The C-terminal t peptide of acetylcholinesterase forms an a helix that supports homomeric and heteromeric interactions Suzanne Bon 1 , Jean Dufourcq 2 , Jacqueline Leroy 1 , Isabelle Cornut 2 and Jean Massoulie ´ 1 1 Laboratoire de Neurobiologie Cellulaire et Mole ´ culaire, Ecole Normale Supe ´ rieure, Paris, France; 2 Centre de Recherche Paul Pascal, Pessac, France Acetylcholinesterase subunits of type T (AChE T ) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetra- mer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChE T , we show that the region containing the aromatic residues adopts an a helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix. We also analyzed the formation of disulfide bonds between two different AChE T subunits, and between AChE T subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (Q N )] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with Q N (T 4 –Q N ) 4 , the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD 5 . The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility. Keywords: acetylcholinesterase; amphiphilic alpha helix; disulfide bonds; proline rich domain. The quaternary associations of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are determined by small C-terminal domains that are distinct from the catalytic domain [1,2]. In vertebrates, alternatively spliced exons of the AChE gene 6 encode several C-terminal domains which distinguish different types of subunits. However, only subunits of type T (ÔtailedÕ) exist in the BChE and AChEs of all vertebrates; in mammals they represent the only AChE variant expressed in the adult nervous system and muscles. These subunits possess specific association pro- perties, which depend on their C-terminal t peptide. This peptide is strongly conserved in vertebrates, with 75% identity between cartilagenous fishes (Torpedo) and mam- mals; it contains 40 or 41 residues, with a cysteine at )4from the C-terminus and a series of seven conserved aromatic residues including three tryptophans [3]. Transfected COS cells expressing subunits of type T produce a wide array of catalytically active AChE forms, including monomers, dimers and tetramers [4]. The mono- mers, dimers and some tetramers are amphiphilic, as defined by their interaction with detergent micelles, which modify their sedimentation and their electrophoretic migration in nondenaturing conditions [5]. These amphiphilic molecular forms require detergents to be totally solubilized but are also secreted when expressed in transfected COS cells [4]. The t peptide is necessary for the amphiphilic character of AChE and for the formation of tetramers, as deleted subunits that lack this peptide generate only nonamphiphilic monomers [6]. AChE subunits of type T (AChE T ) can assemble into tetramers with their anchoring proteins ColQ and PRiMA, and these heteromeric associations represent the physio- logically functional species in muscles and brain [7,8]. At the neuromuscular junction, collagen-tailed asymmetric forms are inserted in the basal lamina; in these molecules, one AChE T tetramer (T 4 ) is attached to the N-terminal region of each of the three strands of the triple helical ColQ collagen. In the mammalian brain, the predominant AChE species is a tetramer, anchored at the cell surface through the Correspondence to S. Bon, Laboratoire de Neurobiologie Cellulaire et Mole ´ culaire, CNRS UMR 8544, Ecole Normale Supe ´ rieure, 46 rue d’Ulm, 75005 Paris, France. Fax: + 33 1 44 32 38 87, Tel.: + 33 1 44 32 38 91, E-mail: jean.massoulie@biologie.ens.fr Abbreviations: AChE, acetylcholinesterase; AChE H , AChE subunit of type H; AChE T , AChE subunit of type T (ÔtailedÕ); BChE, butyryl- cholinesterase; BChE T , BChE subunit of type T (ÔtailedÕ); cmc, critical micellar concentration; CTAB, cetyltrimethylammonium bromide; C37, C-terminal cysteine residue at position 37; GPI, glycophospha- tidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PRAD, proline rich attachment domain; Q N , N-terminal fragment of the ColQ protein; SMCC, N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1 carboxylate; t peptide, the C-terminal peptide of AChE T subunits; T, AChE T subunits; WAT, tryptophan amphiphilic tetramerization domain. Note: In this paper the residues of the t peptides of AChE T from different species are numbered from 1 to 40 in order to facilitate comparisons. (Received 31 July 2003, revised 10 October 2003, accepted 23 October 2003) Eur. J. Biochem. 271, 33–47 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03892.x transmembrane protein PRiMA (T 4 –PRiMA). The N-terminal regions of both ColQ and PRiMA contain a proline-rich attachment domain (PRAD) [9], which is responsible for their interaction with AChE T or BChE T subunits; in addition, they contain cysteines that form disulfide bonds with two cholinesterase T subunits in each tetramer, by means of the cysteines located near their C-terminus [10–12]. The t peptide is in fact sufficient for association with a PRAD, as shown by the fact that it can replace a complete AChE T or BChE T subunit in PRAD-associated tetramers, and can induce the formation of PRAD-linked tetramers when added at the C-terminus of foreign proteins such as green fluorescent protein or alkaline phosphatase: it there- fore constitutes an autonomous interaction domain, referred to as the WAT [tryptophan amphiphilic tetra- merization] domain [13]. The t peptide also acts as an enhancer of degradation through the ER-associated degra- dation pathway [14]. In the present study, we analyse the structural basis for the hydrophobic and quaternary interactions of the t pep- tide. In particular, we ask whether hydrophobic interactions result from the structure of the peptide itself or require post- translational modifications, e.g. the addition of lipidic residues. It has been reported that membrane-bound mouse AChE produced in transfected human embryo kidney 293 cells incorporates palmitic acid, but not mevalonate, in spite of the resemblance of its C-terminus with an isoprenylaytion signal [15]. The amphiphilic properties of AChE T subunits suggest that the t peptide constitutes an amphiphilic ahelix, with its seven aromatic residues located in the same sector, forming a hydrophobic cluster [1]. Here, we present evidence that the t peptide actually forms an amphiphilic helix and that it is elongated, rather than folded upon itself in a hairpin as proposed by Giles [16], in AChE T monomers and dimers as well as in tetramers associated with an N-terminal fragment of ColQ (Q N ). We also show that the four t peptides are parallel to each other and antiparallel 7 to the PRAD in the T 4 –Q N heteromeric complex. Materials and methods Materials Egg phosphatidylcholine and its lyso derivative were prepared as described previously [17]. Phosphatidylserine was obtained from Lipid Products (Nutfield, Surrey, UK). The detergents used for the spectroscopic studies were from VWR (Strasbourg, France) and Sigma 8 and were recrystal- lized before use. A lytic tetrameric form (G 4 ) derived from collagen-tailed Electrophorus AChE was purified by affinity chromatography on Sepharose derivatized with hexylamido- carboxyphenyl-dimethylethylammonium, as described pre- viously [18]. Peptide synthesis The t 1)32 peptide was synthesized in the laboratory of J. Vandekerckhove (Laboratorium Genetika, Gent, Bel- gium). It was purified by preparative HPLC and analyzed in a C-18 Vydac column (The Nest Group, Southborough, MA, USA): the preparation contained essentially only the monomeric peptide, with less than 10% dimers, spontane- ously formed upon air oxidation and that could be reduced by dithiothreitol. The t 1)40 peptide, at 85% purity, was synthesized by Neosystem Laboratoires (Strasbourg, France). The t 25)40 peptide was synthesized in the laboratory of J. Igolen (Institut Pasteur, Paris, France) and was puri- fied by preparative HPLC. Whereas the C-terminal cysteine residue at position 37 of t 1)40 (C37) was blocked by an acetamidomethyl group, cysteines were added at the N-ter- minus of t 1)32 and t 1)40 , to allow their linkage to non- amphiphilic AChE tetramers from Electrophorus electric organs, via their N-terminal extremity, as with AChE T subunits. Chemical coupling of peptides with Electrophorus G 4 AChE Each of the t 1)32 ,t 1)40 and t 25)40 peptides were covalently coupled to the G 4 form of Electrophorus AChE by the heterobifunctional reagent N-succinimidyl-4-(N-maleimido- methyl)cyclohexane-1 carboxylate (SMCC). This method involves the reaction of thiol groups from cysteine residues of the peptides with a maleimido group incorporated into AChE after reaction with SMCC. The preparation of AChE–SMCC has been described elsewhere [19]. Subsequently to being dissolved in 0.1 M phosphate buffer, pH 6, the thiol content of the peptides was measured by reaction with 5,5¢-dithiobis(2-nitrobenzoic acid) [20]. Coupling between the peptide and the enzyme was obtained by mixing AChE–SMCC with an excess of thiol groups (the concentration of thiol was  100-fold that of G 4 ). Peptides t 1)32 and t 1)40 were coupled using the added N-terminal cysteine and t 25)40 was coupled through C37. After 3 h at 30 °C, the conjugate was purified by molecular sieve chromatography in a Biogel A0.5 column (Bio-Rad Laboratories), as described previously [21]. We observed no significant loss in enzyme activity during the coupling procedure. Production of antibodies against t 25)40 peptide Anti-(t 25)40 ) polyclonal Ig was raised in rabbit against the t 25)40 peptide covalently coupled to BSA. The t 25)40 –BSA conjugate was obtained by reaction with glutaraldehyde, as described previously [22]. Immunization followed the pro- cedure described by Vaitukatis [23]. Spectroscopic analyses Circular dichroism spectra were obtained in an AVIV 62DS (AVIV, Zu ¨ rich, Switzerland) spectrometer at 25 °C, using cuvettes of 0.1–1 cm path-length according to the concen- tration of peptide. The blank was subtracted in all cases. For evaluation of the molar ellipticity per residue (h) expressed in degÆdmol )1 Æcm 2 , the peptide concentration was calculated by using an absorbance e 280 ¼ 20 000 M –l Æcm –l . Fluorescence spectra were obtained with a Fluoromax SPEX spectrophotometer (Jobin et Yvon, Longjumeau, France)at25°C, with an excitation wavelength of 280 nm and a slit width of 1.7 nm. The spectra corresponding to an average of at least two or three scans were corrected in 34 S. Bon et al. (Eur. J. Biochem. 271) Ó FEBS 2003 emission, and the background fluorescence from buffer and detergent were subtracted. Mutagenesis and transfections cDNA encoding rat AChE subunits was inserted in the pEF-BOS vector, which is under the control of the human EF-10c promotor; this vector was used for mutagenesis and expression in COS cells [4]. All constructs were identical, except for the 3¢ sequence encoding the C-terminal peptides. AChE T subunits were coexpressed with proteins derived from Q N , containing either the natural PRAD motif with its two adjacent cysteines upstream of the proline-rich segment (CC-Q N ), or a modified PRAD, in which these cysteines were replaced by serines, and two cysteines were introduced downstream of the prolines (Q N -CC). A Q N construct from which the PRAD was deleted (residues 70– 86) was used in control cultures, to ensure an identical level of AChE T expression. In a number of experiments we used a construct that contained a C-terminal GPI addition signal derived from Torpedo type H AChE (AChE H ) subunits, so that the resulting complex, (AChE T ) 4 –Q N –GPI, could be recovered from the cell surface by treatment with phos- phatidylinositol-specific phospholipase C (PI-PLC). For transfections, DNA was purified on Nucleobond AX columns (Macherey–Nagel, Hoerdt, France). COS-7 cells were transfected by the diethylaminoethyl-dextran method, as described previously [9]. The cells were maintained at 37 °C and were collected after three days. Preparation of extracts and AChE assay The cells were extracted with TMg buffer [1% (v/v) Triton X-100; 20 m M Tris/HCl pH 7.5; 10 m M MgCl 2 ]at4°C when the AChE T subunits were expressed alone or with Q N , and at 20 °C when they were expressed with a Q N –GPI construct, because the GPI-anchored complex is associated with sphingolipid/cholesterol microdomains which remain partially insoluble in Triton X-100 in the cold. The AChE activity was assayed by the colorimetric method of Ellman [20]. Enzyme samples (10 lL) were added to 0.2 mL of Ellman assay medium and the reaction kinetics were monitored at 414 nm, at 15 s intervals over a 3 min period, using a Multiskan RC microplate reader (Labsystems, Helsinki, Finland). Sucrose gradients and nondenaturing electrophoresis Aliquots of extracts (typically 200 lL) containing 1% (v/v) Brij-96 buffer (10 m M MgCl 2 ,25m M Tris/HCl pH 7) were loaded on 5–20% (w/v) sucrose gradients in 1% (v/v) Brij- 96 buffer. Escherichia coli b-galactosidase (16 S) and alkaline phosphatase (6.1 S) were included as internal sedimentation standards. The gradients were centrifuged for 18 h at 36 000 r.p.m. at 5 °C, in a LE80K centrifuge using an SW-41 rotor (Beckman–Coulter, Villepinte, France). Fractions of 300 lL were collected and assayed for AChE, b-galactosidase and alkaline phosphatase activities. Electrophoresis in nondenaturating polyacryl- amide gels was performed as described previously [24] and AChE activity was shown by the histochemical method of Karnovsky and Roots [25]. Metabolic labeling Two days after cotransfection of AChE T subunits with the Torpedo AChE H C-terminal addition signal, the transfected COS cells were preincubated for 45 min in Dulbecco’s modified Eagle’s medium lacking cysteine and methionine, and then labeled with [ 35 S]methionine–cysteine (Amersham Biosciences) for 3 h. The cells were then rinsed with NaCl/ P i , and chased overnight in a medium containing Nu-serum (BD Biosciences, Bedford, MA, USA). The cell surface GPI-anchored AChE was solubilized by treating intact cells for 2 h at 37 °C with PI-PLC (1 : 600) from Bacillus thuringiensis, kindly provided by I. Silman (Weizmann Institute, Rehovot, Israel). Following centrifugation at 10 000 g for 15 min to remove cell debris, the soluble enzyme (secreted and PI-PLC released) was collected for immunoprecipitation. Immunoprecipitation and SDS/PAGE AChE from cell extracts or medium were immunoadsorbed on protein G immobilized on Sepharose 4B Fast Flow beads (Sigma). The beads were first washed and saturated with 5% (v/v) BSA in a buffer containing 150 m M NaCl, 5m M EDTA, 50 m M Tris/HCl pH 7.4, 0.05% (v/v) NP40. Samples of 1.5 mL of cell extracts or media were incubated with 40 lL of a 10% suspension of beads for 3 h to eliminate nonspecific adsorption and the beads were discarded. The samples were incubated with 1 : 500 anti- (rat AChE) serum A63 [26] overnight at 8 °C, with gentle agitation on a rotating wheel, followed by addition of 80 lL of a 10% suspension of BSA-saturated washed beads and incubation for 1 h. After immunoadsorbtion, the beads were washed and centrifuged three times with 1 mL of buffer containing 1% Triton X-100 and centrifugations at 10 000 g for 5 min. All incubations were performed at 8 °C under mild rotational agitation. For polyacrylamide electrophoresis under denaturing conditions, samples of the washed beads were resuspended in 30 lLof0.125 M Tris/HCl buffer pH 6.8 containing 1% SDS, 0.002% bromophenol blue, 5% 2-mercaptoethanol (v/v/v), heated at 98 °C for 5 min, and centrifuged at 10 000 g for 5 min at room temperature. Aliquots of 10 lL of the supernatant were submitted to electrophoresis in SDS/polyacrylamide gels, and the resulting bands were revealed with the BAS 1000 Fuji Image analyzer (Fujifilm, St Quentin-en-Yvelines, France) or by autoradiography, and analyzed with the Fuji Image GAUGE software. Prediction of secondary structure elements The secondary structure of the C-terminal region of the catalytic domain and of the t peptide was predicted according to Rost [27] using PREDICTPROTEIN at http:// maple.bioc.columbia.edu/predictprotein. Results Modeling of the t peptide as an amphiphilic a helix The primary sequence of the C-terminal region of Torpedo AChE T is shown in Fig. 1A, including the last 12 residues of Ó FEBS 2003 Amphiphilic a helical domain of the AChE T subunit (Eur. J. Biochem. 271)35 the catalytic domain and the t peptide. Secondary structure prediction algorithms show that a large part of this peptide is expected to assume an a helical structure, extending from residue five to residue 26 or 28, with a possible interruption at residues 14–16 that might allow a bend between two helical segments. Giles proposed a similar arrangement, in which a bend at residues 21–22 would bring together the aromatic sectors of the two helices [16]; according to this model, residues located in the N-terminal region of the t peptide would be in close contact with the C-terminal cysteine, C37. If we assume an ahelical structure for the t peptide, a lateral view shows that all the aromatic residues are oriented on the same side (Fig. 1B), and a wheel projection [28] shows that a sector of  100° is totally apolar (Fig. 1B). The polar sector contains five acidic residues (one aspartic and four glutamic acids) and four basic residues (one lysine, two arginines and one histidine), which might form internal salt bridges between residues D4 or E5 and R8, between E7 and K11, and between E13 and R16, as analyzed in a further study (S. Belbeoc’h, J. Leroy, A. Ayon, J. Massoulie ´ & S. Bon, unpublished results). The cluster of hydrophobic side chains in the apolar sector includes the seven aromatic residues that are conserved in all known vertebrate AChEs and BChEs, ranging from cartilagenous fishes (Torpedo)to mammals. In particular, three tryptophans are evenly spaced by seven residues and very close to each other in the wheel diagram (Fig. 1B). This aromatic cluster could be respon- sible for the hydrophobic interactions of AChE T subunits. Chemical grafting of synthetic peptides confers hydrophobic properties on water-soluble AChE To characterize the interactions of the t region while excluding possible effects of putative post-translational modifications, we used chemically synthesized peptides, as shown in Fig. 1C. Peptide t 1)40 corresponds to the whole Torpedo t peptide; peptide t 1)32 corresponds to its first 32 aminoacids and contains all seven conserved aromatic residues. The peptides were grafted onto a water-soluble tetrameric form (G 4 )ofElectrophorus electricus AChE, obtained by tryptic digestion of collagen-tailed forms from the electric organ [29,30]. We used this enzyme preparation because we could obtain it in a highly purified form [18] and because it was very stable, totally nonamphiphilic and could be Fig. 1. Sequence and putative organization of the C-terminal t peptide from AChE T . (A) Primary structure of the last 12 residues of the catalytic domain and of the t peptide. A comparison of the Torpedo and rat sequences shows the high degree of conserva- tion, particularly of the seven aromatic resi- dues, throughout vertebrates. The N-terminal region of the human amyloid Ab peptide is shown to indicate a 12 residue segment which presents some homology with the t peptide (underlined) (B) Proposed helical structure of the N-terminal region of the t peptide: in the side view, the distance of each residue from the helix axis corresponds to the vertical dimen- sion, with the central residue of the aromatic cluster (W17) at the top. The position along the axis corresponds to the horizontal dimen- sion (arbitrary scales). The wheel representa- tion corresponds to a faceview along the helix axis of the segment of the t peptide containing the aromatic residues. (C) Synthetic peptides corresponding to different parts of the t peptide. The underlined residues have been substituted from the wildtype sequence of the Torpedo marmorata tpeptide. 36 S. Bon et al. (Eur. J. Biochem. 271) Ó FEBS 2003 analyzed by the same methods used for the amphiphilic AChE species. Chemical coupling of the synthetic peptides to exposed lysine residues occurred randomly and did not affect enzymic activity. We deduced the mean number of peptides added per tetramer from the apparent increase in molecular mass: the modified Electrophorus G 4 AChE molecules obtained after coupling of the peptides sedimented as fairly homogenous peaks, as illustrated in Fig. 2A. The sedimentation coeffi- cient of G 4 -t 1)32 and of G 4 -t 1)40 was about 12.8 S, as compared to 11.8 S for the original G 4 form (Fig. 2B). Assuming that the mass of this globular protein is propor- tional to S 3/2 , we estimate that the mass of the tetramer increased from 320 kDa to 360 kDa, i.e. 10 kDa per subunit, which corresponds to an average of three grafted peptides per AChE subunit. In the case of G 4 -t 1)40 and G 4 -t 25)40 , the formation of complexes with antibodies raised against t 25)40 confirmed that essentially all the Electrophorus G 4 AChE molecules had been modified (not shown). The G 4 -t 1)32 derivative did not bind the antibodies, indicating that the t 1)32 peptide did not contain the necessary epitopes. The G 4 -t 25)40 derivative, like the original Electrophorus G 4 enzyme, was not amphiphilic: its sedimentation coeffi- cient (12.9 S) was not influenced by the presence of detergent in the gradients. By contrast, the G 4 -t 1)32 and G 4 -t 1)40 derivatives were clearly amphiphilic, as they sedimented more slowly in the presence of Triton X-100 and even more slowly in the presence of Brij-96 (Fig. 2A,B). This amphiphilic character was confirmed by charge-shift electrophoresis under nondenaturing conditions. The t-peptide–AChE conjugates migrated in opposite directions in the presence of the negatively and positively charged detergents, cetyltrimethylammonium bromide (CTAB) and Na + deoxycholate (not shown). The fact that the short t 1)32 peptide and the long t 1)40 peptide confer amphiphilic properties to Electrophorus AChE tetramers, whereas the t 25)40 peptide does not suggests that the 1–32 region, containing an a helix with seven aromatic residues, is sufficient to support hydropho- bic interactions. Characterization of t peptide–lipid interactions by use of circular dichroism Figure 3 shows the CD spectrum in the far UV of the t 1)32 peptide under various conditions. In organic solvents, such as methanol, the spectrum presents the characteristic features of an a helical structure, with double minima at 210 nm and 222 nm. The h 222 value of )31 600 degÆdmol )1 Æ cm 2 indicates that about 85% of the polypeptide is a helical. We obtained a similar proportion of a helical structure by reconstituting the whole spectrum as a sum of the contri- butions of different secondary structures, derived from a set of known proteins [31]. This high a helical content is comparable to that of amphiphilic peptides of similar length, which have been characterized by various methods as monomeric  20-residue a helical rods [32,33]. When the peptide was dissolved in an aqueous buffer, the minima at 210 nm and 222 nm displayed ellipticities of only h ¼ )12 210 degÆdmol )1 Æcm 2 and h ¼ )9770 degÆdmol –l Æcm 2 respectively, indicating a much lower a helical content of  35%. Fig. 2. Effect of detergents on the sedimentation of Electrophorus AChE tetramers, chemically coupled with the t 1)40 peptide. (A) Sedimentation patterns of a conjugate of Electrophorus AChE G 4 species with the t 1)40 peptide, obtained in sucrose gradients containing no detergent; 0.1% Triton X-100 or 0.1% Brij-96. (B) Sedimentation coefficients obtained in these different conditions for G 4 AChE and its conjugates. The conjugated enzymes containing peptides t 1)32 and t 1)40 sedi- mented faster without detergent than in the presence of Triton X-100 or Brij-96, indicating that they bind detergent micelles, in contrast with conjugated enzyme containing peptide t 25)40 and the nonconjugated enzyme, which sedimented in the same way under all three conditions. Fig. 3. Far UV dichroic spectrum of peptide t 1-32 . Peptide (5 l M )in 1m M Tris/HCl buffer, pH 7.5, using a 1 cm path-length cuvette (dotted line); the same solution after addition of lysolecithin micelles, with a lipid/peptide molar ratio of 20 (thin line); 50 l M peptide in methanol, using a 0.1 path-length cuvette (bold line). Ó FEBS 2003 Amphiphilic a helical domain of the AChE T subunit (Eur. J. Biochem. 271)37 The CD spectrum was markedly modified by addition of lysolecithin micelles. It approached that observed in meth- anol when the lysolipid/peptide molar ratio was about 10, and was not modified further at higher micelle concentra- tions (Fig. 3). Under these conditions, the helical content was about 68%, corresponding to 18–22 residues per peptide organized into an a helix. Thus, lipid micelles can induce an a helical conformation in the t peptide. Intrinsic fluorescence of the t peptide The t 1)32 peptide displays intrinsic fluorescence due to the fact that it contains three tryptophans; W10, W17 and W24, and two tyrosines, Y20 and Y31. When dissolved in aqueous buffer and excited at 280 nm, its emission spectrum was centered at 345 nm. The shape of the emission spectrum was identical when excitation was at 295 nm, a wavelength at which tyrosine residues do not absorb. Thus the fluorescence of the peptide is totally due to tryptophan residues: Y20 and Y31 are either totally quenched or very efficiently transfer their energy to tryptophan residues in their neighbourhood. In aqueous solution, the fluorescence of the tryptophan residues showed a blue shift of 6 nm relative to N-acetyltryptophanylamide, indicating that they are only slightly buried. The blue shift was increased by about 2 nm when dithiothreitol was omitted. Addition of methanol, which decreased the polarity of the medium, prevented aggregation and increased the a helical content; this pro- duced an increase in quantum yield and a slight shift of the maximum emission wavelength, indicating that the trypto- phan residues were more exposed to the solvent. We obtained similar results with the t 1)40 peptide, except that it was more aggregated in aqueous solution; the t 1)32 peptide also aggregated above 1 l M , as indicated by an increase in the light scattering. On the contrary, reducing the concentration below 0.2 l M induced a progressive red shift of the emission k max for both peptides; however, this never reached 350 nm, which would correspond to total exposure of tryptophan residues. Interaction of peptides with detergents and phospholipids as followed by fluorescence We followed changes of the intrinsic tryptophan fluores- cence by addition of phospholipids (Fig. 4) and detergents (Fig. 5). The induced blue shifts in the k max of emission and intensity changes were similar for t 1)32 and t 1)40 . Figure 4 shows that addition of lipid vesicles to an aqueous solution of the t 1)32 peptide at pH 7.5 (5 l M ) produced changes both in intensity and wavelength of fluorescence. For the zwitterionic egg lecithin vesicles, the changes did not reach a plateau even at Ri values greater than 150, indicating a low affinity of the peptides for the lecithin–water interface. In contrast, we observed a stronger blue shift and more pronounced quenching upon addition of negatively charged phosphatidylserine vesicles, and both effects reached a plateau below an Ri value of 100. The emission maximum at the plateau, 327 nm, indicates that the tryptophan residues were in a very hydrophobic environment. Figure 5 shows that addition of 32 l M lysolecithin to peptide t 1)32 (3.2 l M ) shifted the emission maximum close to 330 nm, and increased the intensity twofold. The affinity of the peptide was much higher for lysolecithin micelles than for lecithin vesicles, indicating that insertion of the peptide is easier in the more fluid and dynamic lysolecithin micelles than in the bilayer of lecithin vesicles, as observed for other amphiphilic peptides [34]. At lower concentrations of the peptide (0.5 l M ), lysolecithin induced a similar shift in k max but a more complex variation of the intensity, which first decreased, reaching a minimum at a lipid : peptide molar ratio (Ri) of  30–40 and then increased again (not shown). Such biphasic curves were previously observed for lipid–peptide interactions occurring in the concentration range of the critical micellar concentration (cmc) [17]. For lysolecithin, the cmc is 20 l M , corresponding to Ri values of 5–6 and 30–40, for peptide concentrations of 3.2 and 0.5 l M respectively. These observations show that the t 1)32 peptide interacts with lysolecithin both below and above the cmc. CTAB is a positively charged detergent with a cmc of 0.2–0.3 m M , and SDS is negatively charged and has a cmc of  1–2 m M [35]. At neutral pH, addition of CTAB to 3.2 l M peptide shifted k max down to 334 nm, reaching a plateau for Ri ¼ 20, and induced a large increase in the intensity at 334 nm, attaining 260% for Ri values above 100, i.e. above the cmc that corresponds to Ri values of 60–90 (Fig. 5). In contrast, SDS did not induce any significant change in fluorescence up to Ri ¼ 150; above this value, we noted a gradual shift of k max downto330nm for Ri ¼ 300–400, i.e. above the cmc of the detergent. We obtained similar results at pH 5.7, in spite of a reduction in Fig. 4. Effects of phospholipid vesicles on the intrinsic fluorescence of peptide t 1)32 . The peptide concentration was 5 l M ,in20m M Tris/ acetate buffer pH 7.5 containing 5 m M dithiothreitol to avoid the formation of disulfide bonds, under a nitrogen stream, at 25 °C. (A) Variation of the wavelength of maximum emission (k max ) as a function of the molar ratio of lipids to peptide (Ri). (B) Relative variation of emission intensity at 333 nm (DI/I 0 ) as a function of Ri. (m)egg lecithin vesicles; (s) phosphatidylserine vesicles. 38 S. Bon et al. (Eur. J. Biochem. 271) Ó FEBS 2003 the negative charge of the peptide. Thus, the zwitterionic and positively charged detergents readily interact with the peptides even below the cmc, while the negatively charged detergent interacts only when approaching the cmc. Formation of disulfide bonds in homomeric oligomers with cysteines at various positions in the C-terminal region of rat AChE T subunits The preceding studies were performed on isolated peptides or on conjugates in which peptides were chemically coupled at the surface of a protein. However, the t peptide is normally linked to the C-terminus of the catalytic domain of AChE T subunits and it contains a cysteine (C37) which allows their dimerization through an intersubunit disulfide bond. The crystallographic structure of AChE dimers [36,37] or monomers [38] shows that the catalytic domain terminates with an a helix (helix a 10 ) constituted by residues )18 to )1. Secondary structure predictions suggest that this helix is separated from the a helical portion of the t peptide by a short loop (around residues )1to2),andmaypresenta break around residues 15–16 (Fig. 1A). An interrupted helix could form a hairpin, as proposed by Giles [16], who suggested that the aromatic-rich sectors of two a helical segments would constitute a compact aromatic cluster. According to this model, a bend at residues 21 and 22 would bring the N-terminal and C-terminal ends into close proximity. To obtain information on the articulation between the catalytic domain and the t peptide, we analyzed the formation of intercatenary disulfide bonds by cysteine residues located at the end of the catalytic domain of rat AChE T or at the beginning of its t peptide, in the )5to6 interval; in these mutants, the original cysteine was either retained or replaced by a serine (C37S). We also introduced cysteine residues near the middle of the t peptide, in the predicted a helical region containing aromatic residues (at positions 19 and 21), and in its C-terminal region, which is not predicted to be a helical, at positions 34 to 36. The AChE T cysteine mutants were expressed in transi- ently transfected COS cells. In the absence of any cysteine in the C-terminal region of rat AChE, we obtained mainly monomers, with a small proportion of tetramers, as reported previously in the case of human AChE [39] and rat AChE [40]. Therefore, the presence of dimers, as observed in the case of the other mutants, indicates the formation of an intercatenary disulfide bond. In the hypothesis of a hairpin structure, an intracatenary disulfide bond might be formed in mutants containing the original cysteine or another C-terminal cysteine, together with a cysteine in the N-terminal region of the t peptide; this would preclude the formation of dimers, which requires an intercatenary disulfide bond. However, we did not observe this in any combination of N-terminal and C-terminal cysteines (not shown). Therefore, the t peptide almost certainly adopts an elongated conformation in AChE T monomers and dimers. When the original cysteine was mutated to serine (C37S), all mutants containing a single cysteine at positions )5to6, 19, 21, or 34 to 36, produced active AChE which was secreted at variable levels (Fig. 6A). The cellular and secreted enzymes contained different proportions of dimers, sometimes with a small amount of tetramers, as indicated by nondenaturing electrophoresis (Fig. 6B). Sedimentation patterns illustrating the amounts of mono- mers, dimers and tetramers are shown in Fig. 7 for cysteines in the )5 to 6 interval. The proportion of dimers produced was very low with cysteines in the )5to)3 interval, a region which is predicted to be a helical. The distances between pairs of alpha carbons corresponding to residues )5to)2 can be determined from the crystallographic structure of a catalytic dimer [36]: they are 8.6, 13, 15 and 8.9 A ˚ respectively. A small proportion of AChE T subunits were dimerized with cysteines at position )5and)2, for which the distance is smallest but still appears too high for establishment of a disulfide bond, which is normally < 6 A ˚ . This indicates that, in AChE T subunits, the distal part of the catalytic domain is sufficiently flexible to allow the forma- tion of a disulfide bond in this segment, between the two subunits in a dimer. The production of dimers was higher than for the wildtype with cysteines at positions )2to3, suggesting that this region, which is predicted to form a coil, constitutes a flexible hinge between the catalytic domain and the amphiphilic helix of the t peptide; it was lower at positions 4 and 5 and increased again at position 6. As these three residues are probably included in the N-terminal region of the helix, the observed variations in the efficiency of dimerization may be due to their orientation relative to the Fig. 5. Effects of zwitterionic and charged detergents on the intrinsic fluorescence of peptide t 1-32 . The peptide concentration was 3.2 l M ,in 20 m M Tris/HCl buffer containing 5 m M dithiothreitol to avoid the formation of disulfide bonds. (A) Variation of the wavelength of maximum emission (k max ) as a function of the molar ratio of detergent to peptide (Ri). (B) Relative variation of emission intensity at 333 nm (DI/I 0 ) as a function of Ri. (h, j)SDS;(s, d) cetyl-trimethyl- ammonium bromide (CTAB); (n, m) lysolecithin. Filled symbols (j, d, m), pH 7.5; open symbols (h, s, n), pH 5.7. Ó FEBS 2003 Amphiphilic a helical domain of the AChE T subunit (Eur. J. Biochem. 271)39 aromatic sector: residue 6 is in the aromatic sector, while residues 4 and 5 are on the opposite side. We also studied the production of dimers with cysteine residues located at positions 19 and 21, in the center of the predicted amphiphilic a helical region but in opposite sectors. With a cysteine at 19, the cellular enzyme contained dimers but their secretion was very low (Fig. 6B), suggesting that the presence of a disulfide bond at this position induced their degradation. In contrast, a cysteine at position 21, within the sector containing aromatic residues, appeared much more favorable for dimerization and secretion. In contrast with dimers containing disulfide bonds in the N-terminal or C-terminal regions of the t peptide, the M21C/C37S dimers did not interact with detergent micelles (not shown), indicating that the two aromatic clusters occluded each other. Dimers were as efficiently produced and secreted with cysteines located at positions 34, 35 or 36 as with the original cysteine (at position 37) suggesting that this C-terminal region of the t peptide is flexible. It is noteworthy that the level of cellular activity was markedly higher with a cysteine at 35, corresponding to an increased amount of monomers; the presence of a cysteine instead of an aspartic acid at this position seems to increase the retention or decrease the degradation of monomers. Figures 6B and 7 show that the production of tetramers varied with the position of the cysteine and was not proportional to that of dimers. Tetramer production was systematically higher with C-terminal cysteines (34–37) than with cysteines in the N-terminal region of the t peptide ()2 to 3). This suggests that the relative organization of the t peptides and of the catalytic domains is more favorable for tetramerization when dimers are joined through a C-terminal disulfide bond. Hetero-oligomerization: orientation of the PRAD and t peptides in the T 4 –Q N complex The Q N protein possesses two adjacent cysteine residues (C70 and C71) located immediately upstream of the proline- Fig. 6. Effect of cysteines at various positions in the C-terminal region of rat AChE T subunits. Cysteines were introduced into rat AChE T subunits at various positions at the junction of the catalytic domain and the t peptide ()5to 6), in the middle of the t peptide (19 or 21), and in the C-terminal part of the t peptide (34 to 36); in these mutants, the original cysteine (C37) was replaced by a serine, so that all mutants possessed a single cysteine. (A) Cel- lular and secreted AChE activities: all mutants produced and secreted active AChE when expressed with or without Q N .Whencysteines were present in the )5 to 6 interval, we used a modified Q N (Q N -CC) with cysteines down- stream of the proline-rich region; with the other mutants, we used the Q N protein con- taining cysteines upstream of the proline-rich region. Activities are expressed as percentage of the wildtype; the bars indicate the standard errors of two to three independent experi- ments. The shaded and hatched rectangles correspond to mutants expressed without and with Q N , respectively. (B) Nondenaturing electrophoresis of AChE oligomers produced by rat AChE T subunits containing a single cysteine at different positions, expressed without Q N . 40 S. Bon et al. (Eur. J. Biochem. 271) Ó FEBS 2003 rich motif (Fig. 8A,B) such that the disulfide linkage of two AChE T subunits with one Q N protein produces a Ôheavy dimerÕ that can be distinguished by SDS/PAGE under nonreducing conditions from Ôlight dimersÕ consisting of only two disulfide linked AChE T subunits [12] (Fig. 8C). In order to study the formation of these disulfide bonds, AChE T mutants were coexpressed with the natural Q N protein possessing two adjacent cysteines C70 and C71 upstream of the PRAD (CC-Q N ), and with a Q N mutant in which the original cysteines were mutated to serines and two cysteines were introduced downstream of the PRAD, at positions 87 and 88 (Q N -CC), as shown in Fig. 8A. The C37S mutant that formed no intercatenary disulfide bonds but was recruited into T 4 –Q N complexes, served as a control. Figure 8Ca illustrates the fact that CC-Q N formed disulfide bonds with AChE T mutants that contained a cysteine in the C-terminal region of the t peptide (as expected, given that this corresponds to the wildtype situation), but not with AChE T mutants containing an upstream cysteine (Fig. 8Cb); in the latter case, all AChE T subunits were disulfide-linked in homodimers. Recipro- cally, disulfide bonds could be formed, although less efficiently, between Q N -CC and some of the AChE T mutants that contained a cysteine in the N-terminal region of the t peptide (Fig. 8Cd) but not in the C-terminal region (Fig. 8Cc). This indicates that the N- and C-terminal extremities of the t peptides are distant in the complex, eliminating the possibility that the peptides would be folded in hairpins as suggested above for the free t peptides; the same reasoning shows that the PRAD is also elongated. Taking into consideration that both the t peptides and the PRAD are elongated in the heteromeric complexes, we Fig. 7. Formation of homo-oligomers of rat AChE T subunits with cysteines at various posi- tions in their C-terminal region. Sedimentation patterns in sucrose gradients, for mutants of the )5 to 6 interval. The patterns obtained for the wildtype (solid line) and for the C37S mutant with no C-terminal cysteine (C37S) (dotted line) are shown for comparison. The monomers, dimers and tetramers are indicated (T 1 ,T 2 ,T 4 respectively). The areas under the sedimentation profiles are proportional to the cellular and secreted activities, so that the areas of the peaks represent the relative amounts of the corresponding molecular forms. Ó FEBS 2003 Amphiphilic a helical domain of the AChE T subunit (Eur. J. Biochem. 271)41 can then question their respective orientations: the forma- tion of intercatenary disulfide bonds shows that the four t peptides are all parallel, and are oriented in the opposite direction to the PRAD, as the N-terminal extremity of the PRAD can only be disulfide linked to the C-terminal region of two t peptides, and vice versa. Exploring the association of t peptides and PRAD in the T 4 –Q N complex by the formation of heterophilic intercatenary disulfide bonds Figure 9A shows an analysis of the complexes formed between the various AChE T cysteine mutants and Q N ,in Fig. 8. Disulfide bonds between Q N and two AChE T subunits, in the T 4 –Q N complex. (A) Schematic representation of the constructs used. The T 4 –Q N complex was formed when AChE T subunits possessing a cysteine near the N- or C-terminus of the t peptide were expressed with Q N constructs containing pairs of cysteines located either upstream or downstream of the PRAD. The arrows indicate the N-terminal to C-terminal orientation. (B) Schematic representation of the different combinations of cysteine mutants; the PRAD is shown as a thick central line and the t peptides as zigzags; the cysteines are indicated by circles and the disulfide bonds by thick lines. Scheme a corresponds to the wildtype 44 situation; b corresponds to an association of wildtype t peptides with Q N -CC; c and d correspond to associations of t peptides containing an upstream cysteine (L3C/C375) with CC-Q N and Q N -CC, respectively. (C) Analysis of disulfide-linked species by SDS/PAGE after metabolic labeling. Lanes a, b, c, d correspond to the four diagrams in panel 45 (B). ÔHeavy dimersÕ (composed of one Q N protein linked to two AChE T subunits) were produced only when cysteines were at opposite ends of the t peptide and PRAD. Fig. 9. Effect of the position of cysteines in the C-terminal region of AChE T on the formation of hetero-oligomers (T 4 -Q N ). (A) Nondenaturing electrophoresis of AChE oligomers secreted by cells expressing AChE T subunits with the appropriate Q N construct (Q N -CC for AChE T subunits containing a cysteine in the )5 to 6 interval, CC-Q N for AChE T subunits containing a cysteine at positions 19, 21 and in the 34 to 37 interval). (B) Analysis of disulfide bonds between AChE T subunits and Q N (Ôheavy dimersÕ), by nonreducing denaturing electrophoresis after metabolic labeling. There were no heavy dimers with cysteines at 19 or 21, with either the CC-Q N or Q N -CC construct (not shown). 42 S. Bon et al. (Eur. J. Biochem. 271) Ó FEBS 2003 [...]... versa between N-terminal cysteines of the t peptide and C-terminal cysteines in the PRAD (QN-CC) This excludes the hypothesis that the two ends of the t peptide would be in close contact, so that the t peptide appears to adopt an elongated structure in the complex, as also shown without a PRAD In addition, the organization of intercatenary disulfide bonds in the T4 –QN complex implies that the four t peptides... N-methyl-D-aspartate receptors in the guinea-pig hippocampus [58] This indicates that fragments derived from the t peptide may possess distinct conformations and biological activities The C-terminal t peptide of AChE forms an amphiphilic a helix In this study, we present spectroscopic evidence that at least part of the C-terminal t peptide of acetylcholinesterase AChET subunits may adopt an a helical structure,... bond All subunits that possessed a cysteine in the 34 to 37 G4 T2 5-40, which only contains the last two aromatic interval efficiently formed dimers and tetramers, indicating residues, does not interact with detergents This indicates that this C-terminal region is flexible and that the geometry that at least part of the first 32 residues of the t peptide of these dimers is favorable for their assembly into... (T1 -32) may form an amphiphilic helix that is sufficient to tetramers explain the hydrophobic interactions of AChET subunits, and that the distal eight residues are not necessary in this respect Association of four t peptides with a PRAD in the T4 –QN complex Self assembly of t peptides and intercatenary disulfide The most interesting and physiologically important propbonds erty of the t peptide is its... level of secreted dimers was higher with a normal biosynthesis of AChET subunits and contribute to cysteine at position 6 than at positions 4 and 5 may result from a more appropriate orientation, relative to the their hydrophobic properties In fact, it has been reported aromatic sector of the helix that palmitate was incorporated into a membrane-bound The effect of a cysteine in the central region of the. .. the t peptide, but not to a fragment containing the ÔreadthroughÕ r peptide [50] AChE may also participate in the pathogenesis of Alzheimer’s disease as it induces the aggregation of Ab amyloidogenic peptide and increases its neurotoxicity [53,54] It is possible that the t peptide itself, or a fragment of the t peptide without the catalytic domain, may be involved in this pathology As illustrated in... of the t peptide can adopt an a helical structure in which all seven aromatic residues are grouped in a hydrophobic sector, and explains the fact that the T4 –QN complex is nonamphiphilic The presence of a flexible hinge between helix a1 0 of the catalytic domain and the helical part of the t peptide, as predicted by secondary structure algorithms, may therefore be crucial for the assembly of AChE tetramers... any modification of the t peptide subunits [37] and in monomers of the truncated subunits, However, this does not exclude the possibility that postreduced to the catalytic domain [38] (c) Residues 4 to 6 are translational modifications, such as acylation by fatty acids probably included in the a helical region of the t peptide; the or addition of other lipidic moieties may occur during 36 fact that the. .. of AChET subunits is necessary for association with the collagen protein ColQ to produce collagen-tailed forms, and with the transmembrane protein PRiMA to produce hydrophobic-tailed tetramers [2] The physiological importance of these associations is illustrated by the fact that mutations in the human COLQ gene which prevent this association or modify the structure of the collagen tail result in AChE-deficient... with QN-CC, although less than half of the AChET subunits were included in the ÔheavyÕ dimers The formation of disulfide bonds between cysteines at positions 3 and 6 of the t peptide and cysteines introduced at positions 87 and 88 of QN suggests that the PRAD can slide over the corresponding distance, in the cylinder of t peptides Cysteines at positions 19 and 21, located in opposite sectors of the a . putative organization of the C-terminal t peptide from AChE T . (A) Primary structure of the last 12 residues of the catalytic domain and of the t peptide. A comparison of the Torpedo and rat sequences. introduced into rat AChE T subunits at various positions at the junction of the catalytic domain and the t peptide ()5to 6), in the middle of the t peptide (19 or 21), and in the C-terminal part. cysteines (34–37) than with cysteines in the N-terminal region of the t peptide ()2 to 3). This suggests that the relative organization of the t peptides and of the catalytic domains is more favorable for