Molecular genetic approach to the study of mammalian choline acetyltransferase

Brain Research Bulletin, Vol. 22, pp. 147-153. * Pergamon

Press

plc,

1989. Printed

0361-9230189

in the U.S.A.

$3.00 + .OO

Molecular Genetic Approach to the Study of Mammalian Choline Acetyltransferase SYLVIE BERRARD, ALEXIS BRICE AND JACQUES MALLET’ Dkpartement de GPnPtiqueiMol&culaire,Laboratoire de Neurobiologie Cellulaire et Moltkulaire Centre National de la Recherche Scientifique, F-91198 Gif-sur- yvette, France

BERRARD, S., A. BRICE AND J. MALLET. Molecular genetic approach to the study of mammalian choline acetyltransferase. BRAIN RES BULL 22(l) 147-153, 1989.-The enzyme choline acetyltransferase catalyses the biosynthesis of the neurotransmitter acetylcholine and constitutes a specific marker of cholinergic system. To date, there is very limited information about the structure of the mammalian enzyme. More detailed understanding of this enzyme is particularly desirable because of the importance of the cholinergic system in neurotransmission, as well as the possible involvement of this system in certain neurotogical disorders. In this article, recent studies concerning the isolation of a cDNA encoding the complete sequence of the porcine enzyme are reported and the potential applications of this probe are discussed. Choline acetyltransferase

Xenopus oocytes

Rabbit reticulocyte lysate

Complete amino acid sequence

(14). Proteins were measured according to the method of Bradford (4). Results are given as pmol of ACh formed per min and per mg of protein. In injected oocytes, ChAT activity was measured by using the technique described by Smith et al. (36). The enzymatic activity is given as fmol of ACh formed per min and per oocyte. Some assays were carried out in the presence of the ChAT specific inhibitor NVP [4-(I-naphthylvinyl)pyridine~ (C~biochem~ at the ~on~entration of 0.5 mM (38).

THE control of motor behavior constitutes one of the most important functions of the central nervous system. Numerous regions of the brain are involved in this process that is integrated ultimately in the motoneurons of the spinal cord, the “final common path” in the control of movement. These neurons, which lie in the ventral horn, exhibit a cholinergic phenotype and therefore express choline acetyltransferase (ChAT; E.C 2.3.1.6.), the enzyme synthesizing acetylcholine (ACh), which is a specific marker of the cholinergic system. Despite the importance of this enzyme both in peripheral and central nervous system (CNS), only limited information is, to date, available on its structure, its mechanism of action as well as on the regulation of its expression [for review see (24,33)]. These problems can now be more easily approached by means of the language of molecular genetics. Here, we summarize our recent studies on the isolation, from a ventral spinal cord cDNA library, of a cDNA encoding the complete sequence of the porcine enzyme and we discuss the potential applications of this probe.

Oligonucleotide Screening of a Randomly Primed cDNA Library

A cDNA library was prepared in the bacteriophage lambda gtf0 from porcine ventral spinal cord poly(A)+RNA (3). Briefly, the first strand cDNA synthesis was primed by random DNA sequences of 20 to 50 nucleotides. The amplified library contained 1.2~ lo6 independent recombinant phages with inserts longer than 500 bp. About lo6 recombinant phages were screened on duplicate filters with a mixture of eight 29mer oligonudeotides corresponding to the N-terminal sequence of the porcine protein (5). Phage DNA was prepared from positive clones according to Maniatis et al. (26), and the cDNA insertions were excised by digestion with EcoRI (Boehringer Mannheim) .

METHOD

Isolation of Poly(A) ‘RNA and Oocyte Injection

Male Wistar rats were killed by decapitation, and pigs were electrocuted. Tissues were rapidly dissected, frozen and stored at -80°C. Total RNA was extracted as described by Lomedico and Saunders (23). Poly(A)+RNA was purified by oligo(dT)cellulose (Boehringer Mannheim) chromatography. Oocytes were treated and injected as described (17).

In Vitro Synthesis

of ChA T

ChAT cDNA insertion was subcloned in the transcription plasmid pSPT 18 (Promega Biotec), which contains SP6 and T7 promoters in opposite orientation. Complementary RNA was synthesized by T7 RNA polymerase as described (19,28) and was translated in rabbit reticulocyte lysate (Amersham) (32). Immunoprecipitations were carried out with a polyclonal antibody raised against porcine brain ChAT (11,12).

ACh Synthesis Assay

ChAT assay has previously been described in (2). In rat tissues, ChAT activity was determined as described by Fonnum

‘Requests for reprints should be addressed to J. Mallet, Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS, 1 avenue de la Terrasse, F-91 198 Gif-sur-Yvette, France.

147

BERRARD,

148 RESULTS

AND DISCUSSION

Comparison of ChA T Activity and ChA T mRNA Levels in Several Regions of Rat Central Nervous System (2) The success in the isolation of a particular cDNA depends, at least in part, on the appropriate choice of the starting material. This step is particularly important when dealing with molecules such as ChAT which are expressed at a very low abundance. Available data indicate that, in the CNS of mammals, a purification factor of more than 1,OOO,OOO is necessary to obtain a pure enzyme (12,33). In a first series of experiments, the ChAT activity was assayed in several regions of the rat CNS, which are known to contain cholinergic neurons. As shown in Table lA, the highest activity was found in striatum and was 1.6-fold that of the ventral spinal cord (VSC). Most importantly, we then compared the corresponding mRNA levels taking advantage of the ability of Xenopus oocytes to synthesize proteins from exogenous mRNA (17). The oocytes were injected with poly(A)+RNA purified from the same tissues as above and the activity of in ovo synthesized ChAT was assayed by the sensitive technique described by Smith et al. (36). The results presented in Table 1B indicate that whole brain, striatum and VSC RNA direct the synthesis of an active ChAT. VSC RNA yielded the most prominent activity, which was 2.5- and IO-fold higher than with brain and striatum mRNA, respectively. It is at first sight surprising that VSC contains IO-fold more ChAT mRNA than striatum, whereas the corresponding enzymatic activity is 1.6-fold lower. However, these results can be explained by the neuroanatomical differences between these two structures. The long axons of cholinergic motor neurons

TABLE

1

ChAT ACTIVITY IN SEVERAL RAT TISSUES AND IN OOCYTES INJECTED WITH 50 ng OF THE CORRESPONDING POLY(A)+RNA

(A) ChAT Activity of Tissues SA (pmol/min/ mg prot.) Striatum Ventral

1805?95(9)

% of Striatum Activity

(B) ChAT Activity of Injected Oocytes SA (fmol/min/ oocyte)

% of Striatum Activity

100

24

100

61 64 36 20 3 0

234 91 0 0 -

915 _ 379 0 0 _

Spinal

Cord Brainstem Brain Hippocampus Cerebellum Liver

1111k42(6) 1159*38(2) 644t57(2) 362+60(2) 57+4 (4) 0 (2)

(A) ChAT specific activity (SA) was determined in rat tissues by the method of Fonnum (14). Mean values of several experiments numbered in parentheses are shown. SA is given as pmol of acetylcholine formed per min and per mg proteins. The specificity of the assay was proven by a 85-95% inhibition of ChAT activity measured in these tissues in the presence of the ChAT inhibitor NVP [4-(1-naphthylvinyl)pyridinel (38). (B) In injected oocytes, ChAT was measured as already described in (36). Mean values of duplicates of a single experiment are shown. SA

is given as fmol of acetylcholine formed per min and per oocyte.

BRICE AND MALLET

from the spinal cord provide a dense innervation of the skeletal muscles; therefore, a large pool of ChAT mRNA in the cell bodies is required to maintain a high level of synthesis of the enzyme, which has to be transported over long distances. In contrast, the cholinergic neurons of striatum are interneurons and the ChAT activity is more closely related to the corresponding mRNA level in this structure. These observations clearly indicated that VSC was the material of choice to generate the cDNA library from which a ChAT cDNA clone could be isolated.

Isolation of a cDNA Clone Containing the Complete Sequence of Porcine ChA T (3) The sequence of the first N-terminus 13 amino acids of porcine brain ChAT was determined as described by Braun et al. (5). Based on this information, the isolation and identification of a cDNA clone encoding the entire amino acid sequence of porcine ChAT involved the following steps. 1) A mixture of eight 29-mer oligonucleotide probes corresponding to the sequence of 10 of these amino acids were synthesized, in which desoxyinosine (27) was inserted in every position where codon ambiguity involved all 4 nucleotides (Fig. 1). 2) These oligonucleotides, reflecting all codon combinations, were used to screen a lambda gtl0 cDNA library generated from porcine VSC poly(A)+RNA. To ensure that the sequence corresponding to the coding 5’ end of the mRNA would be represented in the library, the first strand cDNA synthesis was primed by short DNA fragments randomly hybridized to the RNA template. Among lo6 recombinant clones tested, only one clone, designated pChAT-I, contained a sequence which matched perfectly with the ChAT N-terminus region (Fig. 1). 3) pChAT-1 cDNA was subcloned in a transcription plasmid pSPT18 allowing the synthesis of the corresponding RNA (19,28). This RNA produced in rabbit reticulocyte lysate a protein of an apparent molecular weight of 68 kDa as determined by SDS-PAGE (Fig. 2, lane 2). This value agrees with the estimated size of the purified porcine protein (11,12). Furthermore, this protein corresponding to pChAT-I is immunoprecipitated with a polyclonal antibody (Fig. 2, lane 4) and a monoclonal antibody (Fig. 2, lane 5) prepared against porcine ChAT (12). In contrast, no protein of this molecular weight could be detected after translation of either antisense RNA (not shown) or after translation of sense RNA and precipitation with nonimmune sera (Fig. 2, lanes 3 and 6). 4) Finally and most importantly, pChAT-1 RNA directed, both in Xenopus oocytes and in rabbit reticulocyte lysate, the synthesis of an active ChAT enzyme. The specificity of the ChAT assay was ascertained by testing the activity in presence of NVP, a ChAT specific inhibitor (Table 2). Interestingly the observation that ChAT mRNA directs the synthesis of an active enzyme in rabbit reticulocyte lysate suggests that posttranslational modifications are not required to yield ChAT activity.

Analysis of the Sequence of Porcine ChA T The complete

nucleotide

sequence of pChAT-1

is shown on

Fig. 3. It contains an open reading frame of 1,953 nucleotides encoding a protein of 640 amino acids, with a calculated molecular weight of 71,517 and an isoelectric point of 7.72.

These values are in good agreement with that of 68 kDa for the apparent molecular weight of the purified porcine enzyme (12) and that of 8.1 for the isoelectric value determined for the

MAMMALIAN

CHOLINE ACETYLTRANSFERASE

Cl-AT

149

cDNA

N-terminal

amino

:

acids

H2N

code:

Genetic

PRO

ILE

LEIJ GLU

LYS

TAR

PRO

PRO

LYS

MET

ALA

ALA

LYS

CCU

AUU

UUA

GAA

AAA

ACU

CCU

CCU

AAA

AUG

GCU

GCU

AAA

C

C

G

G

G

C

C

C

G

C

C

G

A

A

CUU

A

A

A

A

A

C

G

0

G

G

G

G

A G

Synthetic

GAA AAA AC1 CC1 CC1 AAA ATG GCI GCI AA

oligonucleotides:

G

0

GAA

AAA

G

pChAT-1 cDNA sequence

ACT CCC CCT AAG ATG GCA GCA AAA

FIG. 1. Sequences of (first line) the N-terminus of porcine ChAT, (second line) the corresponding genetic code, (third line) the complementary mixture of 29-mer oligonucleotides and (fourth line) pChAT-1 cDNA and corresponding amino acid sequence. Deoxyinosine was inserted in every third position where codon ambiguity allowed for all 4 nucleotides. Note that the ChAT protein sequence determined from the purified enzyme was identical to that obtained from pChAT-1 cDNA analysis.

TABLE 2 ChAT ACTIVITY GENERATEDFROM pChAT-1 RNA IN Xenopus OOCYTE AND IN RABBIT RETICULOCYTE LYSATE SYSTEMS Control cpm No RNA (A) Oocytes (B) Lysate

150 120

TPH-RNA 160 140

pChAT-l cpm -NVP +NVP 13,070 14,330

520 310

% Inhibition by NVP 97 99

(A) ChAT activity was measured as described in (36) in 15 oocytes each injected with 25 ng of pChAT-1 RNA. Eggs were homogenized in 50 ~1 of 50 mM phosphate buffer pH 7.4, 0.5% Triton X-100. Cpm correspond to the (i4C) ACh synthesized in 5 ~1 homogenate after 5 min reaction. (B) ChAT was assayed after translation of 200 ng of pChAT-I RNA in rabbit reticulocyte lysate. Cpm correspond to the total amount of ACh synthesized. Control experiments were performed without RNA and with RNA encoding rat tryptophan hydroxylase (Darmon et al., personal communication).

(6). Further the amino acid composition reported for porcine ChAT (5) is also in consonance with our data. When the total of these 640 amino acids is used as a base of calculation, most of the amino acids that can be reliably determined by the o-phthaldialdehyde method are within 10% of the sequence data. The hydropathy profile of porcine ChAT was calculated using amino acid hydrophobicity values (20) averaged over a sliding window of 7 residues (Fig. 4). Predicted secondary structure based on the algorithm of Garnier and Robson (15) human

enzyme

shows that 36% of the amino acids are organised in o-helix whereas 13% are assigned to P-sheet regions (data not shown). ChAT has been reported to exist both in a soluble and in a membrane bound form in Torpedo, rat, mouse and human (1,13). Inspection of the amino acid sequence reveals no obvious hydrophobic region and the present data does not allow to discriminate between these two forms. Both could arise from the same ChAT premessenger through alternative splicing. This possibility can now be tested by Sl mapping experiments and genomic clones analysis. Attachment to the membrane may also result from posttranslational modifications (8,35). Whether pChAT- 1 encodes the membrane and/or soluble form of ChAT may now be approached by transfection experiments. This last methodology can also be used to test whether the protein is phosphorylated. Indeed the sequence reveals the presence of serine or threonine residues which are found to be aligned as consensus sequences for phosphorylation (7,3 1). These modifications have been found, in several instances, to play a role in short term regulation (37). The low abundance of ChAT has so far precluded such investigation which could be conveniently addressed with a cell line expressing relatively high amount of the enzyme. Comparison of Porcine and Drosophila ChA T Sequences

melanogaster

The primary structure of porcine ChAT and the partial Drosophila melanogaster ChAT sequence (18) were optimally

aligned with insertion of 11 gaps, and their comparison revealed 32% identity and 51% homology (Fig. 5). However, six domains located on porcine ChAT at positions 22-80, 125-181, 241-285, 292-344, 369-430 and 545-605, are more highly conserved and display homology ranging from 64 to 79%. These domains are likely to play a role in the conformation of the

BERRARD, BRICE AND MALLET

150

34

5

6

92.5-

69-

46-

FIG. 2. Translation of pChAT-1 RNA in rabbit reticulocyte iysate and immunoprecipitation of translation products. pChAT-1 cDNA was subcloned in the transcription plasmid pSPT18, sense RNA was synthesized and translated in rabbit reticuiocyte lysate. Lane 1: translation of reticulocyte lysate endogenous RNA. Lane 2: translation product of 20 ng of pChAT-1 RNA. Translation products of 300 ng of pChAT-I RNA were immunoprecipitated by a polyclonal antibody (lane 4) or a monoclonal antibody (lane 5). Lanes 3 and 6 represent the proteins precipitated by the corresponding nonimmune sera. Molecular weights shown x lo3 were calculated by reference to the mobilities of standard proteins.

protein and particularly of its active site. One of them contains

2 conserved histidine residues. As the imidazole group of a histidine has been reported to play a crucial role in the enzymatic activity of ChAT (25), this domain probably participates in the catalytic reaction. This hypothesis can be tested by mutagenesis experiments, in conjunction with expression of the corresponding protein in Xenopus oocytes or rabbit reticulocyte lysate. Perspectives The identification of a cDNA clone encoding a complete mammalian ChAT enzyme opens new avenues in the study of

the cholinergic system. Regardless of the possible existence of both a membrane and a soluble form of ChAT, it will be of practical interest to examine whether the enzyme exists in multiple forms generated from the alternative splicing of a single premessenger RNA. Such a situation has recently been found to occur in the case of human tyrosine hydroxylase (TH) (16,21). The possibility to differentially express various forms of the enzyme that exhibit distinct specific activities represents an attractive mean to regulate neurotransmitter availability at particular synapses. Such a possible diversity could also be developmentally regulated or be tissue specific (29). The porcine ChAT cDNA hybridizes with the rat and human

FACING PAGE FIG. 3. Nucleotide and predicted amino acid sequences of porcine ChAT as deduced from pChAT-I cDNA clone. pChAT-I cDNA was sonicated and fragments were subcloned in the vector M13mpS (10) and both strands of the cDNA were sequenced as described by Sanger et al. (34). Nucleotides are numbered in the 5’ to the 3’ direction starting with the first residue following the EcoRl cloning site. The N-terminal proline and the C-terminat proline are numbered 1 and 640 respectively. Peptide sequence derived from purified porcine ChAT is underlined. Arrowheads represent the putative SER or THR phosphoryfa&ion sites.

MAMMALIAN

CHOLINE ACETYLTRANSFERASE

123 AT6 CCC AK CTG GA%A

ACT CCC CCT d3:TG

NET PRO 213

cDNA

151

GCA GCA AAA AG,:':cc AGC AGT GAG da&G CC1 W CT0 CC?% CTC CC1 GTG CCC SER PRO SER SER GLU GlU GLU PRO GLV LLU PRO LVS LEU PRO VAL PRO

230 273 290 243 260 CcA nG UG CAG ACC CTG GCC KC TAC CTG CGG TGC AT6 CAG W CT6 GTA CC1 GAG GAG CAA TTT AGG AGG AGC CAG GCC ATT GTG CAG PRO LEU GLN GCN THR LEU ALA THR TVR LEU ARG CYS NET GLN HIS LEU VAL PRO GLU GLU GLN PHE ARG ARG SEA GLN ALA ILE IL GLN

305 CAG TTT GGG GCC CC:%T GGC CT1 GGC d3kc CTG CAG C#iG M:?TC CTG GAA CGG CA:'& CAG ACA GCT M?&G GTG TCT GAG TAC GLN PHE GLY ALA PRO GLY GLV LEU GLY GLU THR LEu GLN GLN LVS LEU LEU GLU ARG GLN GLU GLN THR ALA ASN TRP VAL SER GLU TVR 393 TGG CTG MC TRP LEU MN

435 470 410 440 423 GAC ATG TAT CTC MT MC CGT CTG CCC CTG CC1 GTC MC TCC AGC CCA GCT GTG ATT TTT GCC CGG CAG CAC TTC CAA GAC ASP NET TVR LEU ASN ASN ARG LEu ALA LEU PRO VAL ASN SER SER PRO ALA VAL ILE PHE ALA ARG GLN HIS PHE GLN ASP

483 560 ACE MT GAC CAG CTAmkG TTT GCA Cc M?hT ATE TCT GGT GT::TC AGC TAC AAG GC?:TG CTG GAC AGC CAC TCC ATC CCC ATT wx: THR ASN ASP GLN LEU ARG PHE ALA ALA ASN LEU ILE StR GLY VAL LEU SER T'fR LYS ALA LEU LEU ASP SER HIS SER ILE PRO ILE ASP

373

390

605

620

633

630

TGT GCC MG GGC CAG CTG TCA GGA CAG CC1 CTC TGT ATG MG CM TAC TAT GGG CT1 TTC TCC TCT TAC CGG CTC CCT GGC CAC ACC CAG CVS ALA LYS GLV GLN LEU SER GLY GLN PRO LEu CYS MET LVS GLN TYR TVR GLV LEU PHE SER SER TYR ARG LEU PRO GLY HIS THR GLN 663 GM: Kc CTG GTA GC!%G MG AGC AGT fi~c6'kG ccc GAG CCL GArf'dC GTC ATC GTG GC~2&C TGC MC CAG TT%T GTC TTG GAT GTT ASP THR LEU VAL ALA GLN LYS SER $07 VAL RET PRO GLU PRO GLU HIS VAL ILE VAL ALA CYS CYS ASN GLN PHE PHE VAL LEU ASP VAL 733 770 783 GTC ATT MT TTC CGC CGT CTC AGT GAG GGG GA1 CTG TTC ACT CAf?TG AGA MG ATA GT&A ATG GCT TCC AAt3&G GAT GAA CGt T-rG VAL ILE ASH PHE ARG ARG LEU SLR GLU GLY ASP LEU PHt THR GLN LEU ARG LVS ILE VAL ARG NET ALA Sr" ASN GLU ASP GLU Affi LEU 843 CC1 CCA ATC GGC CT?!TG ACG TCA WC GGi':GG AGC GAG TGG GC%G CCC AGG ACG Gf':TC GTG AM GAC d2:CC MT CGG GAC TCT PRO PRO ILE GLY LEU LEU TtiR SER ASP GLV ARG SER GLU TRP ALA GLU ALA ARC THR VAL LEU VAL LYS ASP SER THR ASN ARG ASP SER

930 965 CTG UT ATG ATC GIG CGG TGC ATC TGC CTG 676 TGC CTG GAT GCECT ffiA GGC ATG GA:':TC AGC GAC ACC ~~1~~ GCG UC CAG CTT LEU ASP NET ILE GLU ARG CYS ILL CYS LEU VAL CYS LEU ASP ALA PRO GLY GLV KT GLU LEU SER ASP THR ASN AR% ALA LEU GLN LEU

933

1100 1083 1035 1070 1040 CT1 UC GGC '%A GGC TGC AGC MC MT ffiA 'XC MC CGC TW TAC GAC MC TCC CTA CAG TTT GTG GTG ffiC CGA CAT GGC ACC TGC GGC LEU HIS GLY GLY GLV CYS SER LYS ASN GLY ALA ASN ARC TRP TVR ASP LYS SER 1EU GLN PHE VAL VAL GLY ARG ASP GLY TIR CYS GLV

1023

1175 1145 llb0 1190 1130 1113 GTG GTG TGC GM CAC TCC CCT TTT GAT GGC ATT GTC CTG GTG tAG TGC ACG GAG CAT CTG CTC AM CAC ATG GTG AAG AGC AGC AAG MG VAL VAL CYS GLU HIS SER PRO PHE ASP GLY ILt VAL LLU VAL GLN CYS THR GlU HIS LEU LEU LYS HIS NF.T VAL LYS SER SER LYS LVS 1263 1225 12% 1233 1220 1203 ATG GTC CGA GCT GAC TCG GTC Au: GAG CTC CCA GCA CCC AGA AGG CTG AGG TGG MG 167 TCC CCG GAA ATC CAA GGC CTC TTA GCT Tee NET VAL ARC ALA ASP 5.. VAL SER GLU LLU PRO ALA PRO ARG ARC CEU ARG TRP LYS CYS SER PRO GLU ILE GLN GLY LEU LEU ALA SER 1340 1323 1335 1370 1310 1293 TCG GCA GM AAA CTC CM CM ATA GTC MG MT CTT wu: TTC ACT GTT TAT MA TTT GAC GAC TAT GGG MG ACT TTC ATT AAG CAG CAG SER ALA GLU LYS LEU GLN GLN ILE VAL LYS ASN LEU ASP PHE THR VAL TYR LYS PHE ASP ASP TYR GLY LYS THR PHE ILE LYS GLN GLN 1415 1443 1430 1400 1460 1385 MA TGC AGC CCC GAT G&C TTT ATT CAG GTG GCC CTC CAG CTA GCC TTC TAC AGG CTC CAT GGG AGA CTC GTG CC1 ACC TAT GAG AGc GCG LYS CYS SER PRO ASP ALA PHE ILE GLN VA1 ALA LEU GLN LEU ALA PHE TYR ARC CEU HIS GLY ARC LEU VA1 PRO THR TYR GLu SER AU 1520 1490 1305 1333 1473 1350 TCC ATC CGC CGA TTC CAC GAG GGA CGG GTG GAC MC ATT CGA KG GCC ACT CCG GAG GCA CTG CAT 171 GTG AAA GCC ATT ACT GAC CAT SER ILL ARG AC PHE HIS GLU GLV ARG VAL ASP ASN ILE ARG SLR ALA 1:" PRO GLU ALA LEU HIS PHE VAL LYS ALA ILE THR ASP HIS 1610 1380 1395 1623 1363 1640 GCA TCT GCC ATG CCG GA1 KG GAG AAG CTG CTG CTC CTG AAG GAT GCC ATC CGA GCC CAG ACC CAG TAC ACA GTC ATG GCC ATC ACG GGG ALA SfR ALA MET PRO ASP SER GLU LYS LEU LEU LEU LEU LYS ASP ALA ILE ARG ALA GLN T;R GLN TYR THR VAL MET ALA ILE THR GLY 1633 ATG GCC AK MET ALA ILE

1670 1683 1700 1715 1730 GAC AAC CAC CTG CTG GGG CTG CGG GM CTG GCC CGA GAA GTG TGC AAG GM CTG CC1 GAG ATG TTC ACG GAT W ACA TAC ASP ASN HIS LEU LEU GLY LEU ARG GLU LfU ALA ARG GLU VAL CYS LYS GLU LEU PRO GLu NET PHE THR ASP GLU THR TYR

1760 1775 1790 1743 1803 1820 CTG ATG AGC MC CGC TTT GTC CTC TCC ACC AGC CAG GTG CCC ACC KC ATG GAG ATG TTT TGC TGC TAT GGT CCT GTG GTA Ccc MT &G LEU NET SER ASN ARG PHE VA1 LEU SER THR SER GLN VAL PRO THR THR MET GLU MET PHE CYS CYS TYR GLV PRO VAL VAL PRO ASN'GLY 1830 1865 1880 1893 1833 1910 TAC CGT GCC TGC TAC AK CCC CAG CCA GAG AGE ATC CTT TTC TGC ATC TCC AGC TTT CAC GGC TGC AAA GM ACE TCT TCA ACC AA6 TTT TYR GLY ALA CYS TYR ASN PRO GLN PRO GLU SER ILE LEU PHE CYS ICE SER SER PHE HIS GLY CYS LYS GLU THR SER SER THR LYS PHE 1940 1935 1970 1923 1985 2oM) GCA MA GCT GTG GAA GM AU: TTT ATT GM ATG AM GGT CTC TGC AGT CTG TCC CAG TCT GGC ATG GGC MG CCC CTG GCA ACA MG GM ALA LYS ALA VAL GLU GLU SER PHE IlE GLU MET LYS GLY LEl! CYS SER LEU SER GLN SER GLV MET GLY LYS PRO LEU ALA THR LYS GLU 2013 2030 2045 2060 2075 2090 2103 AM GTA ACA AGG CCT AGC CAG GTA CAC CAA CCT TGACTGCTGCCGCTCAGTTTCGCCTCCCCAAACCU\GCACTCTG~GCTGC~GACCCT~TGMCCCCTGCTCT LYS VAL THR ARG PRO SER GLN VAL HIS GLN PRO 640

2120

152

BERRARD.

BRICE AND MALLET

Cell lines which express high levels of ChAT can now be obtained through transfection experiments, thereby providing model systems which will greatly facilitate the biochemistry and pharmacology of this enzyme. This approach will also allow the identification of the elements necessary to produce cells which express and release acetylcholine. Ultimately, these cells may be grafted in the CNS and may prove efficient in compensating cholinergic deficit. Finally, the analysis of the ChAT gene and its expression, in conjunction with that encoding TH, might reveal important clues concerning the molecular basis of the phenotypic plasticity of cholinergic versus catecholaminergic neurons (22,30) and more generally to the question of plasticity of neurotransmitter expression in the central nervous system. 192

128

64

256

384

320

amino

446

512

640

576

ACKNOWLEDGEMENTS

acids

The authors are grateful to Yves-Alain Barde, Axe1 Braun and Friedrich Lottspeich for providing the N-terminal sequence of porcine brain ChAT and to Julian Smith for helpful discussion. We also thank C. BrCant, J. Clot, D. GrCgoire, G. Peudevin and D. Samolyk for technical assistance; S. Vyas for critical reading of the manuscript; J. P.

FIG. 4. Hydropathy profile of the predicted amino acid sequence of porcine ChAT. Hydropathy values for a span of seven amino acid residues were calculatedaccording to Kyte and Doolittle(20).

Bouillot for photographs. This work was supported by grants from Institut de la Recherche sur la Moelle Epinitre, CNRS, INSERM, Rhane-Poulenc Sante, Fondation pour la Recherche MCdicale, Ministizre de la Recherche et de I’Enseignement Suptrieur. S. Berrard received fellowships from the Association Claude Bernard. A. Brice is a fellow of the Fondation pour la Recherche Medicale and the Fonds d’Etude du Corps MCdical des Hopitaux de Paris.

ChAT mRNAs, which will facilitate the isolation of the corresponding gene in these two species. These probes will be more appropriate to analyse the regulation of the expression of the ChAT gene under various physiological conditions as well as in pathological situations such as in Alzheimer’s disease and amyotrophic lateral sclerosis, for which there are no animal models.

IPDPKGANVASNEASTSAAGSGPESAALFSKLRSFSIGSGPNSPQRVVSNLRGFLTHRLSNITPSDTGWKDSI

FGAPGGLGETLQQKLLERQEQTANwvsEYwLNDMYLNNRLALPvNSSPAvIF~RQHFQDT120

PILEKTPPKMAAKSPSSEEEPGLPKLPVPPLQQTLATYLRCMQHLVPEEQFRRSQAIV

: I

:111:111:1::1:1

I

I:1

:: :

I

I:

73

::

I:11

I:1

I I II::

:11:11

I

I::

II

LsIpKKwLSTAESVDEFGFPDTLPKVPVPALDETMADyIRALEPITTPAQLER~K~LIRQFSAPQGIGARLHQYLLDKREARITGPITTGSTRCTWIFAFPLPINSNPGIGv~AAsLQDR

193

NDQLRFAANLISGVLSYKALLDSHSIPIDCAKGQLSGQPLCMKQYYGLFSSYRLPGHTQDTLVAQKSSVMP-EPEHVIVACCNQFFVLDVVlNFRR---LSEGDLFTQLRKIVRMASNED

236

PRRAHFAARLLDGIL~HREMLD~GEL~LF~L-AEKNQ~LC~Q~YRLLG~CRR~G~KQD~QFL~~RERLNDEDRH~~~ICANQM--YK~~LQA~DRGKL~E~EIA~QILYVLSDAPCLP

310

:I11

I: I:11

: :I11

:I:

I

11111

Ill

I :I I II

:

II:

I

II:1

I II

II::

111:::

:I:

::

I:

ERLPPIGLLTSDGRSE~AEART~L~KD~TNRD~LD~IERCICLVCLDAPGGMELS------------------DTNRALQLLHGGGC~KNGANR~~DK~LQF~~GRDGTCG~~C~H~~FD

:

1:1111:::11

II

I :I

I

I

I::11

::I11

I : :

:I1

I :::I111

1::111:11::1

::

Ill

I:

Ill

:

AK~~~~GLLTAE~R~T~ARDREMLQEDERNQRNLELIETAQVVLCLDEPLAGNFNARGFTGATPTVHRAGDRDETNMAHEMIHGGG~EYN~GNRWFDKTMQLIICTDGTWGLCYEH~C~E

GIVLVQCTEHLLKHMVKSSKKMVRADSV---SELPAPRRLRWKCSPEIQGLLASSAEKLQQIVKNLDFTVYAFDDYGKTFIKQQKCspDAFIQvALQLAFYRL~GRL-------vp~YEs

II :I1

II :: I

II I: :I::1

II:1

I

I ::

::

: :I11

II :: 1111111

Ill

:11:1

:

:

I,

I

GIAVVQ----LLEKIYKKIEEHPDEDNGLPQHHLPPPERLEWHVGPQLQLRFAQASKSVDKCIDDLDFYVYRYQSYGKTFIKSCQVSPDVYIQLATATGsLQvvRTsGGHLRKCv~sTIs

ASIRRFHEGRVDNIRiATPEALHFVKAITDHASAMPDSEKLLL~KDAIR--------------AQTQYTVMAIT~MAIDN~LL-------GLRELAR~VC~E~P~MFTDETYLMS~RF~L

I

II I::

I

I

II

I:1

II:

I

*II

II

III

I,

ARPRRLHQSGQHGGI~V~QAMCQGEGANVPLESDREDEEESRK~KFSIYSKDHLRELFRCAVARQTEVMVRISWAMASTS~CWPARGQYRGHRRDARAVQ~R~L~QC-------SQCNLL

,I

STSQVPTTMEMFCCYGPVVPNGYGACYNPQPESILFCISSFHGCKETSSTKFAKAVEESFIEMKGLCSLSQSGMGKPLATKEKVTRP~QVHQP

Ill

I: : : I Ill1

I Ill

Ill

II 1:11:1:1

:I :11::::11::::1

I: I

~T~G~AC~TD~FMG~G~~T~RGYGC~Y~~H~EQI~FC~~AFY~CEDT~A~RYAK~LQD~LDI~~RDLLQN

FIG. 5. Comparison of porcine (top line) and Drosophila rn. (18) (bottom line) ChAT amino acid sequences. Vertical bars and discontinuous represent identical residues and homologous amino acids (9) respectively. Amino acid position is indicated on the right side.

338 430

448 546

547 659

640 728

traits

MAMMALIAN

CHOLINE ACETYLTRANSFERASE

cDNA REFERENCES

1. Benishin, C. G.; Carroll, P. T. Multiple forms of choline-o-acetyltransferase in mouse and rat brain: Solubilization and characterization. J. Neurochem. 41:1030-1039; 1983. 2. Berrard, S.; Faucon Biguet, N.; Gregoire, D.; Blanot, F.; Smith, J.; Mallet, J. Synthesis of catalytically active choline acetyhransferase in Xenopus oocytes injected with messenger RNA from rat central nervous system. Neurosci. Lett. 72:93-98; 1986. 3. Berrard, S.; Brice, A.; Lottspeich, F.; Braun, A.; Barde, Y-A.; Mallet, J. cDNA cloning and complete sequence of porcine choline acetyltransferase: In vitro translation of the corresponding RNA yields an active protein. Proc. Nat]. Acad. Sci. USA 84:92809284; 1987. 4. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254; 1976. 5. Braun, A.; Barde, Y-A.; Lottspeich, F.; Mewes, W.; Thoenen, H. N-terminal sequence of pig brain choline acetyltransferase purified by a rapid procedure. J. Neurochem. 48:16-21; 1987. 6. Bruce, G.; Wainer, B. H.; Hersch, L. B. Immunoaffinity purification of human choline acetyltransferase: comparison of the brain and placental enzymes. J. Neurochem. 45:61 l-620; 1985. I. Cohen, P. The role of protein phosphorylation in the hormonal control of enzyme activity. Eur. J. Biochem. 151:439-448; 1985. 8. Cross, G. A. M. Eukaryotic protein modification and membrane attachment via phosphatidylinositol. Cell 48:179-181; 1987. 9. Dayhoff, M. 0.; Schwartz, R. M.; Orcutt, B. C. In: Dayhoff, M. O., ed. Atlas of protein sequence and structure. vol. 5, suppl. 3. Washington, DC: National Biomedical Research Foundation; 1978:345-352. 10. Deininger, P. L. Random subcloning of sonicated DNA: Apphcation to shotgun DNA sequence analysis. Anal. Biochem. 129:216223; 1983. 11. Eckenstein, F.; Barde, Y-A.; Thoenen, H. Production of specific antibodies to choline acetyltransferase purified from pig brain. Neuroscience 6:993-1000; 1981. 12. Eckenstein, F.; Thoenen, H. Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterisation and use for identification of cholinergic neurons. EMBO J. 1:363-368; 1982. 13. Eder-Colli, L.; Amato, S. Membrane-bound choline acetyltransferase in Torpedo electric organ: a marker for synaptosomal plasma membranes? Neuroscience 15:577-589; 1985. 14. Fonnum, F. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24:407-409; 1975. 15. Garnier, J.; Osguthorpe, D. J.; Robson, B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120:97-120; 1984. 16. Grima, B.; Lamouroux, A.; Boni, C.; Julien, J. F.; Javoy-Agid, F.; Mallet, J. A single human gene encoding multiple tyrosine hydroxylases with different predicted functional characteristics. Nature 326:707-711; 1987. 17. Gurdon, J. B.; Lane, C. D.; Woodland, H. R.; Marbaix, G. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:177-182; 1971. 18, Itoh, N.; Slemmon, J. R.; Hawke, D. H.; Williamson, R.; Morita, E.; Itakura, K.; Roberts, E.; Shively, J. E.; Crawford, G. D.; Salvaterra, P. M. Cloning of Drosophila choline acetyltransferase cDNA. Proc. Natl. Acad. Sci. USA 83:4081-4085; 1986. 19. Krieg, P. A.; Melton, D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12:7057-7070; 1984.

20. Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132; 1982. 21. Le Bourdelles, B.; Boularand, S.; Boni, C.; Horellou, P.; Dumas, S.; Grima, B.; Mallet, J. Analysis of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms. J. Neurochem. 50:988-991; 1988. 22. Le Douarin, N. M. The ontogeny of the neural crest in avian embryo chimaeras. Nature 286:663-669; 1980. 23. Lomedico, P. T.; Saunders, G. F. Preparation of pancreatic mRNA: cell-free translation of an insulin-immunoreactive polypeptide. Nucleic Acids Res. 3:381-391; 1976. 24. McGeer, P. L.; McGeer, E. G.; Peng, J. H. Minireview. Choline acetyhransferase: purification and immunohistochemical localization. Life Sci. 34:2319-2338; 1984. 25. Malthe-Sorenssen, D. Choline acetyltransferase-evidence for acetyl transfer by a histidine residue. J. Neurochem. 27:873-881; 1976. 26. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1982. 27. Martin, F. H.; Castro, M. M. Base pairing involving deoxyinosine: implications for probe design. Nucleic Acids Res. 13:8927-8938; 1985. 28. Melton, D. A.; Krieg, P. A.; Rebagliati, M. R.; Maniatis, T.; Zinn, K.; Green, M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes for plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:70357056; 1984. 29. Padgett, R. A.; Grabowski, P. J.; Konarska, M. M.; Seiler, S.; Sharp, P. A. Splicing of messenger RNA precursors. Annu. Rev. Biochem. 55:1119-1150; 1986. 30. Patterson, P. H. Environmental determination of autonomic neurotransmitter functions. Annu. Rev. Neurosci. l:l-17; 1978. 31. Pearson, R. B.; Woodgett, J. R.; Cohen, P.; Kemp, B. E. Substrate specificity of a multifunctional calmodulin-dependent protein kinase. J. Biol. Chem. 260:14471-14476; 1985. 32. Pelham, H. R. B.; Jackson, R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67: 247-256; 1976. 33. Rossier, J. Choline acetyltransferase: a review with special reference to its cellular and subcellular localization. Int. Rev. Neurobiol. 20:283-337; 1917. 34. Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chain-terminating inhibitors,. Proc. Natl. Acad. Sci. USA 74:54635467; 1977. 35. Sefton, B. M.; Buss, J. E. The covalent modification of eukaryotic proteins with lipid. J. Cell Biol. 104:1449-1453; 1987. 36. Smith, J.; Fauquet, M.; Ziller, C.; Le Douarin, N. M. Acetylcholine synthesis by mesencephalic neural crest in the process of migration in vivo. Nature 282:853-855; 1979. 37. Tachikawa, E.; Tank, A. W.; Yanagihara, N.; Mosimann, W.; Weiner, N. Phosphorylation of tyrosine hydroxylase on at least three sites in rat pheochromocytoma PC12 cells treated with 56 mM K+: determination of the sites on tyrosine hydroxylase phosphorylated by cyclic AMP-dependent and calcium/calmodulin dependent protein kinases. Mol. Pharmacol. 30:476-485; 1986. 38. White, H. L.; Cavallito, C. J. Choline acetyltransferase. Enzyme mechanism and mode of inhibition by a styrylpyridine analogue. Biochem. Biophys. Acta 206:343-358; 1970.