peptides, produced only monomers, sedimenting around 4S (not shown). The levels of cellular activity were lower for these mutants than for the wild-types but secretion was increased (Fig. 2A,B), in agreement with our previous conclusions that t peptides induce a partial misfolding of the polypeptides, as well as an intracellular degradation of a fraction of active subunits [3]. Cells expressing wild-type human AChE (Aa) secreted approximately 15% of their content per hour and produced mostly monomers and dimers, with a small proportion of tetramers (less than 10% of the A B Fig. 1. Structures of AChE and BChE t peptides. (A) Sequences of the C-terminal t peptides of human AChE and BChE. These peptides (a and b) are encoded by 3¢ exons from the cholinesterase genes; in the present study, we numbered from their first residue. The seven aromatic residues, which are conserved in all vertebrate cholinesterases, are shown in blue; acidic residues are shown in red and basic resi- dues in green; the cysteines are indicated by orange arrowheads and the residues that have been mutated to cysteines in the present study are underlined. Residues that differ between peptides a and b and were mutated in b are indicated by vertical lines above the sequence (those which were mutated as a group are joined by an horizontal line). (B) En face view of the a helices formed by the N-terminal regions of peptides a and b . Colours are as in (A), except that cysteines are shown in orange and residues that were mutated to cysteines are indi- cated by orange circles. Oligomerization and secretion of AChE and BChE D. Liang et al. 96 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works secreted activity). For human BChE (Bb), the rate of secretion was only approximately 5% of the cellular content per hour. This enzyme formed a higher pro- portion of oligomers, mostly dimers in the cells, and tetramers represented approximately 30% of the secreted enzyme, together with comparable proportions of dimers and monomers (Fig. 3A). The fact that Bb forms a higher proportion of olig- omers, but is less efficiently secreted than Aa, is quite paradoxical because secretion generally increases with the degree of oligomerization. Clearly, the assembly of tetramers is not restricted by the fact that each BChE subunit possesses nine N-linked glycans [35], whereas AChE has only four. This was confirmed by 050100150200250 0 50 100 150 200 250 - A- Aa Aa S19C Aa S38C - Ab Ab SSVGL Ab N19C Ab N19C N18S Ab N19C MD22VH Ab N19C N18S MD22VH - B- Ba Ba S19C - Bb Bb SSVGL Bb A12C Bb H15C Bb N19C Bb D23C Bb N26C Bb S37C Bb N19C N18S Bb N19C MD22VH Bb N19C N18S MD22VH - Cellular activity A B Secreted activity 0 1 2 3 4 5 6 A- Aa Aa S19C Aa S38C Ab Ab SSVGL Ab N19C Ab N19C N18S Ab N19C MD22VH Ab N19C N18S MD22VH B- Ba Ba S19C Bb Bb SSVGL Bb A12C Bb H15C Bb N19C Bb D23C Bb N26C Bb S37C Bb N19C N18S Bb N19C MD22VH Bb N19C N18S MD22VH Ratio of secreted to cellular activity (% of the wild type) Fig. 2. Cellular and secreted activities produced by human AChE, BChE and mutants used in the present study. (A) Cellular and secreted activities. A and B represent AChE and BChE from which the t peptides were deleted; Aa and Bb represent the wild-type enzymes with their t peptides, Ab and Ba represent chimeras in which the t peptides were exchanged; mutations in the t peptides are indicated. For each mutant, the cellular and secreted activities are shown by bars to the left and the right. AChE and BChE activities were determined by the Ellman assay with acetylthiocholine and butyrylthiocholine as substrates, respectively: AChE activities are indicated as grey bars and BChE activities as hatched bars. AChE and BChE activities were normalized to the wild-type enzymes (Aa and Bb, respectively). (B) Ratio of secreted to cellular activity. Note that the double mutation M22V ⁄ D23H is abbreviated as MD22VH. D. Liang et al. Oligomerization and secretion of AChE and BChE FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 97 the fact that mutants lacking some of the N-glycosyl- ation sites, which were provided by O. Lockridge [36], did not produce a higher proportion of tetramers (not shown). For the chimeras Ab and Ba, the rates of secretion were approximately 5% and 15% of the cellular con- tent per hour, respectively, and therefore appeared to be mainly determined by the t peptides. By contrast, Fig. 3B shows that the sedimentation profiles were very similar for Aa and Ab, and for Ba and Bb, except that the BChE species sedimented faster than their AChE counterparts, in agreement with the higher mass of BChE subunits [37]. This indicates a predominant influence of the catalytic domain on oligomerization. Role of the C-terminal cysteine Mutation of the cysteine located at )4 from the C-ter- minus to a serine in the a or b peptides suppressed the formation of Aa or Bb dimers, but not the production of a small proportion of tetramers (not shown). These mutations increased the ratio of secreted to cellular activity in both cases (Fig. 2). However, in the case of Bb, the cellular activity was decreased and secretion was increased, suggesting that the presence of this cys- teine retains the enzyme intracellularly. In case of Aa, the cellular activity of AChE was also decreased by approximately 50% but secretion was not modified, suggesting that degradation was increased by suppres- sion of the cysteine. Thus, it appears that the effect of a C-terminal cysteine on the trafficking of cholinesterase in the secretory pathway largely depends on the nature of the preceding catalytic domain. Oligomerization might be affected by the distance between the aromatic core and the C-terminal cysteine, which forms inter-catenary disulfide bonds. There are five residues between Y31 and this cysteine in peptide a, and six in peptide b, because of an additional residue, T32. To evaluate the possible influence of this residue on oligomerization, we deleted T32 in Bb and we mutated peptides a (CSDL to SCDL) and b (SCVGL to CSVGL), to modify the number of residues between the cysteine and the aromatic core. We found that these mutations had no effect on either the levels of cellular and secreted activities, or on the distribution of oligo- meric forms (not shown). Similarly, these mutations did not modify the secretion or the oligomerization of mutants possessing a cysteine at position 19 (see below). Thus, the addition or subtraction of one residue in the interval between the aromatic residues and the cysteine had no influence, suggesting that this peptidic segment represents a flexible spacer, in agreement with previous studies on Torpedo AChE [38]. Role of cysteines in oligomerization – effects of introducing an additional cysteine In a previous study, we found that mutating residue 19 in the t peptide of Torpedo AChE considerably increased the production and secretion of tetramers Cell extract Medium G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 3 G 6 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 Aa Bb Ba Ab Aa AChE activity (arbitrary units)BChE activity (arbitrary units) Sedimentation coefficients 55101015 15 Wild-type t peptides AB With added cysteine 19C Bb Ba Ab 19C 19C 19C Fig. 3. Sedimentation profiles indicating the proportions of oligo- meric forms produced by AChE, BChE, chimeras and mutants. (A) Left panels: Aa, Ab, Ba, Bb. (B) Right panels: mutants containing a cysteine at position 19 (S19C in peptide a, N19C in peptide b). The profiles corresponding to cell extracts are shown with filled sym- bols (d, AChE; , BChE) and a continuous line, and those corre- sponding to the medium with empty symbols (s, AChE; h, BChE) and a dashed line. The peaks corresponding to tetramers, dimers and monomers are indicated as G 4 ,G 2 and G 1 , respectively. Note that the molecular forms of BChE and its mutants sediment slightly faster than the corresponding AChE molecular forms. Oligomerization and secretion of AChE and BChE D. Liang et al. 98 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works [4]. We therefore introduced similar mutations in a and b, and analyzed the resulting activities and molecular forms produced by expressing the four cholinesterase combinations in COS cells. This mutation did not modify the level of secretion for Aa 19C , increased it for Ba 19C , and decreased it for Ab 19C and Bb 19C (Fig. 2). The fact that the cellular activity was unchanged or decreased, whereas sec- retion was decreased, indicates that the N19C mutation in peptide b induced an intracellular degra- dation of Ab 19C and Bb 19C . As observed in the pre- ceding section, the ratio of secreted to cellular activity again appeared to depend essentially on the t peptides: it was much higher for Aa 19C and Ba 19C than for Ab 19C and Bb 19C . The 19C mutations enhanced the difference between the two peptides because the secreted ⁄ cellular ratio was increased with peptide a 19C compared to a and decreased with pep- tide b 19C compared to b. By contrast to the oligomeric patterns obtained without a cysteine at position 19, we observed a much stronger similarity between enzymes possessing the same C-terminal peptide (a 19C or b 19C ) than between those possessing the same catalytic domain (Fig. 3B). Thus, mutation S19C in a 19C strongly increased the proportion of tetramers, which became the predominant secreted species for both Aa 19C and Ba 19C : these mutants produced very similar patterns of molecular forms. The effect of mutation N19C in peptide b 19C had little effect on the distribution of secreted molecular forms of Bb 19C . In the case of Ab 19C , the effect was more complex: the cells con- tained mostly 4S monomers but secreted a variety of oligomers, mostly monomers, dimers, trimers, tetra- mers and hexamers (see below). The fact that the oligomeric forms were very low or undetectable in the cells suggests that they were secreted very rapidly after their assembly. The results were identical when the 19C mutations were combined with mutations that modified the distance between the C-terminal cysteine and the aromatic residues, as indicated above (not shown). Taken together, these results show that the t pep- tides possessing a cysteine at position 19 had a stron- ger effect on the secretability of cholinesterases than wild-type t peptides, and exerted a dominant influence on oligomerization. Effects of introducing cysteines at different positions in BChE Our previous study of Torpedo AChE showed that the pattern of oligomerization depended critically on the position at which a cysteine was introduced in the t peptide [4]. Because the presence of a cysteine induced tetramerization at position 19 of peptide a, but not at position 19 of peptide b, we explored the effects of cysteines at other positions in BChE. We mutated residues that, similar to N19, are located within the aromatic-rich segment of peptide b, but are oriented in the opposite sector of the a helix, produc- ing mutants A12C, H15C, D23C and N26C (Fig. 1B). We also added a second cysteine near the C-terminus (S37C), changing the C-terminal peptide from SCVGL to CCVGL. These mutations had little effect on the cellular or secreted activities compared to wild-type BChE, except that the secreted ⁄ cellular ratio presented a minimum with a cysteine at position 19, and was notably increased in the mutant possessing two C-terminal cysteines (S37C). As shown in Fig. 4, the sedimenta- tion profiles of cellular enzyme varied mostly in the proportions of monomers and dimers, whereas tetra- mers remained low. The ratio of dimers to monomers was markedly increased with cysteines in the N-termi- nal region of peptide b: b 12C and even more for b 15C . We previously reported a similar observation in the case of mutants of Torpedo AChE [4]. The proportion of tetramers was higher in the medium, and was maxi- mal with mutation N19C. Therefore, position 19 appears to be the most favorable for tetramerization, as previously observed in the case of Torpedo AChE. Is the difference between oligomerization and secretion caused by individual residues that differ between peptides a and b? Because a and b peptides only differ at a few positions, we introduced point mutations to reduce these differ- ences. We made these mutations in Ab 19C because the level of activity, secretion and molecular forms of this mutant were strikingly different from those of Aa 19C (Fig. 1B). We thus mutated the first three residues of peptide b (GNI) as a group; W8 and E9 together and separately; G13, N18, M22 and D23 together and sepa- rately; and N29 and D30 together, replacing these residues by the corresponding ones in a. We also mutated KES to QDR, and VG to DL at the C-termi- nus. We observed no marked effect of any of these mutations on the cellular or secreted activities, except that mutation W8R increased both cellular activity and secretion, in agreement with the notion that aromatic residues induce degradation of AChE through an endo- plasmic reticulum associated degradation process [3]. In all these mutants, the cellular extracts contained only a trace of tetramers, as observed for Ab and D. Liang et al. Oligomerization and secretion of AChE and BChE FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 99 Ab 19C (Fig. 5). The sedimentation profiles of the secreted enzyme were similar to those obtained with Ab 19C , except that the proportion of tetramers (G 4 ) was somewhat increased with N18S. The M22V muta- tion mostly increased the 13.5S species, and the D23H mutation did not increase G 4 by itself, but their combi- nation, M22V ⁄ D23H, induced a significant increase in the proportion of secreted tetramers. Hoping to obtain a higher yield of secreted tetra- mers, we then combined the N18S and M22D ⁄ D23H mutations in Ab 19C . The combination of mutations N18S, N19C, M22D and D23H, abbreviated as S, pro- duced the highest proportion of secreted G 4 tetramers and the highest secreted ⁄ cellular activity ratio. Because these mutations appear to favor the production of tetramers with the b peptide, we introduced them, separately and together, in Bb 19C . However, the resulting Bb S mutant did not produce a higher proportion of tetramers than Bb 19C (Fig. 5). Stokes radius and mass of oligomers We wished to further characterize the oligomers of Ab 19C and other mutants, some of which sedimented faster than tetramers, at 12.3S and 13.5S. Because cholinesterase oligomers may be associated with elongated proteins such as collagen ColQ, their mass cannot be simply deduced from their sedimentation coefficient, but rather from a combination of their Stokes radius and sedimen- tation coefficient [39]. We therefore used gel filtration chromatography to determine the Stokes radius of oligo- mers secreted by the mutant Ab N19C-N29D-D30H , which was chosen because it produces the complete variety of Ab oligomers (Fig. 6A). The major oligomers were iso- lated from gradient fractions. By comparison with the standard proteins b-galactosidase and alkaline phospha- tase, we obtained Stokes radii values, as indicated in Fig. 6B. We then determined the mass of these oligomers, assuming that it is proportional to the product of the sed- imentation coefficient and Stokes radius, as expected for proteins of similar density. The values thus obtained indi- cated a globular structure because the mass was in fact proportional to S 3 ⁄ 2 . This relationship allowed us to determined the mass of the minor components, sediment- ing at 8.5S and 12.3S (Fig. 6A). Figure 6C shows that Cell extract Medium G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 G 1 G 2 G 4 Bb A12C BChE activity (arbitrary units) Sedimentation coefficients 51015 Bb H15C Bb N19C Bb D23C Bb N26C Bb S37C Fig. 4. Sedimentation profiles of mutants of human BChE (Bb) with cysteines at positions 12, 15, 19, 23 and 26. The profiles obtained for Bb 19C , also shown in Fig. 3, are repeated here for comparison with the other mutants. The symbols are as in Fig. 3. Tetramers, dimers and monomers are indicated as G 4 ,G 2 and G 1 , respectively. The mutations replacing various residues by cysteines in the C-terminal peptide are indicated. Oligomerization and secretion of AChE and BChE D. Liang et al. 100 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works the masses of the six observed oligomers represent multi- ples of the smaller one, demonstrating that they represent monomers (G 1 ), dimers (G 2 ), trimers (G 3 ), tetramers (G 4 ), pentamers (G 5 ) and hexamers (G 6 ). Trimers, pentamers and hexamers were only formed with an additional cysteine (S19C). Thus, mutants of Ab can associate into these different multimers, illustrating the versatility of associations between t peptides possessing a cysteine at position 19. As noted above, most of these oligomers were observed in the medium but not in cells. By contrast, BChE only formed monomers, dimers and tetramers. Discussion The C-terminal t peptides do not influence cholinesterase activity The catalytic domain of cholinesterases is associated with two major types of C-terminal peptides: the h peptides contain a signal for the post-translational addition of a glycolipid anchor and cysteines that allow the formation of disulfide-linked dimers, and the t peptides allow the formation of a variety of oligo- mers. These peptides are not required for catalytic activity because truncated enzymes, which are reduced to their catalytic domains, are fully active. Previous studies showed that oligomers of AChE T subunits pos- sessed the same turnover rate per site, but this did not exclude a possible influence of the nature of C-terminal peptides. To examine this question, we titrated the active sites of truncated, wild-type and chimeric cho- linesterases with the irreversible inhibitor DEPQ, and compared their activities with the substrates acetylthio- choline and butyrylthiocholine. We found that the cat- alytic rate per active site only depends on the catalytic domain: it was identical for truncated enzymes (A or B) and with enzymes possessing either a or b C-termi- nal peptides, in agreement with previous studies [40] showing that the variants AChE T , AChE R and a trun- cated mutant possessed the same K m value and excess substrate inhibition. These results also show that the catalytic activity is not influenced by the oligomeric state of the enzymes, and thus justifies quantitative comparisons between the activities of the various mutants investigated in the present study. Effect of the C-terminal t peptides on folding and secretion The t peptides of cholinesterases form amphiphilic a helices with a sector containing their seven conserved aromatic residues. This organization is critical for the association of cholinesterase tetramers with anchoring proteins containing a PRAD, and most probably also for the assembly of homomeric tetramers. However, we have previously shown that the presence of aro- matic residues in the t peptide reduces the production and secretion of AChE, at two distinct checkpoints [41]. First, it induces a partial misfolding of newly synthesized polypeptides; this effect depends on the G 1 G 2 G 4 G 3 G 6 G 1 G 2 G 4 G 6 cell extract medium G 1 G 2 G 4 G 6 G 1 G 2 G 4 G 1 G 2 G 4 G 3 G 6 G 1 G 2 G 4 G 3 G 6 G 1 G 2 G 4 G 3 Sedimentation coefficients 51015 Ab N18S N19C Ab N19C M22V Ab N19C D23H Ab N19C M22V D23H Ab N18S N19C M22V D23H G 1 G 2 G 6 G 1 G 2 G 6 Bb N18S N19C Bb N18S N19C M22V D23H Bb N19C M22V D23H AChE activity (arbitrary units) BChE activity (arbitrary units) 51015 Ab N19C Fig. 5. Effect of mutations suppressing differences between a and b, on the distribution of oligomeric forms. (A) Left panels and top right panel: Ab 19C . (B) Lower right panels: Bb 19C . Sedimentation patterns are shown as in Fig. 3. The sedimentation profiles of the Ab 19C mutant (top right panel) are repeated for comparison with those obtained with additional mutations, which suppressed some of the differences with peptide a. Note that the effects of muta- tions M22V and D23H are not additive. D. Liang et al. Oligomerization and secretion of AChE and BChE FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 101 hydrophobic character of these residues because the same effect was observed when they were replaced by leucines [5]. Second, they target a fraction of active AChE subunits towards degradation by endoplasmic reticulum associated degradation rather than secretion; this effect depends on the presence of aromatic resi- dues, rather than on hydrophobicity. This quality con- trol process may ensure that only correctly assembled subunits are efficiently exported from the cells. The present results confirm that the production, secretion and oligomerization of human AChE and BChE are strongly influenced by their t peptides. In agreement with previous results, secretion was consid- erably increased for both enzymes when the t peptides were deleted. Using chimeras in which these peptides were exchanged (Ab, Ba), we further showed that the ratio between secreted and cellular activities, which may be taken as an index of secretability, was essen- tially determined by the t peptide. The rate of secretion with the t peptide from AChE (a) was more than two- fold higher than with the t peptide from BChE (b). With modified t peptides possessing a cysteine near the center of the aromatic cluster (S19C in a and N19C in b), this difference became more than six-fold. The respective roles of the catalytic domains and t peptides in oligomerization Although the truncated A and B mutants only pro- duced monomers, the Aa, Ab, Ba and Bb enzymes all formed oligomers, including tetramers. Because these tetramers were obtained without co-expression with a PRAD-containing protein, they most probably repre- sent homotetramers, in which the four t peptides may form a coiled coil complex with all aromatic residues oriented inwards, but without a central PRAD. This hypothesis is supported by the fact that, although the presence of a PRAD only induces the assembly of tet- ramers, expression of some mutants without a PRAD produces tetramers together with other oligomers, including molecular forms sedimenting as trimers, pen- tamers and hexamers. The odd-numbered complexes are not likely to represent heteromeric associations containing other proteins because they only occur with some Ab mutants with an added cysteine, and their masses correspond exactly to those expected for multi- ples of AChE subunits. Because the formation of these unusual oligomers appears to depend strictly on the presence of an additional cysteine, they are probably stabilized by a network of inter-catenary disulfide bonds, linking all subunits together. The Ab 19C chimera formed all oligomeric forms from monomers to hexamers, illustrating the versatility of oligomeric associations based on the t peptide, in association with the catalytic domain. It should be noted that hexamers have been observed in transfected COS cells expressing wild-type rat AChE, and appeared as a transient mode of association, which could be dissociated into monomers, dimers and tetra- mers (e.g. in the presence of Triton X-100) [7]. By Ab N19C N29D-D30H secreted activity G 1 G2 G3 G4 G5 G6 Sedimentation coefficients 51015 Arbitrary units G6 0 0.2 0.4 0.6 0.8 1 Arbitrary scales Elution coefficient (Ve-Vo)/(Vt-Vo) -galactosidase Alkaline phosphatase G4 G2 G1 01234567 0 100 200 300 400 500 4.3 S 6.5 S 10.5 S 13.5 S 8.5 S 12.3 S Masses of oligomers (kDa) Numbers of subunits G6 G5 G4 G3 G2 G1 AB C Fig. 6. Determination of the Stokes radius and mass of AChE B oligomers. (A) Oligomers were isolated from sucrose gradients of medium from cells expressing the Ab 19C-29D-30H mutant. The profile of cellular activity was identical to that shown in Fig. 3B for the Ab 19C mutant. (B) Elution of oligomers in gel filtration chromatography. The elution parameters were defined as K e =(V e – V o ) ⁄ (V t – V o ), where V o corre- sponds to the exclusion volume (blue dextran) and V t is the total volume (potassium ferricyanide). The Stokes radii were determined from the linear relationship between the Stokes radius and the square root of [)log (K e )] using the standards b-galactosidase (6.9 nm, 16S, 464 kDa) and alkaline phosphatase (3.3 nm, 6.1S; 87 kDa). (C) The masses of the different oligomers were determined by their proportional- ity to the product of the Stokes radius with the sedimentation coefficient. The masses of the minor 8.5S and 12.3S species were deter- mined from the linear relationship with S 3 ⁄ 2 , observed for the other oligomers. The masses are found to be proportional to discrete degrees of oligomerization, from 1 to 6, showing that the oligomers correspond to monomers (G 1 ), dimers (G 2 ), trimers (G 3 ), tetramers (G 4 ), penta- mers (G 5 ) and hexamers (G 6 ). Oligomerization and secretion of AChE and BChE D. Liang et al. 102 FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works contrast, Bb 19C only formed the classical monomers, dimers and tetramers, possibly because of steric constraints due to the catalytic domain or to its associ- ated N-glycans. Although the nature and proportions of oligomers depended on the presence of the t peptides and their cysteines, the catalytic domains also influenced the oligomerization patterns. The cellular and secreted oligomers formed by Aa and Ab were very similar, as well as those formed by Ba and Bb, suggesting a pre- dominant influence of the catalytic domains on oligo- merization. This may be due in part to the difference in N-glycosylation of AChE and BChE, which carry four and nine N-glycan chains, respectively [35]; we therefore compared the oligomeric patterns of wild-type BChE and mutants in which part of the N-glycosylation sites were mutated [36], but observed no difference (not shown). The relative influence of the C-terminal t peptide appeared to be strongly increased when a cysteine was added at position 19 because the patterns obtained for Aa and Ba were almost the same, except for a shift in the sedimentation coefficients, which are higher for BChE than for AChE. By contrast to Aa and Ab, oligomers of Ba and Bb represented a significant proportion of cellular activity, indicating that AChE oligomers were secreted more rapidly after assembly than BChE oligomers. It is remarkable that the Ab 19C oligomers were observed in the medium but not in cell extracts. This could be related to the presence of the peptide b 19C which reduces secretion but may be masked in the oligomers. Thus, both the catalytic domain and the C-terminal t peptides contribute to the control of oligomerization and secretion, in a complex interplay. Origin and significance of the difference between the t peptides of AChE and BChE The a and b peptides present a considerable sequence similarity, with 60% identical residues, including the seven aromatic residues and the cysteine, which play a key role in the interaction properties of the t peptides. In addition, both peptides are predicted to possess the same tendency to form amphiphilic a helices. It was therefore unexpected to observe a strong difference in their influence on the oligomerization and secretion of AChE and BChE. We tried to assign this difference to some of the residues that distinguish the a and b peptides. Because oligomerization also depends on the catalytic domains, as indicated by the difference between the molecular forms produced by Ab 19C and Bb 19C , the linkage between the two domains might well play a crucial role in the quaternary associations of the cholinesterase subunits. The first three residues of peptides a and b are indeed different, but their replace- ment in Ab 19C (GNI to DTL) had little effect on either secretion or oligomerization. It is also noteworthy that the effects of the combined mutations M22D ⁄ V23H could not be simply accounted for by the effects of the separate mutations M22V and D23V. This suggests that the secretory trafficking of molecules containing peptides a and b depends on global properties of the peptides rather than on individual residues. AChE and BChE are expressed differentially during embryogenesis [42–44]. They appear to play distinct roles, which may be based on their catalytic activity, but also on protein–protein interactions [45], because their catalytic domain is homologous to adhesion pro- teins such as neuroligin [46,47]. For example, AChE may be involved in neurite extension during brain development [40,48,49]. Both catalytic and noncatalytic functions clearly require appropriate oligomeric orga- nization and localization and therefore depend on the C-terminal t peptides, which may be directly involved in distinct interactions with partner proteins. The two cholinesterases present a complex relation- ship with the development of Alzheimer’s disease, which may be partly related to their C-terminal t peptides. Both AChE and BChE are associated with senile pla- ques in Alzheimer’s disease [50] but they appear to play antagonistic roles: AChE promotes amyloid aggregation and increases the neurotoxicity of the Ab peptide in vitro, suggesting that it may participate in the patho- genesis of the disease [51,52]; this appears to depend on interactions of Ab peptides with the peripheral site of AChE and not on its C-terminal t peptide, which has no effect on Ab aggregation [53]. By contrast, the C-termi- nal t peptide of BChE (peptide b) was found to reduce Ab aggregation, possibly because of the presence of its additional tryptophan (W8) located opposite to the aro- matic cluster of the amphiphilic helix (Fig. 1B), so that BChE might have a protective effect against Alzheimer’s disease [53,54]. In this respect, it is worth recalling that, although the human AChE t peptide (here termed peptide a) is organized as an a helix, its AEFHRWS- SYMVHWK fragment, which resembles a portion of the amyloid Ab peptide (AEFRHDSGYEVHHQK), was found to organize into b sheets and to form fibrils; by contrast, the homologous fragment from BChE (AGFHRWNNYMMDWK) did not possess this property [55–57]. AChE and BChE probably arose from a gene dupli- cation in the lineage of vertebrates and it is remarkable that sequence differences between their t peptides are strongly conserved, suggesting that they correspond to distinct molecular interactions and the oligomerization D. Liang et al. Oligomerization and secretion of AChE and BChE FEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 103 [...]... and constructs The coding sequences of human AChET (T variant, Aa) and BChET (Bb), inserted in the pGS vector, were generously provided by O Lockridge The residues of the t peptides are numbered from the first residue following the catalytic domain To exchange the C-terminal t peptides, BsiWI restriction sites were introduced at the junction between regions encoding the catalytic domains and the t peptides. .. lamellipodin [14] In conclusion, the catalytic domains of cholinesterases and their C-terminal t peptides constitute modules of quaternary interactions that are only partially independent The conservation of these peptides during the evolution of vertebrates probably reflects their subtly distinct functions, associated with the respective roles of AChE and BChE in synaptic and nonsynaptic contexts Experimental... peptides Fragments encoding the t peptides were cut between these sites and a downstream SacII site in the vector, purified in agarose gels, and religated with the appropriate complementary fragment The nucleotides separating the coding sequences of the catalytic domains and C-terminal peptides were then removed by site-directed mutagenesis with the method of Kunkel et al [60] Other mutations were performed... (2002) The origin of the molecular diversity and functional anchoring of cholinesterases NeuroSignals 11, 130–143 ´ 2 Massoulie J & Bon S (2006) The C-terminal T peptide of cholinesterases: structure, interactions, and in uence on protein folding and secretion J Mol Neurosci 30, 233–236 ´ 3 Belbeoc’h S, Massoulie J & Bon S (2003) The C-terminal T peptide of acetylcholinesterase enhances degradation of. .. marsupials), including the seven conserved aromatic residues and the C-terminal cysteine Thus, the differences between the t peptides of AChE and BChE are conserved in vertebrates, suggesting that they are functionally significant It is possible that, although the physiological localization of AChE in cholinergic tissues depends on its association with its anchoring proteins ColQ and PRiMA, the major function of. .. rather depends on other interactions, particularly on the formation of soluble tetramers circulating in the bloodstream Production of recombinant AChE or BChE tetramers We have shown that mutation S19C in the C-terminal peptide of human AChE (Aa19C) allows the production of recombinant secreted AChE homotetramers This may be useful to obtain a stable form of AChE that could serve in the decontamination... degradation and secretion of acetylcholinesterase J Biol Chem 280, 878–886 ´ 6 Massoulie J, Bon S, Perrier N & Falasca C (2005) The C-terminal peptides of acetylcholinesterase: cellular trafficking, oligomerization and functional anchoring Chem Biol Interact 157-158, 3–14 ´ 7 Bon S & Massoulie J (1997) Quaternary associations of acetylcholinesterase I Oligomeric associations of T subunits with and without the. .. between man and Maccaca mulatta, and the K variant of human BChE, which occurs with high frequency in European and American populations, consists of the replacement of A6 by a threonine [58], but it does not affect the assembly of tetramers [59] Quite surprisingly, the human t peptide shares 34 common residues with that of chicken and only 13 of these residues are common to both AChEs and BChEs (ten.. .Oligomerization and secretion of AChE and BChE D Liang et al of these sister enzymes The t peptides of higher mammals, including rat, mouse, rabbit, horse, bovine, dog, cat and primates, are identical and share 22 common residues with the t peptide of chicken AChE (however, there are more differences with marsupials) (Fig 7) The t peptides of BChE show more variation between mammalian species: there... between peptides a and b This was unexpected because soluble BChE homotetramers were considered to represent the major species of this enzyme in the bloodstream This suggests that the physiological assembly of BChE tetramers depends on the presence of a PRAD-containing protein or peptide, in agreement with the recent discovery that human plasma BChE contains a proline-rich peptide from lamellipodin [14] In . Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase Dong. 3). The C-terminal t peptides do not in uence the catalytic activity of AChE and BChE We examined the possible in uence of the C-terminal peptides on the