Báo cáo khoa học: Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase potx

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Respective roles of the catalytic domains and C-terminaltail peptides in the oligomerization and secretorytrafficking of human acetylcholinesterase andbutyrylcholinesteraseDong Liang1,2, Jean-Philippe Blouet1, Fernanda Borrega1, Suzanne Bon1and Jean Massoulie´11 Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supe´rieure, Paris, France2 Key Laboratory of Brain Functional Genomics, MOE&STCSM, Shanghai Institute of Brain Functional Genomics, East China NormalUniversity, ChinaIn vertebrates, butyrylcholinesterase (BChET) and theT splice variant of acetylcholinesterase (AChET)consist of a catalytic domain of approximately 500residues, 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 cysteinelocated at position )4 from the C-terminus. Thet peptide plays an important role in the biosynthesis ofcholinesterases, particularly their folding and export.For example, it has been shown that it induces themisfolding of a significant fraction of newly synthe-sized AChE polypeptides, and that this effect dependson hydrophobicity since it was maintained when thearomatic residues were replaced by leucines. The t pep-tide also reduces export, as indicated by the fact thatKeywordsacetylcholinesterase; butyrylcholinesterase;cysteines; oligomers; secretionCorrespondenceJ. Massoulie´, Laboratoire de Neurobiologie,CNRS UMR 8544, Ecole NormaleSupe´rieure, Paris, FranceFax: +33 1 44 32 38 87Tel: +33 1 44 32 38 91E-mail: jean.massoulie@biologie.ens.fr(Received 13 August 2008, revised25 September 2008, accepted24 October 2008)doi:10.1111/j.1742-4658.2008.06756.xButyrylcholinesterase (BChE) and the T splice variant of acetylcholinester-ase that is predominant in mammalian brain and muscles (AChET) possessa characteristic C-terminal tail (t) peptide. This t peptide allows theirassembly into tetramers associated with the anchoring proteins ColQ andPRiMA. Although the t peptides of all vertebrate cholinesterases areremarkably similar and, in particular, contain seven strictly conservedaromatic residues, these enzymes differ in some of their oligomerizationproperties. To explore these differences, we studied human AChE (Aa) andBChE (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 thet peptides, and that it was more efficient with a than with b. The patternsof 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 ofa cysteine within the aromatic-rich segment of the t peptides modified theoligomeric patterns: with a cysteine at position 19, the proportion of tetra-mers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesserextent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms,from monomers to hexamers. These results indicate that both the catalyticdomains and the C-terminal t peptides contribute to the capacity of cho-linesterases to form and secrete various oligomers. Sequence comparisonsshow that the differences between the t peptides of AChE and BChE areremarkably conserved among all vertebrates, suggesting that they reflectdistinct functional adaptations.AbbreviationsAchET, T splice variant of acetylcholinesterase; BChE, butyrylcholinesterase; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide;Nbs2, 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 worksthe ratio of secreted to cellular AChE was stronglyincreased when it was deleted; this effect was sup-pressed by mutation of the aromatic residues toleucines [3–6].However, the major function of the t peptides is thatthey allow the assembly of tetramers of AChET[7] andof BChET[8] and their association with the structuralproteins ColQ and PRiMA [9,10]. These heteromericstructures are based on a tight association betweenfour t peptides, also named tryptophan (W) amphi-philic tetramerization domains, and the poly-prolinemotifs, or proline-rich attachment domains (PRADs)of ColQ and PRiMA [11–13]. In addition, the BChEtetramers that circulate in the blood plasma haverecently been shown to incorporate a similar proline-rich peptide derived from the protein lamellipodin [14].Crystallographic analyses of a complex of synthetic tand PRAD peptides showed that four a-helical t pep-tides form a coiled-coil around the PRAD, which isarranged in a poly-proline II helix [15].The assembly of cholinesterase homo-tetramers orPRAD-associated tetramers is entirely conditioned bythe presence of a t peptide because truncated AChEsubunits lacking the t peptide only produce secretedmonomers [16]. This peptide constitutes an autono-mous interaction module, necessary and sufficient fortetramerization and association with PRAD-containingproteins, because addition of a t peptide at the C-ter-minus of green fluorescent protein or alkaline phos-phatase allowed the formation of tetramers associatedwith an N-terminal fragment of ColQ [17]. However,the catalytic domains are also involved in quaternaryinteractions that certainly participate in the formationand stability of these oligomers. In particular, the tet-ramers are formed of two pairs of subunits, in whicha7,8and a10helices from each subunit form a fourhelix bundle, with a hydrophobic contact zone [16,18].The respective contributions of the catalytic domainsand the t peptides in oligomers has not been evaluated.The formation of AChETtetramers associated withPRAD-containing proteins is physiologically importantbecause it ensures their functional localization by ColQin the basal lamina at neuromuscular junctions [19], aswell as by PRiMA in cell membranes, particularlyin the brain [20]. Similarly, the formation of BChETtetramers conditions the secretion of this enzyme andits stability in the bloodstream.Injection of AChE or BChE offers a very efficientprotection against poisoning by anti-cholinesteraseagents, such as organophosphorous pesticides, butmonomers and dimers are much more rapidly elimi-nated than tetramers after injection in the circulation[21–27]. Although the half life of recombinantenzymes, even monomers, in the bloodstream can beconsiderably increased by derivatization with polyeth-ylene glycol [28–33], it may be interesting to producethese 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 theC-terminus do not form stable dimers, but can formtetramers, particularly in the presence of a PRAD-con-taining protein. It is likely that transient dimerizationoccurs as a first step in the assembly of tetramers,either associated or not with a PRAD. We recentlyfound that addition of a second cysteine at an appro-priate position in the t peptide of Torpedo AChEgreatly increased the formation and secretion of homo-tetramers [4]. We therefore explored the possibility thatmutations in the t peptides of human AChETandBChETmight induce their assembly into tetramers.Because these two enzymes differ in their capacity toform oligomers, we investigated the respective roles ofthe catalytic domain and of the t peptides. For thispurpose, we constructed chimeric proteins, in whichwe associated the catalytic domain of each enzymewith the t peptide of the other. For convenience, thelarge catalytic domains are designated by capital letters(A and B), whereas the small t peptides are designatedby lower case letters (a and b), so that the wild-typeAChE and BChE are Aa and Bb, and the chimeras areAb and Ba. Comparisons of wild-type enzymes andchimeras, as well as of various mutants, show thatboth domains contribute critically to the oligomeriza-tion and to the efficiency of secretion.ResultsExchange of t peptides between human AChEand BChEThe T variants of human AChE and human BChE arecomposed of a catalytic domain of approximately 500residues, followed by small C-terminal t peptides of 40and 41 residues, respectively. In the present study, thecatalytic domains are indicated by capital letters ( A andB) and the C-terminal peptides by lower case letters (aand b), so that the wild-type enzymes are abbreviated asAa and Bb. The C-terminal t peptides of human AChET(a) and BChET(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 immediatelyfollowing the catalytic domain. Some of the differencesD. Liang et al. Oligomerization and secretion of AChE and BChEFEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 95between the peptides a and b might be important for theprocessing and the activity of AChE and BChE, notablythose involving charged residues, the presence in peptideb of an additional tryptophan (W8) and the presence ofsix instead of five residues between the aromatic-richregion and the cysteine. Both peptides are predicted toform 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 thet peptides of human AChE and BChE, we constructedchimeras 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 differentconstructs were expressed in transiently transfectedCOS cells, and we analyzed the cellular and secretedcholinesterase activities (Fig. 2), as well as the oligo-meric patterns, revealed by sedimentation profiles insucrose gradients (Fig. 3).The C-terminal t peptides do not influence thecatalytic activity of AChE and BChEWe examined the possible influence of the C-terminalpeptides on the AChE and BChE activities bycomparing the catalytic rates per active site. The activesites were titrated with the irreversible inhibitor7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide(DEPQ) (see Experimental procedures). The slopes ofresidual activity, plotted as a function of the amountof DEPQ, were identical for A, Aa and Ab, withacetylthiocholine as substrate, as well as for B, Baand Bb, using either acetylthiocholine or butyrylthio-choline as substrates. Because of excess substrate inhi-bition, AChE presented a maximal rate forapproximately 2 mm acetylthiocholine. The rates ofhydrolysis of acetylthiocholine and butyrylthiocholine(at 6 mm) by BChE were approximately 14% and24% of the rate of hydrolysis of acetylthiocholine(at 2 mm) by AChE.Influence of the C-terminal t peptides on activity,secretion and oligomerizationAs expected, the truncated mutants A and B, withoutt peptides, produced only monomers, sedimentingaround 4S (not shown). The levels of cellular activitywere lower for these mutants than for the wild-types butsecretion was increased (Fig. 2A,B), in agreement withour previous conclusions that t peptides induce a partialmisfolding of the polypeptides, as well as an intracellulardegradation of a fraction of active subunits [3].Cells expressing wild-type human AChE (Aa)secreted approximately 15% of their content per hourand produced mostly monomers and dimers, with asmall proportion of tetramers (less than 10% of theABFig. 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 sevenaromatic 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 studyare 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 regionsof 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 workssecreted activity). For human BChE (Bb), the rate ofsecretion was only approximately 5% of the cellularcontent per hour. This enzyme formed a higher pro-portion of oligomers, mostly dimers in the cells, andtetramers represented approximately 30% of thesecreted enzyme, together with comparable proportionsof dimers and monomers (Fig. 3A).The fact that Bb forms a higher proportion of olig-omers, but is less efficiently secreted than Aa, is quiteparadoxical because secretion generally increases withthe degree of oligomerization. Clearly, the assemblyof tetramers is not restricted by the fact that eachBChE subunit possesses nine N-linked glycans [35],whereas AChE has only four. This was confirmed by0501001502002500 50 100 150 200 250-A-AaAa S19CAa S38C-AbAb SSVGLAb N19CAb N19C N18SAb N19C MD22VHAb N19C N18S MD22VH-B-BaBa S19C-BbBb SSVGLBb A12CBb H15CBb N19CBb D23CBb N26CBb S37CBb N19C N18SBb N19C MD22VHBb N19C N18S MD22VH-Cellular activityABSecreted activity0123456A-AaAa S19CAa S38CAbAb SSVGLAb N19CAb N19C N18SAb N19C MD22VHAb N19C N18S MD22VHB-BaBa S19CBbBb SSVGLBb A12CBb H15CBb N19CBb D23CBb N26CBb S37CBb N19C N18SBb N19C MD22VHBb N19C N18S MD22VHRatio of secreted to cellularactivity (% 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 secretedactivities. A and B represent AChE and BChE from which the t peptides were deleted; Aa and Bb represent the wild-type enzymes withtheir t peptides, Ab and Ba represent chimeras in which the t peptides were exchanged; mutations in the t peptides are indicated. For eachmutant, the cellular and secreted activities are shown by bars to the left and the right. AChE and BChE activities were determined by theEllman assay with acetylthiocholine and butyrylthiocholine as substrates, respectively: AChE activities are indicated as grey bars and BChEactivities as hatched bars. AChE and BChE activities were normalized to the wild-type enzymes (Aa and Bb, respectively). (B) Ratioof 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 BChEFEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 97the 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 secretionwere approximately 5% and 15% of the cellular con-tent per hour, respectively, and therefore appeared tobe mainly determined by the t peptides. By contrast,Fig. 3B shows that the sedimentation profiles werevery similar for Aa and Ab, and for Ba and Bb,except that the BChE species sedimented faster thantheir AChE counterparts, in agreement with thehigher mass of BChE subunits [37]. This indicates apredominant influence of the catalytic domain onoligomerization.Role of the C-terminal cysteineMutation of the cysteine located at )4 from the C-ter-minus to a serine in the a or b peptides suppressed theformation of Aa or Bb dimers, but not the productionof a small proportion of tetramers (not shown). Thesemutations increased the ratio of secreted to cellularactivity in both cases (Fig. 2). However, in the case ofBb, the cellular activity was decreased and secretionwas 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 byapproximately 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-terminalcysteine on the trafficking of cholinesterase in thesecretory pathway largely depends on the nature of thepreceding catalytic domain.Oligomerization might be affected by the distancebetween the aromatic core and the C-terminal cysteine,which forms inter-catenary disulfide bonds. There arefive 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 residueon oligomerization, we deleted T32 in Bb and wemutated peptides a (CSDL to SCDL) and b (SCVGL toCSVGL), to modify the number of residues between thecysteine and the aromatic core. We found that thesemutations had no effect on either the levels of cellularand secreted activities, or on the distribution of oligo-meric forms (not shown). Similarly, these mutations didnot modify the secretion or the oligomerization ofmutants possessing a cysteine at position 19 (see below).Thus, the addition or subtraction of one residue in theinterval between the aromatic residues and the cysteinehad no influence, suggesting that this peptidic segmentrepresents a flexible spacer, in agreement with previousstudies on Torpedo AChE [38].Role of cysteines in oligomerization – effects ofintroducing an additional cysteineIn a previous study, we found that mutating residue19 in the t peptide of Torpedo AChE considerablyincreased the production and secretion of tetramersCell extractMediumG1G2G4G1G2G4G1G2G4G1G2G4G3G6G1G2G4G1G2G4G1G2G4G1G2G4Aa Bb Ba Ab Aa AChE activity (arbitrary units)BChE activity (arbitrary units)Sedimentation coefficients55101015 15Wild-type t peptidesABWith added cysteine19CBb Ba Ab 19C19C19CFig. 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 acysteine at position 19 (S19C in peptide a, N19C in peptide b). Theprofiles 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, dimersand monomers are indicated as G4,G2and G1, respectively. Notethat the molecular forms of BChE and its mutants sediment slightlyfaster 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 aand b, and analyzed the resulting activities andmolecular forms produced by expressing the fourcholinesterase combinations in COS cells. Thismutation did not modify the level of secretion forAa19C, increased it for Ba19C, and decreased it forAb19Cand Bb19C(Fig. 2). The fact that the cellularactivity was unchanged or decreased, whereas sec-retion was decreased, indicates that the N19Cmutation in peptide b induced an intracellular degra-dation of Ab19Cand Bb19C. As observed in the pre-ceding section, the ratio of secreted to cellularactivity again appeared to depend essentially on thet peptides: it was much higher for Aa19Cand Ba19Cthan for Ab19Cand Bb19C. The 19C mutationsenhanced the difference between the two peptidesbecause the secreted ⁄ cellular ratio was increased withpeptide a19Ccompared to a and decreased with pep-tide b19Ccompared to b.By contrast to the oligomeric patterns obtainedwithout a cysteine at position 19, we observed amuch stronger similarity between enzymes possessingthe same C-terminal peptide (a19Cor b19C) thanbetween those possessing the same catalytic domain(Fig. 3B). Thus, mutation S19C in a19Cstronglyincreased the proportion of tetramers, which becamethe predominant secreted species for both Aa19CandBa19C: these mutants produced very similar patternsof molecular forms. The effect of mutation N19C inpeptide b19Chad little effect on the distribution ofsecreted molecular forms of Bb19C. In the case ofAb19C, the effect was more complex: the cells con-tained mostly 4S monomers but secreted a variety ofoligomers, mostly monomers, dimers, trimers, tetra-mers and hexamers (see below). The fact that theoligomeric forms were very low or undetectable inthe cells suggests that they were secreted very rapidlyafter their assembly. The results were identical whenthe 19C mutations were combined with mutationsthat modified the distance between the C-terminalcysteine 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 thanwild-type t peptides, and exerted a dominant influenceon oligomerization.Effects of introducing cysteines at differentpositions in BChEOur previous study of Torpedo AChE showed that thepattern of oligomerization depended critically onthe position at which a cysteine was introduced in thet peptide [4]. Because the presence of a cysteineinduced tetramerization at position 19 of peptide a,but not at position 19 of peptide b, we explored theeffects of cysteines at other positions in BChE. Wemutated residues that, similar to N19, are locatedwithin the aromatic-rich segment of peptide b, but areoriented 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 SCVGLto CCVGL.These mutations had little effect on the cellular orsecreted activities compared to wild-type BChE, exceptthat the secreted ⁄ cellular ratio presented a minimumwith a cysteine at position 19, and was notablyincreased in the mutant possessing two C-terminalcysteines (S37C). As shown in Fig. 4, the sedimenta-tion profiles of cellular enzyme varied mostly in theproportions of monomers and dimers, whereas tetra-mers remained low. The ratio of dimers to monomerswas markedly increased with cysteines in the N-termi-nal region of peptide b: b12Cand even more for b15C.We previously reported a similar observation in thecase of mutants of Torpedo AChE [4]. The proportionof tetramers was higher in the medium, and was maxi-mal with mutation N19C. Therefore, position 19appears to be the most favorable for tetramerization,as previously observed in the case of Torpedo AChE.Is the difference between oligomerization andsecretion caused by individual residues that differbetween 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 Ab19Cbecause thelevel of activity, secretion and molecular forms of thismutant were strikingly different from those of Aa19C(Fig. 1B). We thus mutated the first three residues ofpeptide b (GNI) as a group; W8 and E9 together andseparately; G13, N18, M22 and D23 together and sepa-rately; and N29 and D30 together, replacing theseresidues by the corresponding ones in a. We alsomutated KES to QDR, and VG to DL at the C-termi-nus. We observed no marked effect of any of thesemutations on the cellular or secreted activities, exceptthat mutation W8R increased both cellular activity andsecretion, in agreement with the notion that aromaticresidues induce degradation of AChE through an endo-plasmic reticulum associated degradation process [3].In all these mutants, the cellular extracts containedonly a trace of tetramers, as observed for Ab andD. Liang et al. Oligomerization and secretion of AChE and BChEFEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 99Ab19C(Fig. 5). The sedimentation profiles of thesecreted enzyme were similar to those obtained withAb19C, except that the proportion of tetramers (G4)was somewhat increased with N18S. The M22V muta-tion mostly increased the 13.5S species, and the D23Hmutation did not increase G4by itself, but their combi-nation, M22V ⁄ D23H, induced a significant increase inthe proportion of secreted tetramers.Hoping to obtain a higher yield of secreted tetra-mers, we then combined the N18S and M22D ⁄ D23Hmutations in Ab19C. The combination of mutationsN18S, N19C, M22D and D23H, abbreviated as S, pro-duced the highest proportion of secreted G4tetramersand the highest secreted ⁄ cellular activity ratio. Becausethese mutations appear to favor the production oftetramers with the b peptide, we introduced them,separately and together, in Bb19C. However, theresulting BbSmutant did not produce a higherproportion of tetramers than Bb19C(Fig. 5).Stokes radius and mass of oligomersWe wished to further characterize the oligomers of Ab19Cand other mutants, some of which sedimented faster thantetramers, at 12.3S and 13.5S. Because cholinesteraseoligomers may be associated with elongated proteinssuch as collagen ColQ, their mass cannot be simplydeduced from their sedimentation coefficient, but ratherfrom a combination of their Stokes radius and sedimen-tation coefficient [39]. We therefore used gel filtrationchromatography to determine the Stokes radius of oligo-mers secreted by the mutant AbN19C-N29D-D30H, whichwas chosen because it produces the complete variety ofAb oligomers (Fig. 6A). The major oligomers were iso-lated from gradient fractions. By comparison with thestandard proteins b-galactosidase and alkaline phospha-tase, we obtained Stokes radii values, as indicated inFig. 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 forproteins of similar density. The values thus obtained indi-cated a globular structure because the mass was in factproportional to S3 ⁄ 2. This relationship allowed us todetermined the mass of the minor components, sediment-ing at 8.5S and 12.3S (Fig. 6A). Figure 6C shows thatCell extractMediumG1G2G4G1G2G4G1G2G4G1G2G4G1G2G4G1G2G4Bb A12CBChE activity (arbitrary units)Sedimentation coefficients51015Bb H15CBb N19CBb D23CBb N26CBb S37CFig. 4. Sedimentation profiles of mutants of human BChE (Bb)with cysteines at positions 12, 15, 19, 23 and 26. The profilesobtained for Bb19C, also shown in Fig. 3, are repeated here forcomparison with the other mutants. The symbols are as in Fig. 3.Tetramers, dimers and monomers are indicated as G4,G2and G1,respectively. The mutations replacing various residues by cysteinesin 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 worksthe masses of the six observed oligomers represent multi-ples of the smaller one, demonstrating that they representmonomers (G1), dimers (G2), trimers (G3), tetramers(G4), pentamers (G5) and hexamers (G6).Trimers, pentamers and hexamers were only formedwith an additional cysteine (S19C). Thus, mutants ofAb can associate into these different multimers,illustrating the versatility of associations betweent peptides possessing a cysteine at position 19. Asnoted above, most of these oligomers were observed inthe medium but not in cells. By contrast, BChE onlyformed monomers, dimers and tetramers.DiscussionThe C-terminal t peptides do not influencecholinesterase activityThe catalytic domain of cholinesterases is associatedwith two major types of C-terminal peptides: theh peptides contain a signal for the post-translationaladdition of a glycolipid anchor and cysteines thatallow the formation of disulfide-linked dimers, and thet peptides allow the formation of a variety of oligo-mers. These peptides are not required for catalyticactivity because truncated enzymes, which are reducedto their catalytic domains, are fully active. Previousstudies showed that oligomers of AChETsubunits pos-sessed the same turnover rate per site, but this did notexclude a possible influence of the nature of C-terminalpeptides. To examine this question, we titrated theactive sites of truncated, wild-type and chimeric cho-linesterases with the irreversible inhibitor DEPQ, andcompared their activities with the substrates acetylthio-choline and butyrylthiocholine. We found that the cat-alytic rate per active site only depends on the catalyticdomain: it was identical for truncated enzymes (A orB) and with enzymes possessing either a or b C-termi-nal peptides, in agreement with previous studies [40]showing that the variants AChET, AChERand a trun-cated mutant possessed the same Kmvalue and excesssubstrate inhibition. These results also show that thecatalytic activity is not influenced by the oligomericstate of the enzymes, and thus justifies quantitativecomparisons between the activities of the variousmutants investigated in the present study.Effect of the C-terminal t peptides on foldingand secretionThe t peptides of cholinesterases form amphiphilica helices with a sector containing their seven conservedaromatic residues. This organization is critical for theassociation of cholinesterase tetramers with anchoringproteins containing a PRAD, and most probably alsofor the assembly of homomeric tetramers. However,we have previously shown that the presence of aro-matic residues in the t peptide reduces the productionand secretion of AChE, at two distinct checkpoints[41]. First, it induces a partial misfolding of newlysynthesized polypeptides; this effect depends on theG1G2G4G3G6G1G2G4G6cell extractmediumG1G2G4G6G1G2G4G1G2G4G3G6G1G2G4G3G6G1G2G4G3Sedimentation coefficients51015Ab N18SN19CAb N19CM22VAb N19CD23HAb N19CM22V D23HAb N18S N19CM22V D23HG1G2G6G1G2G6Bb N18SN19CBb N18S N19CM22V D23HBb N19CM22V D23HAChE activity (arbitrary units)BChE activity (arbitrary units)51015Ab N19CFig. 5. Effect of mutations suppressing differences between a andb, on the distribution of oligomeric forms. (A) Left panels and topright panel: Ab19C. (B) Lower right panels: Bb19C. Sedimentationpatterns are shown as in Fig. 3. The sedimentation profiles of theAb19Cmutant (top right panel) are repeated for comparison withthose obtained with additional mutations, which suppressed someof 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 BChEFEBS Journal 276 (2009) 94–108 Journal compilation ª 2008 FEBS. No claim to original French government works 101hydrophobic character of these residues because thesame effect was observed when they were replaced byleucines [5]. Second, they target a fraction of activeAChE subunits towards degradation by endoplasmicreticulum 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 assembledsubunits are efficiently exported from the cells.The present results confirm that the production,secretion and oligomerization of human AChE andBChE are strongly influenced by their t peptides. Inagreement with previous results, secretion was consid-erably increased for both enzymes when the t peptideswere deleted. Using chimeras in which these peptideswere exchanged (Ab, Ba), we further showed that theratio between secreted and cellular activities, whichmay be taken as an index of secretability, was essen-tially determined by the t peptide. The rate of secretionwith 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 thecenter of the aromatic cluster (S19C in a and N19Cin b), this difference became more than six-fold.The respective roles of the catalytic domainsand t peptides in oligomerizationAlthough the truncated A and B mutants only pro-duced monomers, the Aa, Ab, Ba and Bb enzymes allformed oligomers, including tetramers. Because thesetetramers were obtained without co-expression with aPRAD-containing protein, they most probably repre-sent homotetramers, in which the four t peptides mayform a coiled coil complex with all aromatic residuesoriented inwards, but without a central PRAD. Thishypothesis is supported by the fact that, although thepresence of a PRAD only induces the assembly of tet-ramers, expression of some mutants without a PRADproduces tetramers together with other oligomers,including molecular forms sedimenting as trimers, pen-tamers and hexamers. The odd-numbered complexesare not likely to represent heteromeric associationscontaining other proteins because they only occur withsome Ab mutants with an added cysteine, and theirmasses correspond exactly to those expected for multi-ples of AChE subunits. Because the formation of theseunusual oligomers appears to depend strictly on thepresence of an additional cysteine, they are probablystabilized by a network of inter-catenary disulfidebonds, linking all subunits together.The Ab19Cchimera formed all oligomeric formsfrom monomers to hexamers, illustrating the versatilityof oligomeric associations based on the t peptide, inassociation with the catalytic domain. It should benoted that hexamers have been observed in transfectedCOS cells expressing wild-type rat AChE, andappeared as a transient mode of association, whichcould be dissociated into monomers, dimers and tetra-mers (e.g. in the presence of Triton X-100) [7]. ByAb N19CN29D-D30Hsecreted activityG1G2G3G4G5G6Sedimentation coefficients51015Arbitrary unitsG60 0.2 0.4 0.6 0.8 1Arbitrary scalesElution coefficient (Ve-Vo)/(Vt-Vo)-galactosidase Alkaline phosphataseG4G2G10123456701002003004005004.3 S6.5 S10.5 S13.5 S8.5 S12.3 SMasses of oligomers (kDa)Numbers of subunitsG6G5G4G3G2G1AB CFig. 6. Determination of the Stokes radius and mass of AChEBoligomers. (A) Oligomers were isolated from sucrose gradients of mediumfrom cells expressing the Ab19C-29D-30Hmutant. The profile of cellular activity was identical to that shown in Fig. 3B for the Ab19Cmutant.(B) Elution of oligomers in gel filtration chromatography. The elution parameters were defined as Ke=(Ve– Vo) ⁄ (Vt– Vo), where Vocorre-sponds to the exclusion volume (blue dextran) and Vtis the total volume (potassium ferricyanide). The Stokes radii were determined fromthe linear relationship between the Stokes radius and the square root of [)log (Ke)] 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 S3 ⁄ 2, observed for the other oligomers. The masses are found to be proportional to discrete degreesof oligomerization, from 1 to 6, showing that the oligomers correspond to monomers (G1), dimers (G2), trimers (G3), tetramers (G4), penta-mers (G5) and hexamers (G6).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 workscontrast, Bb19Conly formed the classical monomers,dimers and tetramers, possibly because of stericconstraints due to the catalytic domain or to its associ-ated N-glycans.Although the nature and proportions of oligomersdepended on the presence of the t peptides and theircysteines, the catalytic domains also influenced theoligomerization patterns. The cellular and secretedoligomers formed by Aa and Ab were very similar, aswell 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 differencein N-glycosylation of AChE and BChE, which carryfour and nine N-glycan chains, respectively [35]; wetherefore compared the oligomeric patterns ofwild-type BChE and mutants in which part of theN-glycosylation sites were mutated [36], but observedno difference (not shown). The relative influence of theC-terminal t peptide appeared to be strongly increasedwhen a cysteine was added at position 19 because thepatterns 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 Bbrepresented a significant proportion of cellular activity,indicating that AChE oligomers were secreted morerapidly after assembly than BChE oligomers. It isremarkable that the Ab19Coligomers were observed inthe medium but not in cell extracts. This could berelated to the presence of the peptide b19Cwhichreduces secretion but may be masked in the oligomers.Thus, both the catalytic domain and the C-terminalt peptides contribute to the control of oligomerizationand secretion, in a complex interplay.Origin and significance of the difference betweenthe t peptides of AChE and BChEThe a and b peptides present a considerable sequencesimilarity, with 60% identical residues, including theseven aromatic residues and the cysteine, which play akey role in the interaction properties of the t peptides.In addition, both peptides are predicted to possess thesame tendency to form amphiphilic a helices. It wastherefore unexpected to observe a strong difference intheir influence on the oligomerization and secretion ofAChE and BChE. We tried to assign this differenceto some of the residues that distinguish the a and bpeptides. Because oligomerization also depends on thecatalytic domains, as indicated by the differencebetween the molecular forms produced by Ab19CandBb19C, the linkage between the two domains might wellplay a crucial role in the quaternary associations ofthe cholinesterase subunits. The first three residues ofpeptides a and b are indeed different, but their replace-ment in Ab19C(GNI to DTL) had little effect on eithersecretion or oligomerization. It is also noteworthy thatthe effects of the combined mutations M22D ⁄ V23Hcould not be simply accounted for by the effects of theseparate mutations M22V and D23V. This suggeststhat the secretory trafficking of molecules containingpeptides a and b depends on global properties of thepeptides rather than on individual residues.AChE and BChE are expressed differentially duringembryogenesis [42–44]. They appear to play distinctroles, which may be based on their catalytic activity,but also on protein–protein interactions [45], becausetheir catalytic domain is homologous to adhesion pro-teins such as neuroligin [46,47]. For example, AChEmay be involved in neurite extension during braindevelopment [40,48,49]. Both catalytic and noncatalyticfunctions clearly require appropriate oligomeric orga-nization and localization and therefore depend on theC-terminal t peptides, which may be directly involvedin distinct interactions with partner proteins.The two cholinesterases present a complex relation-ship with the development of Alzheimer’s disease, whichmay 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 playantagonistic roles: AChE promotes amyloid aggregationand increases the neurotoxicity of the Ab peptidein vitro, suggesting that it may participate in the patho-genesis of the disease [51,52]; this appears to depend oninteractions of Ab peptides with the peripheral site ofAChE and not on its C-terminal t peptide, which has noeffect on Ab aggregation [53]. By contrast, the C-termi-nal t peptide of BChE (peptide b) was found to reduceAb aggregation, possibly because of the presence of itsadditional tryptophan (W8) located opposite to the aro-matic cluster of the amphiphilic helix (Fig. 1B), so thatBChE might have a protective effect against Alzheimer’sdisease [53,54]. In this respect, it is worth recalling that,although the human AChE t peptide (here termedpeptide a) is organized as an a helix, its AEFHRWS-SYMVHWK fragment, which resembles a portion ofthe 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 thisproperty [55–57].AChE and BChE probably arose from a gene dupli-cation in the lineage of vertebrates and it is remarkablethat sequence differences between their t peptides arestrongly conserved, suggesting that they correspond todistinct molecular interactions and the oligomerizationD. Liang et al. Oligomerization and secretion of AChE and BChEFEBS 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 BChEWe examined the possible in uence of the C-terminal peptides on the
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Xem thêm: Báo cáo khoa học: Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase potx, Báo cáo khoa học: Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase potx, Báo cáo khoa học: Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase potx