Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide Adam Frederick1, Igor Tsigelny2, Frances Cohenour1, Christopher Spiker1, Eric Krejci3, Arnaud Chatonnet4, Stefan Bourgoin1, Greg Richards1, Tessa Allen1, Mary H Whitlock1 and Leo Pezzementi1 Department of Biology, Birmingham-Southern College, Birmingham, AL, USA Department of Chemistry and Biochemistry, San Diego Supercomputer Center, University of California at San Diego, La Jolla, CA, USA ´ ´ ´ Institut National de la Sante et de la Recherche Medicale U686, Universite Paris Descartes, Biologie des Jonctions Neuromusculaires, Paris, France Institut National de la Recherche Agronomique, Montpellier, France Keywords acetylcholinesterase; asymmetric forms; butyrylcholinesterase; Ciona intestinalis; evolution Correspondence L Pezzementi, Department of Biology, Birmingham-Southern College, Box 549022, Birmingham, AL 35254, USA Fax: +1 205 226 3078 Tel: +1 205 226 4806 E-mail: lpezzeme@bsc.edu Website: http://faculty.bsc.edu/lpezzeme/ Database The nucleotide sequence and derived amino acid sequence data reported for the AChE from Ciona intestinalis are available in the Third Party Annotation Section of the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession no TPA: BK006073 The alignment used to determine the phylogenetic tree for vertebrate and invertebrate cholinesterases presented here is deposited at the EMBL-ALIGN database as ALIGN_001208 To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChET subunit, which should be able to produce all three globular forms of AChE: monomers (G1), dimers (G2), and tetramers (G4), and assemble into asymmetric forms in association with the collagenic subunit collagen Q Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium In cell extracts, amphiphilic monomers (G1a) and non-amphiphilic tetramers (G4na) are found Amphiphilic dimers (G2a) and non-amphiphilic tetramers (G4na) are secreted into the medium Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A12 form of the enzyme Collagenase digestion of the A12 AChE produces a lytic G4 form Notably, only globular forms are present in vivo This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms We also performed a phylogenetic analysis of the sequence We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates (Received 14 November 2007, revised January 2008, accepted 15 January 2008) doi:10.1111/j.1742-4658.2008.06292.x Abbreviations a , amphiphilic; AChE, acetylcholinesterase; AChEH, splice variant H; AChET, splice variant T; ATCh, acetylthiocholine; BTCh, butyrylthiocholine; BuChE, butyrylcholinesterase; ChE, cholinesterase; ColQ, collagen Q; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1methylquinolinium iodide; DTNB, 5-(3-carboxy-4nitro-phenyl)disulfanyl-2-nitro-benzoic acid; GPI, glycophosphatidylinositol; HIS buffer, high ionic strength buffer; IC50, half maximal inhibitory concentration; LBA, long branch attraction; na, non-amphiphilic; PPII, polyproline II; PRAD, proline-rich attachment domain; PRiMA, proline-rich membrane anchor; WAT, tryptophan (W) amphipathic tetramerization domain FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1309 AChE from C intestinalis A Frederick et al Gnathostome vertebrates have two evolutionarily related cholinesterases (ChEs), acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) AChE rapidly hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses BuChE appears to act as a scavenger of cholinergic toxins, but may also play a role in synaptic transmission [1,2] These two enzymes appear to be the result of a gene duplication event early in vertebrate evolution [3] ˚ Both enzymes have a 20 A deep catalytic gorge lined with aromatic amino acids [4] AChE has fourteen aromatic residues lining the gorge; in BuChE, aliphatic amino acids replace six of the aromatic moieties In particular, smaller non-aromatic residues in BuChE replace the two phenylalanines of the acyl pocket of AChE (Phe288 and Phe290 in Torpedo californica AChE), a subsite of the enzyme that plays an important role in substrate specificity Amino acid position numbers appearing in parentheses represent the homologous positions in mature AChE from Torpedo californica; residues of the T peptides of AChET from different species are numbered from to 48 to facilitate comparisons As a result, BuChE can accommodate larger and more diverse substrates and inhibitors compared to AChE [5] By contrast to the dichotomous acyl pocket situation of ChEs in the vertebrates, invertebrates have a wider diversity in the structure of this subsite In approximately 90% of invertebrate ChEs, the acyl pocket is formed in a fundamentally different way [6] Instead of Phe288 and Phe290 forming the pocket, it is formed by phenylalanines at positions homologous to Phe290 and Val400 For example, in ChE2 from the cephalochordate amphioxus, which is very specific for the substrate acetylthiocholine (ATCh), the acyl pocket is composed of Phe312 (Phe290) and Phe422 (Val400) [6] One of the exceptions to this invertebrate pattern is found in the sequence for a putative AChE from the urochordate Ciona intestinalis [7,8], a deuterostome invertebrate that is a close relative to the vertebrates In this enzyme, phenylalanines homologous to those of the acyl pocket of vertebrates appear to form the acyl pocket [6] Previously, based on substrate and inhibitor specificity, it was reported that C intestinalis possesses an AChE in vivo [9–11] However, that work was conducted before the techniques of molecular biology were available, precluding the definitive identification of the enzyme Another difference between vertebrate and invertebrate ChEs is that vertebrates possess both globular and asymmetric forms of the enzymes, but invertebrates apparently possess only globular forms The globular forms of ChEs are monomers (G1), dimers 1310 (G2), and tetramers (G4) of catalytic subunits The asymmetric forms are comprised of one (A4), two (A8), or three (A12) tetramers attached to a triplestranded collagenic tail (collagen Q; ColQ) [12,13] The asymmetric forms associate with the basal lamina [14] Alternative splicing of the AChE gene in the vertebrates produces a number of carboxyl termini [15], resulting in the multiple molecular forms mRNAs containing the H-terminus (AChEH) are translated into glycophosphatidylinositol-membrane-anchored (GPI) G2 forms of AChE By contrast, transcripts containing the alternatively spliced T-terminus (AChET) are capable of forming all globular forms: amphiphilic monomers (G1a), amphiphilic dimers (G2a), and nonamphiphilic tetramers (G4na), but not GPI-membraneanchored G2a More importantly, AChET, via its tryptophan (W) amphipathic tetramerization domain (WAT) sequence [16], can associate with the prolinerich attachment domain (PRAD) of the collagenic subunit ColQ to form asymmetric enzyme [17,18] or with the proline-rich membrane anchor (PRiMA) protein [19] AChET appears to be rare in invertebrates, where AChEH predominates AChET has been reported for AChE1 from the nematodes Caenorhabditis spp [20,21] and Meloidogyne spp [22], where it forms G1a and a G4 form that may associate with a structural subunit [20,21] Meedel reported that C intestinalis larvae have G1, G2, and G4 forms of AChE, implying the presence of AChET in the invertebrate, but did not find any asymmetric forms [11] The cloning, in vitro expression, and characterization of this putative AChE from C intestinalis should identify the nature of the enzyme and provide additional information about the evolution of the ChEs, including the origins of the acyl pocket, the T exon, and the asymmetric forms of ChE in the vertebrates Results The sequence of the ChE from C intestinalis suggests that the enzyme is an AChET The sequence for C intestinalis AChE contains 618 amino acids (see supplementary Fig S1) The members of the catalytic triad of AChE are found as Ser 229, Glu 356, and His 471 The three pairs of conserved cysteine residues involved in intrachain disulfide bonding are also found as Cys 94–Cys 121, Cys 293–Cys 297, and Cys 431–Cys 562 Another cysteine (Cys 616) near the carboxyl terminus of the sequence probably mediates interchain disulfide bonding Of FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS A Frederick et al AChE from C intestinalis Table Aromatic amino acids in the catalytic gorge of putative AChEs from C intestinalis and AChE from T californica Numbering for C intestinalis AChE2 starts at first methionine residue in the sequence Conserved aromatic residues are shown in bold Designations of AChE1 and AChE2 are from the ESTHER database [23] to distinguish the AChE described in the present study (AChE1) and another sequence proposed to be an AChE from C intestinalis (GenBank accession no AK112482) Subsite Peripheral site Choline binding site and hydrophobic site Acyl pocket Wall of gorge a C intestinalis AChE1 C intestinalis AChE2 T californica AChE Ile97 Tyr152 Trp311 Trp111 Tyr161 Tyr359 Phe360 Phe317 Phe319 Val430 Trp145 Trp262 Tyr363 Trp463 Tyr473 Ser124 Phe176 Ala336 –a Tyr185 Phe387 Ile388 Tyr342 Glu346 Phe450 Trp169 Tyr289 Val391 Cys484 Ser498 Tyr70 Tyr121 Trp279 Trp84 Tyr130 Phe330 Phe331 Phe288 Phe290 Val400 Trp114 Trp233 Tyr334 Trp432 Tyr442 There is a deletion in the sequence for AChE2 CLUSTALW alignment in this region of the the fourteen aromatic amino acids that line the catalytic gorge of vertebrate AChE, 13 are conserved in the C intestinalis AChE (AChE1; Table 1) The sequence shows 41% identity with the AChE from T californica The formation of the acyl pocket of C intestinalis AChE may more closely resemble that of vertebrate AChE rather than invertebrate AChE (Fig 1; see molecular modeling below) However, the acyl pockets of C intestinalis and T californica are clearly not identical because, as is the case for other invertebrates, there is a deletion in the region of the acyl pocket of C intestinalis compared to the vertebrate enzyme (Fig 1) Additionally, the carboxyl terminus of the C intestinalis AChE appears to be coded for by a T exon: six of the seven aromatic residues of the T californica AChE WAT domain are conserved; there is a 74% sequence similarity with T californica AChE, and the domain has ability to form an amphipathic helix, characteristic of the T sequence A cysteine that mediates interchain disulfide bonds is also conserved (Fig 2A,B) We found no evidence in the genomic sequence of an upstream H exon in the C intestinalis AChE gene A second gene for AChE in C intestinalis has been proposed [8] (Genbank accession no AK112482; cioinacche2 in ESTHER; AChE2; Table 1) [23] However, Fig Amino acid residues surrounding the acyl pocket of some vertebrate and invertebrate acetylcholinesterases This figure illustrates the differences between the construction of the acyl pocket in vertebrate and invertebrate AChEs The line separates the vertebrate and invertebrate AChEs The numbers at the top of the figure correspond to the amino acids in T californica In the vertebrates, the acyl pocket is composed of Phe288 and Phe290 In the invertebrates, the acyl pocket phenylalanines homologous to the Phe290 and Val400 positions form the acyl pocket (CLUSTALW aligned the amino acid sequences [58]) The alignment of the sequence for C intestinalis AChE was slightly adjusted manually (the QE sequence) to emphasize the similarity with vertebrate AChEs The GenBank accession nos are: Homo sapiens (M55040), Bos taurus (BC123898), Mus musculus (X56518), R norvegicus (S50879), Gallus gallus (U03472), Bungarus fasciatus (U54591), T californica (X03439), Myxine glutinosa (U55003), C intestinalis (TPA: BK006073), B floridae (U74381), S purpuratus (XM_777020; predicted similar to AChE), Drosophila melanogaster (X05893), Anopheles stephensi (228651), C elegans (X75332), Meloidogyne incognita (AF075718), Loligo opalescens (AF065384), and Boophilus microplus (AJ223965) FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1311 AChE from C intestinalis A Frederick et al Fig Amino acid sequences of T peptides found in vertebrates and deuterostome and protostome invertebrates Top, alignment of T amino acid sequences: Vertebrates, H sapiens (M55040), M musculus (X56518), T californica (X03439); deuterostome invertebrates, C intestinalis (TPA: BK006073), S purpuratus (XM_775310); protosome invertebrates, Apysia californica (AASC01147222.1), C elegans (X75332) An alternatively spliced exon codes for the T peptide and numbering starts at the first amino acid of the peptide The six conserved aromatic amino acids of the WAT domain are indicated by ; the one nonconserved aromatic residue by h The S purpuratus sequence is associated with a putative AChE; the A californica sequence has not been associated with AChE Sequences aligned with CLUSTALW Bottom: helical wheel representation of the WAT domain organized as an amphipathic a-helix [62] The conserved aromatic, hydrophobic (green diamonds) cluster at the top of the wheel The arrow points to the nonconserved Tyr in the WAT domain of C intestinalis AChE Green and yellow residues are hydrophobic Red, blue, and orange residues are hydrophilic the derived amino acid sequence shows only 28% identity with the AChE from T californica, and only 30% homology with the C intestinalis AChE described in the present study Although the three pairs of conserved cysteine residues involved in intrachain disulfide bonding in AChEs are found in the sequence, only two members of the catalytic triad are present: serine and glutamate The third residue, histidine, is replaced by a cysteine This replacement would probably inactivate the enzyme; in T californica and human AChEs, respectively, H440Q and H447Q mutants lack activity [24,25] Additionally, of the fourteen aromatic amino acids that line the catalytic gorge of vertebrate AChE, only six are conserved in the sequence (Table 1); however, the sequence shows the invertebrate acyl pocket conformation, which provides a seventh aromatic residue in the gorge Nevertheless, in BuChE, eight of the residues are conserved [1] Particularly important is the absence of the tryptophan of the choline-binding site In human AChE, a W86A mutation increases Km by 660-fold [26] Finally, the sequence clearly does not have a carboxyl terminus coded for by an AChET exon, 1312 as only one of the seven aromatic residues is preserved It is highly unlikely that this protein is an active AChE because it is missing a member of the catalytic triad and the main aromatic residue for binding of substrate Additionally, the protein would not be expected to produce all three globular forms because it does not contain a WAT domain However, it could represent a GPI-anchored protein because it has a putative signal sequence, and a putative hydrophobic C-terminus and cleavage site (not shown) What, if any, role the protein may play in the organism has not yet been determined; although it shows highest homology with ChEs and not other ChE-like adhesion molecules Kinetic characterization of recombinant ChE from C intestinalis expressed in vitro and native enzyme expressed in vivo indicates the enzyme is AChE To determine the nature of the cholinesterase activity of the recombinant C intestinalis enzyme and to compare it with the native AChE, we assayed the hydroly- FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS A Frederick et al AChE from C intestinalis sis of ATCh and butyrylthiocholine (BTCh) by enzyme that was secreted into the medium by the COS-7 cells, enzyme extracted from the cells, and enzyme extracted from adult C intestinalis Only ATCh is hydrolyzed appreciably, as indicated by the low values of VmaxBTCh ⁄ VmaxATCh It proved difficult to determine accurate kinetic parameters for BTCh hydrolysis given the low activity that the enzyme showed for the substrate, and it was not possible to detect BTCh hydrolysis by extracts of adult organisms; nevertheless, the kinetic parameters determined are in reasonable agreement The enzymes also show substrate inhibition (i.e lower enzyme activity at high substrate concentrations, and b parameter values of < 1) (Fig 3; Table 2) The selective hydrolysis of ATCh is characteristic of AChE Pharmacological characterization of the recombinant ChE from C intestinalis expressed in vitro and native enzyme expressed in vivo confirms that the enzyme is AChE To determine further the nature of the cholinesterase activity of the recombinant enzyme, we determined half maximal inhibitory concentration (IC50) values of the enzymes for the inhibitors (3aS-cis)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-ol methylcarbamate (physostigmine), which inhibits all cholinesterases; [4-[5-[4-(dimethyl-prop-2-enyl-ammonio)phenyl]-3-oxo-pentyl]phenyl]-dimethyl-prop-2-enylazanium dibromide (BW284c51), which inhibits AChE preferentially; and 10-(2-diethylaminopropyl) phenothiazine hydrochloride (ethopropazine) and N-[bis(propan-2-ylamino)phosphoryloxy-(propan-2-ylamino)phosphoryl]propan-2-amine (iso-OMPA), which inhibit BuChE at low concentrations Physostigmine and BW284c51 inhibit the enzymes at lm concentrations; by contrast, much higher concentrations of ethopropazine and iso-OMPA are required for inhibition Cholinesterase activity (mAb·min–1) 1200 1000 800 600 400 200 10–7 10–6 10–5 10–4 10–3 10–2 10–1 Substrate (M) Fig Representative experiment showing concentration dependencies for ATCh and BTCh hydrolysis by an extract of COS-7 monkey cells expressing recombinant C intestinalis AChE cDNA Transfected COS-7 cells producing C intestinalis AChE were extracted in HIS buffer and assayed with ATCh (d) or BTCh (s) as described in the Experimental procedures (Fig 4; Table 3) This pattern is characteristic of AChE COS-7 cells transfected with cDNA for AChE of C intestinalis produce all three globular molecular forms of AChE To determine the molecular forms of AChE produced in vitro by COS-7 cells transfected with the catalytic subunit for C intestinalis AChE, we performed velocity sedimentation on sucrose gradients in the presence and absence of Triton X-100 Cell extracts have G1a and G4na because the G1 form shifts to a higher sedimentation coefficient in the absence of detergent The forms of AChE secreted into media are G2a and Table Kinetic parameters for recombinant and native AChE from C intestinalis Data are the mean ± SE of four or more determinations Sources of enzyme: medium, enzyme secreted into the medium, usually 12 mL; cells, enzyme extracted with mL of HIS buffer from the COS-7 cells as described in the Experimental procedures; and organism, enzyme extracted from adult C intestinalis, as described in the Experimental procedures Source VmaxATCh (mAb ⁄ min) KmATCh (lM) KssATCh (mM) bATCh VmaxBTCh (mAb ⁄ min) KmBTCh (mM) KssBTCh (mM) bBTCh VmaxBTCh ⁄ VmaxATCh Medium Cells Organism 495 ± 91 1101 ± 27 132 ± 25 188 ± 53 223 ± 15 100 ± 19 275 ± 78 100 ± 27 501 ± 244 0.19 0.32 0.22 4.15 ± 1.38 3.21 ± 1.06 0b 4.80 ± 3.09 1.57 ± 0.73 – 83 ± 45 171 ± 102 – 0.03 0a – 0.012 ± 0.006 0.003 ± 0.001 a Values of b less than 0.02 are indistinguishable from zero b High concentrations of endogenous reducing compounds in the adult tissue increased the background in the Ellman’s assay and, despite correction, obscured whatever low levels of BTCh hydrolysis there may have been; kinetic parameters for BTCh hydrolysis could not be obtained FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1313 AChE from C intestinalis A Frederick et al 0.16 Fractional AChE activity on gradient Fractional AChE activity 1.00 0.75 0.50 0.25 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 0.00 10–7 10–6 10–5 Inhibitor (M) 10–4 Table IC50 values (lM) for inhibition of recombinant and native AChE from C intestinalis Data are the mean ± SE of three or more determinations Sources of enzyme are the same as in Table Physostigmine BW284c51 Medium 5.09 ± 0.66 Cells 7.35 ± 0.28 Organism 14.1 ± 0.76 Ethopropazine Iso-OMPA 0.93 ± 0.17 768 ± 203 1.91 ± 0.01 650 ± 93 1.23 ± 0.76 741 ± 60 > 3000 > 3000 > 3000 G4na because the sedimentation coefficient of the G2 form also increases in the absence of detergent For extracts of adult C intestinalis, G1a and G4na are seen on the gradients In both extracts and media, the sedimentation coefficient of the G4 form remains unchanged (Fig 5; Table 4) COS-7 cells co-transfected with cDNAs for the catalytic subunit of C intestinalis and ColQ from the rat produce the A12 form of AChE To determine whether the catalytic subunits of C intestinalis AChE catalytic subunits could assemble into asymmetric forms of AChE in the presence of a collagenic tail, we co-transfected COS-7 cells with cDNAs for the C intestinalis catalytic subunit and for R norvegicus ColQ, and analyzed cell extracts on sucrose gradients In addition to peaks corresponding to G1 and G4, a peak of enzyme activity appears at approxi1314 10 15 20 25 10 15 20 Sedimentation coefficient 25 0.14 Fig Representative experiment showing concentration dependencies for inhibition of ATCh hydrolysis by recombinant AChE from C intestinalis Media from transfected COS-7 cells secreting C intestinalis AChE was collected and incubated with inhibitors for 20 prior to being assayed for activity The inhibitors used were BW284c51 (d), physostigmine (s), ethopropazine (.), and isoOMPA (,) Source 10–3 Fractional AChE activity on gradient 10–8 0.12 0.10 0.08 0.06 0.04 0.02 0.00 Fig Velocity sedimentation analysis of the globular molecular forms of C intestinalis AChE produced in vitro and in vivo Medium from COS-7 cells transfected with cDNA for the catalytic subunit for C intestinalis AChE, total HIS (d); extracts of the transfected cells (h) and total HIS extracts of adult C intestinalis tissue ( ) were analyzed on sucrose gradients in the presence (top) and absence (bottom) of Triton X-100 as described in the Experimental procedures mately 16S, which is characteristic of the A12 form of AChE Collagenase digestion at 37 °C converts the putative A12 form to a lytic G4; a shoulder of residual undigested A12 is visible (Fig 6; Table 5) We have not found genes for ColQ or PRiMA in the C intestinalis genome Molecular modeling of C intestinalis AChE also indicates that the catalytic subunit can assemble into asymmetric forms Molecular modeling, in addition to sequence analysis, also indicates that the catalytic gorge of C intestinalis AChE is similar to that of vertebrate AChEs, showing FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS A Frederick et al AChE from C intestinalis Table Sedimentation coefficients of recombinant and native forms of AChE from C intestinalis Data are the mean ± SE of ‡ determinations for recombinant enzyme and three and four determinations for enzyme extracted from adult C intestinalis in the presence and absence of Triton X-100, respectively Sources of enzyme are the same as in Table Sedimentation coefficients Conditions Extract +Triton X-100 )Triton X-100 Molecular form 4.66 ± 0.17 5.67 ± 0.15 G1a Medium 10.78 ± 0.11 10.88 ± 0.15 G4na 6.58 ± 0.14 7.06 ± 0.24 G2a Organism 10.61 ± 0.16 10.80 ± 0.24 G4na 4.97 ± 0.13 6.35 ± 0.25 G1a 11.15 ± 0.15 11.01 ± 0.11 G4na Fig Velocity sedimentation analysis of globular and asymmetric forms of AChE produced by cotransfection with cDNAs for C intestinalis catalytic subunit and rat ColQ Total HIS cell extracts were digested with collagenase and analyzed on sucrose gradients as described in the Experimental procedures Control (d); collagenase digestion (s) the volume of the catalytic gorge for C intestinalis ˚ AChE is 780 A3, whereas the volume of the gorge for ˚ T californica AChE is 986 A3 Molecular modeling of monomeric C intestinalis AChE catalytic subunits with the PRAD domain of ColQ indicates that the WAT domain of the C intestinalis AChE is capable of organizing the subunits into a tetramer through interaction with the PRAD domain of ColQ The [AChET]–ColQ complex model was built based on the PRAD–WAT interaction; inter-subunit interactions involving the catalytic domains were considered secondary [27] As a result, the complex has a quasi-four-fold axis of symmetry (Fig 7A,B) The four WAT domains of the tetramer form a-helices and coil around a single antiparallel PRAD domain, which approximates a left-handed polyproline II (PPII) helical conformation The three tryptophans of the WAT domain orient inwards to interact with ColQ, and come into close contact and stack with the prolines of the PRAD domain (Fig 7C,D) Table Sedimentation coefficients of C intestinalis AChE catalytic subunit co-expressed with ColQ with and without digestion by collagenase Data are the mean ± SE of ‡ determinations Phylogenetic analysis of AChE sequences supports a classical phylogeny for deuterostome invertebrates Fractional activity on gradient 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 10 15 20 Sedimentation coefficient Conditions Sedimentation coefficients )Collagenase +Collagenase Molecular formb 5.10 ± 0.07 5.16 ± 0.12 G1 25 11.48 ± 0.10 11.54 ± 0.08 G4 16.09 ± 0.14 15.65 ± 0.20a A12 Estimated from residual activity b Amphiphilic or non-amphiphilic forms are not designated because the appropriate velocity sedimentation experiments on sucrose gradients in the presence and absence of Triton X-100 were not performed The forms are assumed to be G1a and G4na a an AChE-like acyl pocket, a hydrophobic patch (including the choline binding site), and an oxyanion hole (see supplementary Fig S2) The distance between the acyl pocket phenylalanines, Phe317 and Phe319, is ˚ 3.7 A, the same as for T californica AChE However, A phylogenetic analysis of vertebrate and deuterostome and protostome invertebrate ChEs places C intestinalis AChE intermediate between the echinoderms and the cepalochordate amphioxus (see supplementary Fig S3) This placement is consistent with conventional phylogenetic trees based primarily on morphological data [28] Note, however, that the branch length for C intestinalis AChE is the longest in the tree, and the bootstrap value for the branching between amphioxus and C intestinalis is one of the weakest in the tree Discussion We have expressed in vitro a synthetic recombinant ChE from the urochordate C intestinalis Based on FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1315 AChE from C intestinalis A C A Frederick et al B Fig Modeled structures of C intestinalis AChE [AChET]–ColQ complex (A, B) The [AChET]-ColQ complex modeled on the basis of the [WAT]4–PRAD structure, from the side and bottom respectively Each catalytic subunit is shown in a different color (purple, yellow, blue and orange), as is ColQ (green) (C) Hydrophobic interactions between WAT and PRAD helices The view is down and into the PRAD helix in the center of the figure The four WAT helices are shown colored as in (A) and (B) The magenta space-filled residues are the Trps of the WAT domains, which all face inward and surround the PRAD (D) Cut away view showing the Trps (in space-filling format) of two WAT domains (colored as above) interacting with the PRAD PPII helix The Trp side-chains zipper into the grooves of the PPII helix D substrate and inhibitor specificity, the enzyme is AChE The AChE is AChET because transfected COS-7 cells produce G1, G2, and G4 forms Coexpression of C intestinalis AChE catalytic subunit and rat collagenic tail, ColQ, results in the assembly of the A12 asymmetric form Sequence analysis and molecular modeling support both of these conclusions In some respects, the AChE from C intestinalis more closely resembles the AChE of the vertebrates than any other invertebrate AChE and provides information about the evolution of the ChEs The ChE from the invertebrate C intestinalis is an AChE that resembles vertebrate AChE Our kinetic data are consistent with those of Fromson and Whittaker [9] and Meedel and Whittaker [10], who investigated ChE activity in extracts of larval C intestinalis, and also concluded that the activity is due to AChE They found that the hydrolysis of BTCh was 4.5% of that for ATCh at 25 mm [8], and that high concentrations of ATCh produced substrate inhibition [10] They not show a hydrolysis curve for BTCh and, in the present study, we were unable to detect hydrolysis of BTCh by extracts of adult C intestinalis Our estimates of their values for Km (approximately 100 lm) and Kss (approximately 1316 100 mm) for ATCh hydrolysis data are comparable to our own [10] Our pharmacological results are also consistent with previous studies of C intestinalis demonstrating that physostigmine and BW284c51 were effective inhibitors of the activity, but that iso-OMPA was not [9,10] The only IC50 that can be obtained from these data is for BW284c51 (approximately lm) [9], which is virtually identical to that found in the present study Not only does the congruence of the kinetic and pharmacologic data indicate that C intestinalis possesses AChE, but it also argues that the cDNA expressed in vitro in the present study corresponds to the gene expressed in vivo Sequence analysis of important residues in the catalytic gorge also supports the assertion that the enzyme is AChE Only one of the 14 aromatic amino acids that line the catalytic gorge of T californica and most other vertebrate AChEs is missing in C intestinalis AChE, Tyr70, a member of the peripheral site, which is replaced by Ile97 The Kss of C intestinalis AChE for ATCh is rather high and this substitution could contribute to this value [29] More interesting is the nature of the acyl pocket In all vertebrate AChEs, the acyl pocket is comprised of two phenylalanines close to one another in the primary sequence By contrast, for approximately 90% of invertebrate AChEs, the acyl pocket is composed FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS A Frederick et al of two phenylalanines far apart in the primary sequence, corresponding to Phe290 and Val400 in T californica AChE The only known exception in this subset of invertebrate AChEs is C intestinalis AChE, where the acyl pocket phenylalanines are homologous to those of the vertebrates, suggesting that the C intestinalis acyl pocket is ancestral to that of the vertebrates This conclusion is confounded by the acyl pocket conformations of the two acetylcholinesterases from the cephalochordate amphioxus (Branchiostoma floridae); the cephalochordates have long been considered to be the sister group of the vertebrates ChE2 from this organism shows the typical invertebrate acyl pocket structure, whereas ChE1 apparently has a novel acyl pocket, unlike the typical vertebrate or invertebrate conformations [6,30,31] The echinoderms, urochordates (tunicates, C intestinalis), cephalochordates (amphioxus, B floridae), and vertebrates are members of the deuterostome branch of the animal kingdom, with the echinoderms generally considered as the most basal of the groups, the urochordates intermediate, and the cephalochordates closest to the vertebrates [28,32,33] However, recent data from metaphylogenies and phylogenomics have challenged this view, with Blair and Hedges [34], Delsuc et al [35], and Vienne and Pontarotti [36] proposing that the urochordates are actually the closest living relatives of the vertebrates, with the cephalochordates intermediate to the echinoderms Our phylogenetic analysis of deuterostome AChEs supports the classical phylogeny and is similar to the phylogenetic tree for AChE of various vertebrates and deuterostome invertebrates provided by Vienne and Pontarotti [36] Note, however, that the branch length for C intestinalis AChE is the longest in the tree; this long branch length is typical of many C intestinalis genes and is a result of rapid evolution in the species [34,37] This rapid evolution and the resultant long branch length gives rise to an artifact called long branch attraction (LBA), which has a number of effects Most importantly in this case, LBA results in the grouping of two sequences that evolve more rapidly than the others do: C intestinalis AChE and a putative AChE from the echinoderm Strongylocentrotus purpuratus LBA is also a problem in metaphylogenies, but can be corrected for more easily, and a consensus is forming around the revised deuterostome phylogeny, with the urochordates actually being the sister group to the vertebrates [28,34–36] Not only does LBA compromise our AChE phylogeny, but also the bootstrap value for the branching between amphioxus and C intestinalis AChEs is one of the weakest in the tree, indicating its uncertainty If it is assumed that the AChE from C intestinalis urochordates are the closest living relative of the vertebrates, the acyl pocket of C intestinalis may in fact be ancestral to that of the vertebrates What may have been responsible for the shift in acyl pocket structure during the transition from invertebrates to vertebrates, or nonchordates to chordates and vertebrates, remains a matter of speculation The AChE from C intestinalis is AChET and is able to assemble into asymmetric forms organized by vertebrate ColQ Analysis of the carboxyl terminus sequence indicates that the C intestinalis AChE is AChET, which should be capable of forming the three globular forms: G1a, G2a, and G4na When the catalytic subunit of AChE from C intestinalis was expressed in vitro, G1a, G2a, and G4na forms of enzyme were produced The amphiphilicity of G1 and G2 is due to the exposure of the hydrophobic T peptide of their carboxyl termini, which interact with detergent micelles on the gradients; while the T peptide of G4 is sequestered away from solvent and unable to interact with detergent [38] Extracts of adult C intestinalis contained G1a and G4na forms By contrast, it was reported that extracts of the larvae produce all three globular forms, possibly indicating a developmental difference in AChE assembly between the larvae and adults [11] Nevertheless, all three G forms produced in vivo are also produced in vitro Inspection of the T peptide sequence shows that all of the tryptophans of the WAT domain are conserved in the C intestinalis sequence However, one of the seven aromatic amino acids, Tyr20, is replaced by Ser20 In Torpedo marmorata AChE, the mutations Y20A and Y20P decrease the amphipathic nature of the T peptide a-helix and abolish the assembly of secreted tetramers when catalytic subunits are coexpressed in the presence or absence of a truncated, soluble version of ColQ [39] In the [WAT]4–PRAD model of Dvir et al [18] (PDB ID code 1VZJ), there is an edge-on p-p interaction between the edge of Phe14 in WAT strand A and the face of Tyr20 in chain D This interaction is not observed for the other Phe14Tyr20 combinations The Y20A and Y20P mutations would disrupt this interaction and apparently destabilize the tetramer Clearly, this is not the case for the AChE tetramer of C intestinalis, which forms tetramers in the absence and presence of ColQ The WAT– PRAD interaction of our tetrameric molecular model is in good agreement with the corresponding structure of Dvir et al [18], and indicates that the side-chain of Ser20 in strand D is oriented towards and in close proximity to the edge of Phe14 in strand A One FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1317 AChE from C intestinalis A Frederick et al possibility is that the tetramer is stabilized by the formation of a weak C–HỈO hydrogen bond between the hydroxyl oxygen of Ser20 and a slightly polar C–H group of the aromatic ring of Phe14 Such bonds were first proposed in 1982 for phenylalanines in proteins by Thomas et al [40], and have received considerable attention in recent years [41–44] Co-expression of C intestinalis AChE catalytic subunit with rat ColQ resulted in the production of the A12 asymmetric form of AChE These results confirm our molecular modeling, which indicated that the appropriate interactions between the WAT domain of the catalytic subunit and the PRAD domain of ColQ were present to assemble the catalytic tetramers of the asymmetric forms The A12 form consists of three such tetramers attached to the triple-stranded helix of ColQ This result is the first demonstration of the assembly of catalytic subunits of an invertebrate AChE into asymmetric forms The evolution of the T peptide and tetrameric forms of AChE However, one question arises: what, if anything, assembles the C intestinalis G4 tetramers in the absence of ColQ in vivo or in vitro? T peptide sequences have been identified in vertebrates; deuterostome invertebrates, the urochordate C intestinalis and the echinoderm S purpuratus; and in protostome invertebrates, the mollusk Aplysia californica and various nematodes, including Caenorhabditis elegans, suggesting that the peptide is widespread in nature The presence of the T peptide in both branches of the animal kingdom indicates that it may be as old as and conserved for ‡ 900 million years because it would have had to evolve prior to the protostome-deuterostome split [34] Interestingly, all of the phyla that have the T sequence also have G4 AChE, and use acetylcholine as a neurotransmitter at their neuromuscular junctions, suggesting both are a prerequisite for efficient synaptic transmission at the junctions Given the recent research on the interaction between WAT domains of AChE catalytic subunits and the PRAD domains of ColQ and PRiMA [38,39,45,46], the fact that G4 AChE interacts with a noncatalytic subunit in nematodes [19,20], the recent finding of small PRADcontaining polypeptides associated with soluble tetramers of vertebrate BuChE [47], and the apparent ubiquity of the T domain, we propose that PRADcontaining proteins mediate tetramerization of AChE throughout evolution, with ColQ and PRiMA of the vertebrates comprising just two of the many examples of such proteins 1318 Experimental procedures Materials DMEM, fetal bovine serum, and OptiMEM medium were purchased from Invitrogen (Carlsbad, CA, USA) FuGene was obtained from Roche (Indianapolis, IN, USA) ATCh, BTCh, BW284c51, 5-(3-carboxy-4nitro-phenyl)disulfanyl-2nitro-benzoic acid (DTNB), iso-OMPA, ethopropazine, and physostigmine were purchased from Sigma (St Louis, MO, USA) Type-3 collagenase was obtained from Worthington (Lakewood, NJ, USA) 7-[(diethoxyphosphoryl)oxy]-1methylquinolinium iodide (DEPQ) was a gift from Yacov Ashani Adult specimens of C intestinalis were purchased from The Marine Biological Laboratory (Woods Hole, MA, USA) We thank Andrew Gannon for help with the C intestinalis dissection Gene synthesis and analysis The ci0100132088 gene from the urochordate C intestinalis is now identified in the Department of Energy Joint Genome Institute (DOE JGI) Database (http://genome.jgi-psf org/Cioin2/Cioin2.home.html) as an AChE gene The sequence for this gene is embedded in the C intestinalis genome sequence (GenBank accession no AABS01000124) [7,8] We spliced out the intronic sequences and translated the coding exonic sequences in silico Nucleotide sequence and derived amino acid sequence data reported are available in the Third Party Annotation Section of the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession no TPA: BK006073 These sequence data are also available on the DOE JGI Database The amino acid sequence for the protein has also been deposited in the Esther database as cioin-acche1 (http://bioweb.ensam.inra.fr/ESTHER/general? what=index) [23] A BLAST search was conducted at NCBI with the translated sequence, and it was found to be similar to many AChE amino acid sequences in that database, showing 72% homology with the AChE of Ciona savignyi GenScript Corporation (Piscataway, NJ, USA) synthesized and subcloned a cDNA for the protein into pcDNA3.1 (Invitrogen) after linker sequences containing EcoRI and XbaI restriction sites were added to the 5¢- and 3¢-ends of the cDNA, respectively, for ligation of the cDNA into the expression plasmid Double-strand DNA sequencing confirmed the sequence The recombinant plasmid was then used to transform competent Escherichia coli (XL1Blue; Stratagene, La Jolla, CA, USA) Qiagen maxi-preps (Qiagen, Valancia, CA, USA) were used to obtain plasmid DNA for transfections In vitro expression and extraction of enzymes COS-7 monkey cells (American Type Culture Collection, Manassas, VA, USA) were grown in DMEM containing FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS A Frederick et al 10% fetal bovine serum Cells were plated at a density of 2.5 · 106 cells ⁄ 75 cm2 culture flask, incubated overnight, and transferred to OptiMEM medium FuGene was then used to transfect the cells with 7.8 lg of DNA For co-transfection experiments, 7.8 lg of DNA for the catalytic subunit and for R norvegicus ColQ (GenBank accession no BC107386) was used The cells were then incubated for 48 h at 30 °C, before the medium was harvested and the cells were extracted in 1–5 mL of high ionic strength (HIS) buffer: 10 mm NaHPO4, pH 7, m NaCl, 1% Triton X-100, mm EDTA Extracts were centrifuged at 20 000 g for 20 and the supernatants were assayed for AChE activity The same HIS buffer was used to extract adult C intestinalis tissue but, given the low activity in the adult, equal amounts of tissue and buffer on a weight ⁄ volume basis were used The interstitial fluids in C intestinalis are isosmotic with seawater Typically, specimens of C intestinalis were dissected to separate the outer tunic from the internal organs; subsequently, the digestive system was emptied of its contents The tunic and remaining viscera were then separately flash frozen in liquid nitrogen The viscera, which contained more enzyme, was used for kinetic and sedimentation velocity experiments; the tunic was used for pharmacological experiments For velocity sedimentation on sucrose gradients, extracts were made with HIS buffer containing 10 mm NaHPO4, pH 7, m NaCl, 1% Triton X-100, mm EDTA, 0.02 mgỈmL)1 pepstatin, 0.2 mgỈmL)1 aprotinin, mgỈmL)1 bacitracin, and 0.3 mgỈmL)1 benzamidine [48] Measurement and analysis of AChE activity and inhibition Acetylcholinesterase activity was measured according to the method of Ellman et al [49] as modified by Doctor et al [50] in 100 mm NaHPO4, pH 7, 0.3 mm DTNB, and 167 mm NaCl; the final concentration of Triton X-100 was 0.17% for assays performed with cell extracts or media, and 0.085% for extracts of adults ATCh and BTCh were used as substrates at various concentrations; for pharmacological analyses and assays of sucrose gradients, the concentration of ATCh was mm The kinetic parameters Km, ´ Kss, b, and Vmax, were determined as described by Radic et al [51] and Kaplan et al [29]; the parameter, b, indicates the relative catalytic efficiency of the two-substrate bound complex compared to the single-substrate form If b < 1, the enzyme shows substrate inhibition; if b > 1, the enzyme shows substrate activation, and if b = 1, Michaelis–Menten kinetics is observed sigmaplot (Systat Software, San Jose, CA, USA) was used to fit the kinetic data It was not possible to determine the turnover number kcat (Vmax ⁄ [enzyme]) because DEPQ could not be used to accurately titrate the enzyme, even after overnight exposure Although Triton X-100 can activate AChE, and different concentrations of the detergent can artifactually AChE from C intestinalis alter enzyme activity, total activities for different preparations were never compared, only Vmax ratios for BTCh and ATCh hydrolysis, which were always determined sequentially for the same enzyme preparations Values of IC50 for the inhibitors used were determined by incubating enzymes with various concentrations of drug for 20 and then assaying for enzyme activity in the presence of ATCh sigmaplot was then used to fit the data to a three-parameter logistic function, yielding IC50 Since we were just looking for classical diagnostic differential inhibition, it was not necessary to determine ki, KI, or aKI values for the inhibitors [10,52,53] Velocity sedimentation on sucrose gradients: collagenase digestion The molecular forms of AChE were analyzed by velocity sedimentation in 5–25% isokinetic sucrose gradients prepared in HIS buffer containing mgỈmL)1 BSA Sedimentation was in a Beckman SW 41 rotor at 30 000–37 000 r.p.m for times satisfying the equation [(r.p.m.)2 · t (h)]=2.5 · 1010, as described previously [53] Apparent sedimentation coefficients were calculated relative to the sedimentation of catalase (11.3S) Data were plotted as fractional activity of total AChE activity on the gradient as a function of sedimentation coefficient For collagenase digestion, HIS extracts were adjusted to 10 mm CaCl2 and incubated at 37 °C for h with or without 200 lgỈmL)1 collagenase as described previously [54] Molecular modeling Molecular modeling was performed on an Indigo O2 computer (Silicon Graphics, Sunnyvale, CA, USA) using the discover and insight ii programs (Accelrys, San Diego, CA, USA) The 3D structure of C intestinalis monomeric AChE was built using the Homology module of insight ii and the crystal structure of Torpedo AChE (pdb index 1EA5) as a template The two amino acid sequences were aligned with t-coffee software [55], as clustalw misaligned conserved cysteines involved in intra-molecular disulfide bonding A two-sequence blast confirmed the t-coffee results [56] The structure was minimized for 10 000 iterations of steepest descent in vacuo using the distance-dependent dielectric constant by the discover program (Accelrys) Volumes of active site gorges were calculated with CASTp [57] For modeling of the C intestinalis G4–ColQ complex, the crystal structures of the [WAT]4–PRAD complex (pdb ID 1VZJ) and the mouse [AChET]4–ColQ complex model (a generous gift from D Zhang and J A McCammon) were used and modeling was performed as described previously [27] After modeling, the complex underwent 10 000 iterations of steepest descent minimization FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS 1319 AChE from C intestinalis A Frederick et al Sequence and phylogenetic analysis For analysis of acyl pocket structures, multiple amino acid sequences were aligned with clustalw [58] By contrast to the pairwise alignment, there was no obvious problem with the multiple alignment For phylogenetic analysis, a multiple sequence alignment and phylogenetic tree based on the Neighbour-joining method were generated with clustalx [59,60] Bootstrap values for 1000 replicates are indicated [61] This alignment is deposited at the EMBLALIGN database as ALIGN_001208 10 11 12 Acknowledgements This research was supported by a National Institutes of Health Academic Research Award (no R15 GM072510-01) to LP Additional support was provided by Birmingham-Southern College 13 References ´ Massoulie J, Pezzementi L, Bon S, Krejci E & Vallette FM (1993) Molecular and cellular biology of cholinesterases Prog Neurobiol 41, 31–91 Duysen E, Li B, Darvesh S & Lockridge O (2007) Sensitivity of butyrylcholinesterase knockout mice to (–)huperzine A and donepezil suggests humans with butyrylcholinesterase deficiency may not tolerate these Alzheimer’s disease drugs and indicates butyrylcholinesterase function in neurotransmission Toxicology 233, 60–69 Chatonnet A & Lockridge O (1989) Comparison of butyrylcholinesterase and acetylcholinesterase Biochem J 260, 625–634 Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L & Silman I (1991) Atomic 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4876–4882 60 Jeanmougin F, Thompson JD, Gouy M, Higgins DG & Gibson TJ (1998) Multiple sequence alignment with Clustal X Trends Biochem Sci 23, 403–405 61 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap Evolution 39, 783– 791 62 Zidovetzki R, Rost B, Armstrong DL & Pecht I (2003) Transmembrane domains in the functions of Fc receptors Biophys Chem 100, 555–575 63 Hubbard TJP, Aken BL, Beal K, Ballester B, Caccamo M, Chen Y, Clarke L, Coates G, Cunningham F, Cutts T et al (2007) Ensembl 2007 Nucleic Acids Res 35, D610–D617 Supplementary material The following supplementary material is available online: Fig S1 Alignment of C intestinalis and T californica AChE sequences Fig S2 Molecular model of the catalytic gorge of C intestinalis AChE Some of the key residues comprising the gorge are shown Fig S3 Phylogenetic tree inferred from the alignment of amino acid sequences of vertebrate and invertebrate ChEs This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 1309–1322 ª 2008 The Authors Journal compilation ª 2008 FEBS ... evolution of the ChEs, including the origins of the acyl pocket, the T exon, and the asymmetric forms of ChE in the vertebrates Results The sequence of the ChE from C intestinalis suggests that the. .. other invertebrates, there is a deletion in the region of the acyl pocket of C intestinalis compared to the vertebrate enzyme (Fig 1) Additionally, the carboxyl terminus of the C intestinalis. .. attached to the triple-stranded helix of ColQ This result is the first demonstration of the assembly of catalytic subunits of an invertebrate AChE into asymmetric forms The evolution of the T peptide