255
Ann. Hum.Genet. (1992), 56, 25S265 Printed in Great Britain
Nucleotide sequence and chromosomal assignment of a cDNA encoding the large isoform of human glutamate decarboxylase C. D. KELLY1, Y. EDWARDS3, A. P. JOHNSTONE2, E. HARFST2, A. NOGRAD14, S. S. NUSSEY', S. POVEY3 AND N. D. CARTER' Departments of Child Health and Cellular and Molecular Sciences, St George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 ORE MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NW12HE Department of Anatomy, University College London, Gower Street, London WClE 6BT
SUMMARY
Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter y-aminobutyric acid (GABA). Two forms of human GAD, GAD,, and GAD,,, are encoded by two separate genes. A full length human GAD,, cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD,, gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD,, is not syntenic with the smaller GAD isoform GAD,, which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD,, enabled immunocytological detection of GAD in the rat brain.
INTRODUCTION
The enzyme glutamic acid decarboxylase (GAD ; glutamate decarboxylase L-glutamate-Lcarboxy-lyase, EC 4.11 .15) catalyses the rate limiting step in the synthesis of the inhibitory neurotransmitter GABA from L-glutamic acid. There are two forms of enzymically active human GAD which differ in molecular size, aminoacid sequence, cellular and subcellular location, and interaction with the GAD co-factor pyridoxal phosphate (Erlander & Tobin, 1991). The protein structures are sufficiently similar to allow recognition by the same polyclonal antibody (Katarova et al. 1990) but isoform specific antibodies have also been prepared (Chang & Gottlieb, 1988). The proteins GAD,,, molecular size 64 kDa, and GAD,,, molecular size 67 kDa, are encoded by two separate genes (Erlander et al. 1991). Both are selectively expressed in GABAergic neurons and are primarily concentrated in the nerve terminals (Reetz et al. 1991), although there are differences in their intracellular distribution. Outside the brain, GAD has limited distribution, but is expressed in the ovary and oviduct (Erdo et al. 1989), in spermatozoa (Persson et al. 1990), and at high levels in the islets of the pancreas, specifically in the /3 cells (Okada et al. 1976). Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease for which there is a strong genetic disposition. Autoantibodies are directed against antigenic determinants on the human pancreatic p cells. Antibodies against a 64 kDa polypeptide, now known to be
256
C. D. KELLYAND
OTHERS
GAD (Baekkeskov et al. 1990), have been found in the sera of newly diagnosed patients with IDDM as well as in pre-diabetic individuals several years before the onset of the disease (Baekkeskov et al. 1987). Stiff-man syndrome (SMS) is a rare neurological disorder believed to be due to interference in neuronal pathways involving the neurotransmitter GABA. While the diagnosis of this rare syndrome is often difficult, it appears that most patients have autoantibodies to GABA-secreting neurones and GAD has been determined to be the predominant autoantigen (Solimena et al. 1990). A striking association has been found in these patients between the presence of GAD antibodies and organ-specific autoimmune disease especially IDDM (Solimena et al. 1990). The presence of GAD autoantibodies is the earliest known predictor of IDDM and can also be used as a marker in diagnosis of SMS. In order to examine the role played by autoimmunity to GAD in these two diverse clinical conditions IDDM and SMS, it is important to characterize each isoform structurally and genetically. The cDNA encoding the larger isoform GAD,, has been cloned from the rat (Julien ef al. 1987; Wyborski et al. 1990), mouse (Huang et al. 1990), cat (Kobayashi et al. 1987), and fruit fly (Jackson et al. 1990). Partial cDNA nucleotide sequence derived from cloned human GAL),, is also known (Cram etal. 1991; Michelson et al. 1991; Persson Pt al. 1990). These GAL),, sequences are highly conserved between species such that all amino acid-sequences demonstrate 96% homology. The gene for the smaller human GAD isoform, GAD,, has recently been cloned and sequenced (Karlsen et al. 1991). Comparison of partial amino-acid sequence between the two human isoforms showed less than 65% homology. The GAD,, gene has been firmly allocated to the short arm of chromosome 10 (Karlsen et al. 1991) and it has been proposed, but not confirmed, that the gene for human GAD,, is localized on chromosome 2 (Karlsen et ul. 1991).
We report here the full nucleotide sequence for human GAD,, cDNA and confirm its localization to chromosome 2. Production of polyclonal antibodies to one expressed cDNA clone and its application to immunocytochemical localization of GAD is described.
METHODS AND MATERIALS
Isolation and analysis of human GAD cDNA clones A human frontal cortex cDNA library in AZAP I1 (Stratagene) was screened using a feline GAD cDNA probe (a kind gift from Dr A. J. Tobin, UCLA, California) which was labelled with [32P]dCTPusing the random hexanucleotide primer method (Feinberg & Vogelstein, 1984). The filters were hybridized a t 65 "C in 4 x SSC; 10 x Denhardt's :0*1% SDS; 100 ,ug/ml denatured salmon testes DNA and were subsequently washed to a final stringency of 1 x SSC/Ol O h SDS a t room temperature. Positive recombinant phage were purified to homogeneity and amplified. The pBluescript phagemid containing the cDNA insert was excised from the AZAP vector using R408 helper phage and XLI-Blue host bacteria according to the Stratagene protocol. Phagemid DNA was alkali denatured (Maniatis et al. 1989) and used directly for sequencing using the dideoxynucleotide sequencing method (Sanger et al. 1977), and the Sequenase 2.0 kit (United States Biochemical).
Sequence and assignment of GAD
257
Chromosomal assignment :analysis of somatic cell hybrid DNA by PCR The polymerase chain reaction (PCR) method was used to amplify specifically human GAD,, sequences in DNA extracted from a panel of somatic cell hybrids. The nine hybrids used are listed in Table 1, the human chromosome complement of each hybrid has been assessed by a combination of isozyme and DNA analysis and, in most cases, by karyotyping. Two 25mer oligonucleotide primers : CCT TAG GTT TCA GCT AAG CGA GTC A (reverse) and
CCG CTC CAA GAG AAT TCA CTT TAC C (forward),
corresponding to positions 2985-2961 and 2257-2282, respectively, in the human GAD,, cDNA scyuence and spanning 703 bp of the 3' untranslated region were synthesized. These primer sequences within the 3' noncoding region were chosen as they demonstrated high specificity for the human GAD,, riucleotide sequence (Fig. 2). DNA was prepared from somatic cell hybrids and their parental cell lines as described previously (Edwards et al. 1989). PCR was carried out using 1 pg of parent DNA and 3 pg of hybrid DNA in a total reaction volume of 100 pl. After initial incubation a t 95 "C for 5 min, 2 4 units of Taq polymerase were added to each tube. After 30 cycles of amplification, each consisting of 30 s a t 93 O C , 30 s a t 59 "C and 30 s a t 72 O C , a 10 pl aliquot was rcmoved and analysed on a 1 % agarose gel. DNA products were visualized with ethidium bromide under a U.V.light.
Expression of CDNAclones in baculoviral system and production of GAD antibody Insert DNA from one of the cDNA clones, clone 3blb, isolated from the human frontal cortex library, was introduced into the baculovirus transfer vector pAc373 using BamHI and KpnI sites within Blucscript. This was designed to allow translation to initiate a t the internal ATG site (base 1006), producing a polypeptide corresponding to the C-terminal half of GAD,,. The product from infcctcd SF9 cells was purified by SDS-PAGE and used to immunize rabbits.
Immunohistochemistry and in situ hybridization of GAD on rat brain sections Frozen sections from rat brain and cerebellum were cut (15 pm thickness) and prefixed using 4 YOparaformaldehyde prior to immunocytochemistry and in situ hybridization with cDNA probes. In situ hybridization was carried out using a protocol described by Kato (1990) and the feline GAD,, cDNA clone. Immunohistochemistry was carried out using a 1 :250 dilution of the antibody, incubated with the sections for 3 h at room temperature. Visualization of bound antibody was achieved using a biotinylated goat anti-rabbit antibody and avidin-peroxidase kit. Protocol and reagents were obtained from Vector Laboratories, UK. Controls were carried out using normal rabbit serum. RESULTS
Human GAD cDNA sequence determination Screening of the human frontal cortex cDNA library with feline GAD cDNA as probe resulted in the isolation of 4 cDNA clones (Fig. 1) for human GAD,,: HG2ab (insert length 2867 bp),
C. D. KELLYAND
258
ECOP
OTHERS Hlndlll Hlndlll
EcoRl EcoRl
HG2ab
.
HG23cla
HG12a
Scale I
500bp
‘
Fig. 1 . Restriction map of human GAD,, cDNA and sequencing strategy applied to analyse the isolated cDNA clones.
HG3blb (2251 bp), HG23cla (2768 bp) and HG12a (1034 bp). Sequence analysis demonstrated that the total length of human GAD,, cDNA was 3218 bp, with the initiation codon a t 184 bp from the 5’ end, a termination codon, TAA, a t 1963 bp and 1253 bp of 3’noncoding sequence containing a poly adenylation signal sequence and a poly A tail, A,, (Fig. 2). The open reading frame of 1779 nucleotides encodes a polypeptide of 593 amino-acid residues. Comparison of the coding region of this human GAD cDNA with equivalent regions of feline (Kobayashi et al. 1987) and rat (Wyborski et al. 1990) GAD demonstrates 93 and 91 YOhomology respectively a t the nucleotide level, and 98 and 97% homology respectively a t the amino level (Fig. 3). Homology between human and rat sequences was also found to extend into the 5’ noncoding, 71 YOhomology, and 3’ noncoding, 80% homology, regions. Some areas of the 3’ noncoding region show a similar degree of homology between the two species to that of the coding region. The sequence which encodes the binding site for the GAD co-factor pyridoxal phosphate (Am-Pro-His-Lys) is located a t 1383 bp to 1395 bp. This site is highly conserved between the different GAD molecules of various mammalian and non-mammalian species (Jackson et al. 1990) and is also found to be common amongst other decarboxylases, e.g. pig DOPA decarboxylase (Bossa et ul. 1977),Drosophila DOPA decarboxylase (Eveleth et al. 1986), and is similar to that of plant (Cutaranthus roseus) tryptophan decarboxylase (deLuca et al. 1989), suggesting a common ancestral gene.
Chromosomal location The results of PCR amplification of DNA from somatic cell hybrids using the two primers synthesized from the 3’ noncoding human GAD,, cDNA are shown in Fig. 4 and summarized
Sequence and assignment of GAD
259
T t C G C A G C A G C C T C C A G T A C G C C O C G C A G A l T A C G C C ' I G T C A ~ C C G A G C C G M C G A T C ~ ~ ~ T G ~87A G ~ ~
CCGCAGTGCCCOCCTCCCTCTCCCAOACCCGMCC'IG~TGCGCCGGACCMTCGAGACTCTGOACMTAGA~CCC~ACG 174 ACCGAGC~CGT~~GACCCATITGTCCGCMCCTCA
261
METAlaSerSerThrHisLeuSerAlaThrSerSerAsnAlaGluProAspProAsnThrThrAsnLergProThr A C G T A C G A T A C C T C C T C C O C C G ~ C C C A C O C A T G C A C C A G ~ C T C C T C M G A T C T G C G C ~ ~ W G A C C M C348 m
ThrTyrAspThrTrpCysGlyVa1AlaHisGlyCysThrArgLysLeuGlyLeuLysI1eCyeGlyPheLeuGlnArgThr~nSer C T C G A G A G A A G A G T C G C C T T G A G T C C C T T C M O C A G A O C C M T t C ~ C M G M C C T G C ~ C T G T G A A A A C A G C G A C C ~ A T435
LeuGluGluLysSerArgLeuVa1SerAlaPheLysGluArgGlnSerSerLysAsnLeuLeuSerCysGluAsnSerAspArgAsp G C C C G C T T C C O C C G C A C A G A G A C T C A C T T C T C T M T C T G T T l C C T A G A G A ~ m ~ C G C T M G M C G T G ~ A ~ W C C G T522 G AlaArgPheArgArgThrGluThrAspPheSerAsnLeuPheAl~g~pLeuLeuProAlaLysAenOlyOluOluGluGlnThrVal C
M
T
T
C
C
T
C
C
T
C
C
A
A
C
A
T
A
C
T
C
C
T
t
M
C
T
A
T
509
GlnPheLeuLeuGluValValAspIleLeuLeuAsnTyrValArgLysThrPh~spArgSerThrLy~ValLeuAspPheHi~His C C A C A C C A G T C T C G M G C A T G O A ~ C ' I T C M C ~ A G C T C ~ ' I G A C C A C C C C G M T C ~ T G O M C ~ A T C C T G O T ' K A696 Cm
ProH~sGlnLeuLeuGluGlyMETCluGlyPheAsnLeuGluLeuSerAspHisProOluSerLeuGluGlnIleLeuValAspCy~ AGAGACACCTAAGTATGTTCGCACAGGTCATCCTCGATFITTCMCCAGCTCTCCACTGOATlGGATAlTA~CTAGCT
703
ArgAspThrLeuLysTyrGlyValArgThrGlyHisProArgPhePheAsnGlnLeuSerThrGlyLeuAspIleIleClyLeuAla G C A G M T C G C M M C T C M C G C C A A T A C C M C A T G ~ A C A T A T G A A A ~ C A C C A G T G T T I C T C C T C A T G O M C W T M C A C l T 870
GlyGluTrpLeuAsnSerThrAlaAsnThrAsnKETPheThrTyrGluI1eAlaProValPhoValLeuMETCluGlnIl~ThrLeu M G A A G A T G A G A G A G A T A G T T C C A n ; C T C A A G T A A A G A T C G T G A T A T A ~ ~ C ~ C ~ C A T A T C C M C A T G T A C957 A~
LysLysMETArgGluIleValGlyTrpSerSerLysAspGlyAspGlyIlePheSerProOlyGlyAlaIleSerAsnMETTyrSer ATCATCCCTCCTCGCTACMGTACTTCCCCOCMGTTCMGACAAA~CATGOC~C'IGTGCCTWCTGOTCCTC~ACCTC~M1044 I1eMETAlaAlaArgTyrLysTyrPheProOluValLysThrLysGlyMETAlaAlaVa1ProLysLeuValLeuPheThrSerOlu C A G A G T C A C T A T T C C A T A A A G A A A G C T G G G G C T G C A C T T G G C T T T G G A A C T G A C M T G T G A T T T I G A T W G m M ' I G ~ ~ 11 31
ClnSerHisTyrSerIleLysLysAlaGlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCysAEnG~~gG~y A A A A T A A T T C C A G C T G A T A G G C W l T C T A A G C C A A A C A G A A G G G A T A T G T T C C C M A T G T C M ~ M C m W 1218 LysIleIleProAlaAspPh~luAlaLysIleLeuGluAl~ysGlnLysGlyTyrValProPheTyrValAs~laThrA~aG~y 1305 ACGACTG~ATCCAGCTATCCGATACMGAGA~CAGATATATG'IGAGWTATMCC~~ATG~GA m~C
ThrThrValTyrGlyAlaPheAspProIlaGlnGluIleAlaAspIleCysGluLysTyrAsnLeuTrpLeuHisValAspA~aA~a 1392 ~AGTCTCCTCATCTCCACGAAGCACCGCCATAAACTCMCGCATAGW~CCMCTCMTCACC~MCCCTCAC TrpGlyGlyGlyLauLeuMETSerArgL~sHisArgHisLysLeuAsnGlyIleCluArgAlaAsnSerValThr~~snProHi~ 1479 MGATGATCGTCCTGTCAGTCC~TGCCATTCTCGTCMOC~~TATACTCCMGA'IGCMCCAGATG'IG~A~ ~ T M E T G l y V a l L e u L e u G l n C y s S e r A l a I1eLeuValLysGluLysGlyI 1eLeuGlnGlyCysAsnGlnMETCysAlaGlY TACCTCTTCCAGCCAGACMGCAGTATGATGTCTCCTACGACACCCOOOACMGCMTTCAGTGWCGCCACGTGOATA~
1566
TyrLeuPheClnProAspLysGlnTyrAspValSerTyrAspThrGlyAspLysAlaIleOlnCysGlyArgHisValAspI~ePh~ 5 3T A A G T T C T C G C T C A ' I G T C G A C A A A ~ C A C A G ~ A T T l C ~ C C A G A ~ M C W ' I G C C T G O A ~ T G O C ~ M T A C C T C1T6 A
Ly~PheTrpLeuMETTrpLysAlaLysGlyThrValGlyPheCluAsnGlnI1eAsnLysCysLeuGluLeuA1aGluTyrLeuTy~ G C C A A G A T T ~ C A G A G M G M ~ A G A T C C ~ M T C C C G A G C C T G A G C A C A C ~ C G T C ' I G ~ T A T A ' I T C C A C M1 7 4 0
AlaLysIleLysAsnArgGluGluPheCl~TValPheAsnGlyGluProOluH~sThrAsnVal~sPhe~pTyrIlePr~~n AGCCTCACOOO'IGTGCCAGACAGCCCTCMCGAC~~GCTACACMG~TCC~~WGCCCTGA'IGATGOMTCA
1827
SerLeuArgGlyValProAspSerProOlnArgArgGluLysLeuHisLysValAlaProLy~IleLysAl~e~~~US~~ GTACGACCATGOTTGGCTACCAGCCCCMCOOOACMGCCMC'ITA'ITCCGGATGOTCATCTCCMCCC~C~TACCCAGTCT
1914
GlyThrThrMETVa1GlyTyrGlnProOlnGlyAspLysAlaAsnLeuPheArgMETValIleSerAsnProA~aA~aTh~~n~er G A C A T P C A C T T C C C T C A T P C A G C A G A T A G A A A G A C T O O G C C A G A ~ ~ A ~ C ~ G C A G M C C A T ~ C ~ A2 0~0M1 ~
AspIleAspPheLeuIleGluGluIleGluArgLeuGlyGlnAspLeu GCC~TCCCTCTCCCACTTCCAGMCAAACCTCTATA'IGTPCC'IGAAACACACAGCCA~ATT~CA~WCATMTATCT'K
2088
AAGATATCTTWCCTTCACTMGCTTITGTTCTAGTTCAGCAGWTAGTG~~~G~ACATTCAGMCAGM
2175
TATATATCTACAG'ITATACATACCTCTCTCTATATATACATGTATAG'IGAGTGTCCC~TAGTMTAGATCAC~ATG~2 2 6 2 ~AGCAGTTACCGAGGAGCTAAACATGCTGCCAACCAGCTTGTCCMCAACTCCAGGWCTG~
2349
T ? T C W C G C C A ' I G T C C T A C O O O C C M ~ ~ T G C T G T T G G T G A G M T C G A C C T C A C ' I G T C ~ G ~ T C C A C ~ M G T G A T2 4G3 6 A T C G A T G A G A l U A C A C A C C M ' I G A C A G T C A C A C C T C C C A T T A G T A ~ T G ~ A C O O O ~ T A G T A G C A G M T C A T ' K l T A C ~ T G2T5A 23
CTATCGCTCTATFITTAGAGA'M'AATTlCTGTAGATTGTGTAAATTCCTGT'KTC'IGACCTlGGTGG~~MACTATGTG 2610
T C A T G A ~ M ' I G A ~ T T C m A A T T C T A G T C M T G W T A ~ C l T A ~ A T A ~ A G A G A T G T A C C A ' I G l T ~ A G G c G2T6 9C7 m G T A T T T T C ' I T C C C A T T P C T M T G T A T C ' I T A ~ A T A T A T G A A G T A A G T T C T G A A A A C T G ~ A T G O T A ~ G l C C A T T I C T G2~7 0 4 C W G A G ~ A ~ T A ~ U A ~ T A G ~ A G A T T ~ C T A ~ A T A ~ T M A G T G C C C ~ T W T M T G A ~ ~ A ~ A C T G T C G ~ T ~ M A2 8 7 1
A T T C T M G A T C G T A C A T A A A G ~ A T A T A T A T G O W T C C T G T T A C l T W T A G C A T C m T C ~ l T A C ~ T C T C T G T C W T2 G 958 TACGTCTCCTGTGCTCAATCC~TCTACCAACTCTTGGATMTMCTAGATCTCCTGTM~TAGTAGT'KATGACCM~TC 3045 ~ A A C A ~ C G A A G A C C T C C T G A A G A T C T C A G A T W G T G A C C A G G ~ T C A C A A 3 1C 32 T G ~ ~ " TGMGMOCGAAATTCACAC'IGTGCG~AGAGTATGCAAGAAGAATAT~~~~TA'ITCTCCATGOAGM~MCA
Fig. 2. Nudeotide sequence and deduced amino acid sequence of human GAD,,. Initiation codon, termination cwdon, polyadenylation signal, pyridoxal phosphate binding site and primers synthesized for chromosomal allocation are boxed.
3219
260
C. D. KELLYAND
OTHERS
59 59 60 119 119 120 179 179 18 0 239 239 240 299 299 300 359 3 59 360 4 19 4 19 420 479 479 480 539 539 54 0
Fig. 3 . Deduced amino-acid sequence of human GAL),, compared with published rat and rat sequrnc'e (Julien et 01. 1987, Wyborski et al. 1990)- a single residue deletion in the human sequence is indicated k>y A . Residues in the cat and rat sequence which are identical to human are indicated by -.
in Table 1. Human DNA was specifically amplified to give the expected 703 bp fragment. X o band of this size was seen in the mouse, rat or hamster parent cell lines. The results show that the presence of the human GAD,, gene in the hybrids correlates with the presence of chromosome 2 . There are at least 2 cases of discordance with every human chromosome except chromosome 2 .
Immunohistochemistry and in situ hybridization The incubation of rat brain sections with polyclonal antibody raised to the fusion protein of human GAD,, cDNA clone, HGSblb, demonstrates the localization of GAD,, protein to neurones in the cerebral cortex (interneurones), in the striatum, in the Purkinje cells (Fig. 5a.) and in many other smaller neurones throughout the brain stem (Fig. 5 b ) . Parallel experiments were carried out using cDNA probes, hybridizing [32P]dCTPand [35S]dCTPlabelled HG3cla cDNA and feline GAD cDNA to adjacent sections and these preliminary experiments indicated a similar distribution for GAD,, mRNA (not shown).
Sequence and assignment of CAD
26 1
(D
u3
W
c cv c1
M
t
a L
t
(D
r
cv
C cd
-E
z
Fig. 4.W R analysis. iising oligonucleotides specific for human CAD6,. of human, rodent and various rodent/hunian hyhritl wll lines. The amplified fragment is 703 bp long. M, markers; A. ladder. DISCUSSION
The human CAI),, cDNA nucleotide sequence given here extends the sequence information previously reported for this isoform. While the cDNA appears t o be full length therc is some discrepancy between the size 3219 bp and an estimation of 3700 bp determined as transcript sizr by Nort hcrn analysis. This may simply reflect t,he inaccuracies associated with sizing mRKA after clcctrophoretic separation or may imply that additional untranslated sequences remain to be isolated. Identification of the transcription/initiation site of GAD,, mRNA or isolation of further GAD,, cDNA clones may throw light on this observation. The GAD,, gene has been assigned to chromosome 10 (Karlsen etal. 1991). In this paper we make a firm assignment for the gene for human GAD,, to chromosome 2 using PCR methods. The GAL),, homologue in the mouse has been mapped to mouse chromosome 2. The area of homology between the human and mouse chromosomes includes genes for interleukin-I a and p, sodium-channel type I1 a-polypeptide, cholinergic receptor, nicotinic a-polypeptide, nebulin and horncobox region 4 arid extends from 2 q l l to 2q37. It seems reasonable to suggest that the human GAL),, gene also lies within this distal region of the long arm of chromosome 2, since other regions of human chromosome 2 are homologous to mouse chromosomes 1, 6 and 12. Comparison of human GAD,, nucleotide sequence with that from other species reveals an extremely high degree of homology indicating remarkable sequence conservation. The
Table 1. Segregation of human glutamic acid decarboxylase 67 (GAD6q i n rodentlhumn hybrids
FGlOE8EP2.2 TWIN19F6 FGlOE8EP2 MOG2E5 FST917 MOG2C2 FG lOE8EP2.6 DUR4.3 TWIN19D12
1 2 2 4
+
5
3 6 0 0
1 1 2 5
2 3 1 3
-
+
+ + +-
- + + + -
0 3 3 3
4
+ + ++
3
+ + +- - + + - +
2
+ + + - +- --
-
1
-
-
-
+
2 3 0 3
0 3 3 2
3 1 0 5
0 3 1 1
0 2 3 4
+ - + - -
-
+
-
-
/
-
10
-
/
9
-
+
8
- + - - + / + + / + - + / + + + + +
-
7
+ + + +
I
6
-
-
+
+ + I +
-
+ + + +
-
1
2 2 4
3 3 2
0
2 1 3
1
1 2 5
1 1 4
2
+ + + + + / -
I - + / + -
14 15
+ - +
0
-
-
+ + + / + +
-
-
-
11 12 13
Human chromosome
+ - - - + - + + + - - - + - + + + + + - - + - +
+
-
/
0 3 3 3
1 2 2 4
3 0 0 6
0 5 3 1
1 3 2 2
+ + + - +
-
-
18 19 20
- + + - +
-
16 17
+ + + + I +
+ + + + + / +
/
+
+ + + + +
X
0 2
5
4
1 I 0
3
4
1 1
2
+ + -
22
21
+- _+ +-+
GAD,, chromosome
-
(Note. All of these hybrids are referenced in Wong et al. (1987)except for the FGlOE8EP hybrids which are described in Purdue et al. (1991). The + and - indicate definite scoring of the presence or absence of a human chromosome; / not tested, fragment of chromosome present or equivocal result.)
0
E 5U
I 5
wM
p
0
Sequence and assignment of GAD
263
Fig. 5. Immunohistochemical localization of GAD,,, in rat brain sections. (a)Specific staining in Purkinje cells (arrowed)(magnificationx 250). ( b ) Specific staining of GABAergic neurones (arrowed) in the nucleus of the spinal tract of the trigeminal nerve (N).Also shown are the parts of the medulla oblongata (MO) and the spinal tract of the trigeminal nerve (TN) (magnification x 120).
conservation of the GAD co-factor pyridoxal phosphate binding site was expected but the extensive homology exhibited across all regions of the GAD cDNA sequence suggests the existence of intense selective pressure during phylogeny. There is also a moderate degree of structural homology between the human GAD,, and GAD,, sequences (Karlsen et al. 1991), and it has been shown that the DrosophiZa GAD sequence resembles both forms at different positions (Jackson etal. 1990), suggesting a common parental gene for the two isoforms. Gene duplication, followed by chromosomal translocation and sequence divergence seems a likely evolutionary history for the two human GAD genes. In this paper, we demonstrate localized expression of GAD,, protein in rat brain Purkinje cells (Fig. 5 a ) and in other GABAergic cell types (Fig. 5 b ) . This confirms an earlier study which also demonstrated GAD,, in rat Purkinje cells (Lindefors et al. 1989). The presence of GAD autoantibodies is the earliest predictor of IDDM and is also diagnostic of SMS. The isolation of cDNAs for both human GAD isoforms will surely facilitate the production of pure GAD antigen for the routine detection of GAD autoantibodies.
18
HCR5ti
264
C. D. KELLYAND
OTHERS
Note During the preparation of this manuscript a paper has been published reporting the cloning and deduced protein sequence of both human GAD,, and GAD,, and their assignment to chromosomes lOpll.23 and 2q31, respectively, by in situ hybridization (Bu et al. 1992). The authors would like to thank Dr A. J. Tobin for his kind gift of the feline GAD cDNA clone. This work was partly funded by St George’s Hospital Special Trustees and South West Thames Locally Organised Research Scheme. REFERENCES
BAEKKESKOW, S.,LANDIN, M., KRISTENSEN, J. K., SRIKANTA, S., BRUININQ, G. J., MANDRUP-POULSEN, T., D E BEAUFORT, C., SOELDNER, J. S., EISENBARTH, G., LINDQREN, F., SUNDQUIST, G. & LERNMARK, A. (1987). Antibodies to 64OOO M,human islet cell antigen precede the clinical onset of insulin dependent diabetes. J . Clin. Invest. 79, 926-934. BAEKKESKOV, S., AANSTOOT,H-J., CHRISTQAU, S., REETZ,A., SOLIMENA, M., CASCALHO, M., FOLLI, F., RICHTER-OLESEN, H. & DE CAMILLI,P. (1990). Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesising enzyme glutamic acid decarboxylase. Nature 347, 151-156. BOSSA,F., MARTINI,F., BARRA, D., BOLTATTORNI, C. B., MINELLI,A. & TORANO, C. (1977). The chymotryptic phosphopyridoxyl peptide of DOPA decarboxylase from pig kidney. Biochem. Biophys. Res. Comm. 78, 177-1 84.
Bu, D. F., ERLANDER, M. G., HITZ, B. C., TILLAKARATNE, N. J. K., KAUFMAN, D. L., WAQNER-MCPHERSON, C. B., EVANS, G. A. & TOBIN,A. J. (1992). Two human glutamate decarboxylases 65 KDa GAD and 67 KDa GAD are each encoded by a single gene. Proc. Natl Acad. Sci. USA 89, 21 15-21 19. CHANQ,Y.-C. & GOTTLIEB, D. I. (1988). Characterisation of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase. J. Neurosci. 8, 2123-2130. CRAM,D. S., BARNETT, L. D., JOSEPH, J. L. & HARRISON, L. C. (1991). Cloning and partial nucleotide sequence of human glutamic acid decarboxylase cDNA from brain and pancreatic islets. Biochem. Biophys. Res. Comm. 176, 1239-1244. EDWARDS, Y. H., SAKODA, S., SCHON,E. & POVEY,S. (1989). The gene for human muscle-specific phosphoglycerate mutase PGAM2 mapped to chromosome 7 by polymerase chain reaction. Qenomics 5 , 948-95 1.
ERDO,S. L., Joo, F. & WOLFF,J. R. (1989). Immunohistochemical localisation of glutamic acid decarboxylase in the rat oviduct and ovary: further evidence for non-neuronal GABA systems. Cell. Tiss. Res. 255,431434. ERLANDER, M. G., TILLAKARATNE, N. J. K., FELDBLUM, S., PATEL, N. & TOBIN,A. J. (1991). Two genes encode distinct glutamate decarboxylase. Neuron 7, 91-100. ERLANDER, M. G. & TOBIN,A. J. (1991). The structural and functional heterogeneity of glutamic acid decarboxylase : a review. Neurochem. Res. 16, 215-226. EVELETH, D. D., GIETZ,R. D., SPENCER, C. A., NARQANQ, F. E., HODQETTS, R. B. & MARSH, J. L. (1986). Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants. EMBO J. 5 , 2663-2672. FEINBERQ, A. P. & VOQELSTEIN, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137, 266-267. HUANQ, W. M., REED-FOURQUET, L., Wu, E. & Wu, J.-Y. (1990). Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase. Proc. Natl Acad. Sci. USA 87, 8491-8495. JACKSON, F. R., NEWBY,L. M. & KULKARNI, S. J. (1990). Drosophila GABAergic systems: sequence and expression of glutamic acid decarboxylase. J . Neurochem. 54, 1068-1078. JULIEN, J.-F.,LEQAY, F., DUMAS,S., TAPAZ,M. & MALLET,J. (1987). Molecular cloning, expression and in situ hybridisation of rat brain glutamic acid decarboxylase messenger RNA. Neurosci. Lett. 73, 173-180. KARLSEN, A. E., HAQOPIAN, W. A., GRUBIN,C. E., DUBE,S., DISTECHE, C. M., ADLER, D. A., BARMEIER, H., MATHEWES, S., GRANT, F. J.,FOSTER, D. & LERNMARK, A. (1991). Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc. Natl Acad. Sci. USA 88, 8337-8341. KATAROVA, 2.. SZABO, G., MUQNAINI,E. & GREENSPAN, R. J. (1990). Molecular identification of the 62 KDa form of glutamic acid decarboxylase from the mouse. Eur. J. Neurosci. 2, 19Cb202. KATO,K. (1990). Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in Purkinje cells. FEBS Lett. 271, 137-140. KOBAYASHI,Y., KAUFMAN, D. L. & TOBIN,A. J. (1987). Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J. Neurosci. 7, 2768-2772. LINDEFORS, N., BRENE,M., HERRERA-MARSCHITZ, M., PERSSON, M. (1989). Region specific regulation of
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glutamic acid decarboxylase mRKA expression by dopamine neurons in rat brain. Exp. Brain. Res. 77, 61 1-620. DELUCA, V., MARINEAN,C. & BRISSON,K. (1989). Molecular cloning and analysis of cDKA encoding a plant tryptophan decarboxylase : comparison with animal decarboxylases. Proc. Natl Acad. Sci. lJSA 86, 2582-2586. MANIATIS,T., FRITSCH, E.F. & SAMnRooK, J . (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory. MICHELSON,B. K., PETERSON, ,J. S., BOEL,E., MOLDRUP,A., DYRBERG, T. & MADSEN,0. D. (1991). Cloning, characterisation and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus. Proc. Nut1 Acad. Sci. USA 88, 8754-8758. H. & SHIMADA, C. (1976). High concentration of GABA and high glutamate OKADA,Y., TANIOUCHI, decarboxylase activity in rat pancreatic islets and human insulinoma. Science 194, 62s622. PERSSON, H., PELTO-HIJIKKO, M., METSIS, M., SODER, O., BRENE,S., SKOG, S., HOKFELT, T. & RITZEN,E. M. (1990). Expression of the neurotransmitter-synthesising enzyme glutamic acid decarboxylase in male germ cells. Mol. Cell. Biol. 10, 47014711. PURDUE, P. E., LUMB,M. J., Fox, M., GRIFFO,G., HAMON-BENAIS, C., POVEY, S. & DANPURE, C. J . (1991). Characterisation and chromosomal mapping of a genomic clone encoding human alanine : glyoxylate aminotransferase. Genomics 10, 34-42. REETZ,A,, SOLIMENA, M., MATTEOLI,M.. FOLLI, F., TAKEI,K. & DECAMILLI, P. (1991). GABA and pancreatic 8-cells : colocalisation of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J . 10, 1275-1284. 6.& COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. SANGER, F., NICKLEN, Natl. Acad. Sei. USA 74, 5463-5467. M., FOLLI,F., APARISI,R., POZZA, G. & DECAMILLI, P. (1990). Autoantibodies to GABAergic SOLIMENA, neurons and pancreatic 8-cells in stiff man syndrome. New Eng. J . Med. 322, 1555-1560. I., POVEY, S. & JEFFREYS, A. J . (1987). Characterization of a panel of highly WONIJ, Z., WILSON,V., PATEL, variable minisatellites cloned from human DKA. Ann. Hum. Gen. 51, 26S288. D. I. (1990). Characterisation of a cDNA coding for rat glutamic WYnoRsKr, R. J., BOND,R. W. & GOTTLIEB, acid decarboxylase. Mol. Brain Res. 8, 193-198.
Ann. Hum.Genet. (1992), 56, 25S265 Printed in Great Britain
Nucleotide sequence and chromosomal assignment of a cDNA encoding the large isoform of human glutamate decarboxylase C. D. KELLY1, Y. EDWARDS3, A. P. JOHNSTONE2, E. HARFST2, A. NOGRAD14, S. S. NUSSEY', S. POVEY3 AND N. D. CARTER' Departments of Child Health and Cellular and Molecular Sciences, St George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 ORE MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NW12HE Department of Anatomy, University College London, Gower Street, London WClE 6BT
SUMMARY
Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter y-aminobutyric acid (GABA). Two forms of human GAD, GAD,, and GAD,,, are encoded by two separate genes. A full length human GAD,, cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD,, gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD,, is not syntenic with the smaller GAD isoform GAD,, which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD,, enabled immunocytological detection of GAD in the rat brain.
INTRODUCTION
The enzyme glutamic acid decarboxylase (GAD ; glutamate decarboxylase L-glutamate-Lcarboxy-lyase, EC 4.11 .15) catalyses the rate limiting step in the synthesis of the inhibitory neurotransmitter GABA from L-glutamic acid. There are two forms of enzymically active human GAD which differ in molecular size, aminoacid sequence, cellular and subcellular location, and interaction with the GAD co-factor pyridoxal phosphate (Erlander & Tobin, 1991). The protein structures are sufficiently similar to allow recognition by the same polyclonal antibody (Katarova et al. 1990) but isoform specific antibodies have also been prepared (Chang & Gottlieb, 1988). The proteins GAD,,, molecular size 64 kDa, and GAD,,, molecular size 67 kDa, are encoded by two separate genes (Erlander et al. 1991). Both are selectively expressed in GABAergic neurons and are primarily concentrated in the nerve terminals (Reetz et al. 1991), although there are differences in their intracellular distribution. Outside the brain, GAD has limited distribution, but is expressed in the ovary and oviduct (Erdo et al. 1989), in spermatozoa (Persson et al. 1990), and at high levels in the islets of the pancreas, specifically in the /3 cells (Okada et al. 1976). Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease for which there is a strong genetic disposition. Autoantibodies are directed against antigenic determinants on the human pancreatic p cells. Antibodies against a 64 kDa polypeptide, now known to be
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GAD (Baekkeskov et al. 1990), have been found in the sera of newly diagnosed patients with IDDM as well as in pre-diabetic individuals several years before the onset of the disease (Baekkeskov et al. 1987). Stiff-man syndrome (SMS) is a rare neurological disorder believed to be due to interference in neuronal pathways involving the neurotransmitter GABA. While the diagnosis of this rare syndrome is often difficult, it appears that most patients have autoantibodies to GABA-secreting neurones and GAD has been determined to be the predominant autoantigen (Solimena et al. 1990). A striking association has been found in these patients between the presence of GAD antibodies and organ-specific autoimmune disease especially IDDM (Solimena et al. 1990). The presence of GAD autoantibodies is the earliest known predictor of IDDM and can also be used as a marker in diagnosis of SMS. In order to examine the role played by autoimmunity to GAD in these two diverse clinical conditions IDDM and SMS, it is important to characterize each isoform structurally and genetically. The cDNA encoding the larger isoform GAD,, has been cloned from the rat (Julien ef al. 1987; Wyborski et al. 1990), mouse (Huang et al. 1990), cat (Kobayashi et al. 1987), and fruit fly (Jackson et al. 1990). Partial cDNA nucleotide sequence derived from cloned human GAL),, is also known (Cram etal. 1991; Michelson et al. 1991; Persson Pt al. 1990). These GAL),, sequences are highly conserved between species such that all amino acid-sequences demonstrate 96% homology. The gene for the smaller human GAD isoform, GAD,, has recently been cloned and sequenced (Karlsen et al. 1991). Comparison of partial amino-acid sequence between the two human isoforms showed less than 65% homology. The GAD,, gene has been firmly allocated to the short arm of chromosome 10 (Karlsen et al. 1991) and it has been proposed, but not confirmed, that the gene for human GAD,, is localized on chromosome 2 (Karlsen et ul. 1991).
We report here the full nucleotide sequence for human GAD,, cDNA and confirm its localization to chromosome 2. Production of polyclonal antibodies to one expressed cDNA clone and its application to immunocytochemical localization of GAD is described.
METHODS AND MATERIALS
Isolation and analysis of human GAD cDNA clones A human frontal cortex cDNA library in AZAP I1 (Stratagene) was screened using a feline GAD cDNA probe (a kind gift from Dr A. J. Tobin, UCLA, California) which was labelled with [32P]dCTPusing the random hexanucleotide primer method (Feinberg & Vogelstein, 1984). The filters were hybridized a t 65 "C in 4 x SSC; 10 x Denhardt's :0*1% SDS; 100 ,ug/ml denatured salmon testes DNA and were subsequently washed to a final stringency of 1 x SSC/Ol O h SDS a t room temperature. Positive recombinant phage were purified to homogeneity and amplified. The pBluescript phagemid containing the cDNA insert was excised from the AZAP vector using R408 helper phage and XLI-Blue host bacteria according to the Stratagene protocol. Phagemid DNA was alkali denatured (Maniatis et al. 1989) and used directly for sequencing using the dideoxynucleotide sequencing method (Sanger et al. 1977), and the Sequenase 2.0 kit (United States Biochemical).
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Chromosomal assignment :analysis of somatic cell hybrid DNA by PCR The polymerase chain reaction (PCR) method was used to amplify specifically human GAD,, sequences in DNA extracted from a panel of somatic cell hybrids. The nine hybrids used are listed in Table 1, the human chromosome complement of each hybrid has been assessed by a combination of isozyme and DNA analysis and, in most cases, by karyotyping. Two 25mer oligonucleotide primers : CCT TAG GTT TCA GCT AAG CGA GTC A (reverse) and
CCG CTC CAA GAG AAT TCA CTT TAC C (forward),
corresponding to positions 2985-2961 and 2257-2282, respectively, in the human GAD,, cDNA scyuence and spanning 703 bp of the 3' untranslated region were synthesized. These primer sequences within the 3' noncoding region were chosen as they demonstrated high specificity for the human GAD,, riucleotide sequence (Fig. 2). DNA was prepared from somatic cell hybrids and their parental cell lines as described previously (Edwards et al. 1989). PCR was carried out using 1 pg of parent DNA and 3 pg of hybrid DNA in a total reaction volume of 100 pl. After initial incubation a t 95 "C for 5 min, 2 4 units of Taq polymerase were added to each tube. After 30 cycles of amplification, each consisting of 30 s a t 93 O C , 30 s a t 59 "C and 30 s a t 72 O C , a 10 pl aliquot was rcmoved and analysed on a 1 % agarose gel. DNA products were visualized with ethidium bromide under a U.V.light.
Expression of CDNAclones in baculoviral system and production of GAD antibody Insert DNA from one of the cDNA clones, clone 3blb, isolated from the human frontal cortex library, was introduced into the baculovirus transfer vector pAc373 using BamHI and KpnI sites within Blucscript. This was designed to allow translation to initiate a t the internal ATG site (base 1006), producing a polypeptide corresponding to the C-terminal half of GAD,,. The product from infcctcd SF9 cells was purified by SDS-PAGE and used to immunize rabbits.
Immunohistochemistry and in situ hybridization of GAD on rat brain sections Frozen sections from rat brain and cerebellum were cut (15 pm thickness) and prefixed using 4 YOparaformaldehyde prior to immunocytochemistry and in situ hybridization with cDNA probes. In situ hybridization was carried out using a protocol described by Kato (1990) and the feline GAD,, cDNA clone. Immunohistochemistry was carried out using a 1 :250 dilution of the antibody, incubated with the sections for 3 h at room temperature. Visualization of bound antibody was achieved using a biotinylated goat anti-rabbit antibody and avidin-peroxidase kit. Protocol and reagents were obtained from Vector Laboratories, UK. Controls were carried out using normal rabbit serum. RESULTS
Human GAD cDNA sequence determination Screening of the human frontal cortex cDNA library with feline GAD cDNA as probe resulted in the isolation of 4 cDNA clones (Fig. 1) for human GAD,,: HG2ab (insert length 2867 bp),
C. D. KELLYAND
258
ECOP
OTHERS Hlndlll Hlndlll
EcoRl EcoRl
HG2ab
.
HG23cla
HG12a
Scale I
500bp
‘
Fig. 1 . Restriction map of human GAD,, cDNA and sequencing strategy applied to analyse the isolated cDNA clones.
HG3blb (2251 bp), HG23cla (2768 bp) and HG12a (1034 bp). Sequence analysis demonstrated that the total length of human GAD,, cDNA was 3218 bp, with the initiation codon a t 184 bp from the 5’ end, a termination codon, TAA, a t 1963 bp and 1253 bp of 3’noncoding sequence containing a poly adenylation signal sequence and a poly A tail, A,, (Fig. 2). The open reading frame of 1779 nucleotides encodes a polypeptide of 593 amino-acid residues. Comparison of the coding region of this human GAD cDNA with equivalent regions of feline (Kobayashi et al. 1987) and rat (Wyborski et al. 1990) GAD demonstrates 93 and 91 YOhomology respectively a t the nucleotide level, and 98 and 97% homology respectively a t the amino level (Fig. 3). Homology between human and rat sequences was also found to extend into the 5’ noncoding, 71 YOhomology, and 3’ noncoding, 80% homology, regions. Some areas of the 3’ noncoding region show a similar degree of homology between the two species to that of the coding region. The sequence which encodes the binding site for the GAD co-factor pyridoxal phosphate (Am-Pro-His-Lys) is located a t 1383 bp to 1395 bp. This site is highly conserved between the different GAD molecules of various mammalian and non-mammalian species (Jackson et al. 1990) and is also found to be common amongst other decarboxylases, e.g. pig DOPA decarboxylase (Bossa et ul. 1977),Drosophila DOPA decarboxylase (Eveleth et al. 1986), and is similar to that of plant (Cutaranthus roseus) tryptophan decarboxylase (deLuca et al. 1989), suggesting a common ancestral gene.
Chromosomal location The results of PCR amplification of DNA from somatic cell hybrids using the two primers synthesized from the 3’ noncoding human GAD,, cDNA are shown in Fig. 4 and summarized
Sequence and assignment of GAD
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T t C G C A G C A G C C T C C A G T A C G C C O C G C A G A l T A C G C C ' I G T C A ~ C C G A G C C G M C G A T C ~ ~ ~ T G ~87A G ~ ~
CCGCAGTGCCCOCCTCCCTCTCCCAOACCCGMCC'IG~TGCGCCGGACCMTCGAGACTCTGOACMTAGA~CCC~ACG 174 ACCGAGC~CGT~~GACCCATITGTCCGCMCCTCA
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METAlaSerSerThrHisLeuSerAlaThrSerSerAsnAlaGluProAspProAsnThrThrAsnLergProThr A C G T A C G A T A C C T C C T C C O C C G ~ C C C A C O C A T G C A C C A G ~ C T C C T C M G A T C T G C G C ~ ~ W G A C C M C348 m
ThrTyrAspThrTrpCysGlyVa1AlaHisGlyCysThrArgLysLeuGlyLeuLysI1eCyeGlyPheLeuGlnArgThr~nSer C T C G A G A G A A G A G T C G C C T T G A G T C C C T T C M O C A G A O C C M T t C ~ C M G M C C T G C ~ C T G T G A A A A C A G C G A C C ~ A T435
LeuGluGluLysSerArgLeuVa1SerAlaPheLysGluArgGlnSerSerLysAsnLeuLeuSerCysGluAsnSerAspArgAsp G C C C G C T T C C O C C G C A C A G A G A C T C A C T T C T C T M T C T G T T l C C T A G A G A ~ m ~ C G C T M G M C G T G ~ A ~ W C C G T522 G AlaArgPheArgArgThrGluThrAspPheSerAsnLeuPheAl~g~pLeuLeuProAlaLysAenOlyOluOluGluGlnThrVal C
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GlnPheLeuLeuGluValValAspIleLeuLeuAsnTyrValArgLysThrPh~spArgSerThrLy~ValLeuAspPheHi~His C C A C A C C A G T C T C G M G C A T G O A ~ C ' I T C M C ~ A G C T C ~ ' I G A C C A C C C C G M T C ~ T G O M C ~ A T C C T G O T ' K A696 Cm
ProH~sGlnLeuLeuGluGlyMETCluGlyPheAsnLeuGluLeuSerAspHisProOluSerLeuGluGlnIleLeuValAspCy~ AGAGACACCTAAGTATGTTCGCACAGGTCATCCTCGATFITTCMCCAGCTCTCCACTGOATlGGATAlTA~CTAGCT
703
ArgAspThrLeuLysTyrGlyValArgThrGlyHisProArgPhePheAsnGlnLeuSerThrGlyLeuAspIleIleClyLeuAla G C A G M T C G C M M C T C M C G C C A A T A C C M C A T G ~ A C A T A T G A A A ~ C A C C A G T G T T I C T C C T C A T G O M C W T M C A C l T 870
GlyGluTrpLeuAsnSerThrAlaAsnThrAsnKETPheThrTyrGluI1eAlaProValPhoValLeuMETCluGlnIl~ThrLeu M G A A G A T G A G A G A G A T A G T T C C A n ; C T C A A G T A A A G A T C G T G A T A T A ~ ~ C ~ C ~ C A T A T C C M C A T G T A C957 A~
LysLysMETArgGluIleValGlyTrpSerSerLysAspGlyAspGlyIlePheSerProOlyGlyAlaIleSerAsnMETTyrSer ATCATCCCTCCTCGCTACMGTACTTCCCCOCMGTTCMGACAAA~CATGOC~C'IGTGCCTWCTGOTCCTC~ACCTC~M1044 I1eMETAlaAlaArgTyrLysTyrPheProOluValLysThrLysGlyMETAlaAlaVa1ProLysLeuValLeuPheThrSerOlu C A G A G T C A C T A T T C C A T A A A G A A A G C T G G G G C T G C A C T T G G C T T T G G A A C T G A C M T G T G A T T T I G A T W G m M ' I G ~ ~ 11 31
ClnSerHisTyrSerIleLysLysAlaGlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCysAEnG~~gG~y A A A A T A A T T C C A G C T G A T A G G C W l T C T A A G C C A A A C A G A A G G G A T A T G T T C C C M A T G T C M ~ M C m W 1218 LysIleIleProAlaAspPh~luAlaLysIleLeuGluAl~ysGlnLysGlyTyrValProPheTyrValAs~laThrA~aG~y 1305 ACGACTG~ATCCAGCTATCCGATACMGAGA~CAGATATATG'IGAGWTATMCC~~ATG~GA m~C
ThrThrValTyrGlyAlaPheAspProIlaGlnGluIleAlaAspIleCysGluLysTyrAsnLeuTrpLeuHisValAspA~aA~a 1392 ~AGTCTCCTCATCTCCACGAAGCACCGCCATAAACTCMCGCATAGW~CCMCTCMTCACC~MCCCTCAC TrpGlyGlyGlyLauLeuMETSerArgL~sHisArgHisLysLeuAsnGlyIleCluArgAlaAsnSerValThr~~snProHi~ 1479 MGATGATCGTCCTGTCAGTCC~TGCCATTCTCGTCMOC~~TATACTCCMGA'IGCMCCAGATG'IG~A~ ~ T M E T G l y V a l L e u L e u G l n C y s S e r A l a I1eLeuValLysGluLysGlyI 1eLeuGlnGlyCysAsnGlnMETCysAlaGlY TACCTCTTCCAGCCAGACMGCAGTATGATGTCTCCTACGACACCCOOOACMGCMTTCAGTGWCGCCACGTGOATA~
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GlyThrThrMETVa1GlyTyrGlnProOlnGlyAspLysAlaAsnLeuPheArgMETValIleSerAsnProA~aA~aTh~~n~er G A C A T P C A C T T C C C T C A T P C A G C A G A T A G A A A G A C T O O G C C A G A ~ ~ A ~ C ~ G C A G M C C A T ~ C ~ A2 0~0M1 ~
AspIleAspPheLeuIleGluGluIleGluArgLeuGlyGlnAspLeu GCC~TCCCTCTCCCACTTCCAGMCAAACCTCTATA'IGTPCC'IGAAACACACAGCCA~ATT~CA~WCATMTATCT'K
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CTATCGCTCTATFITTAGAGA'M'AATTlCTGTAGATTGTGTAAATTCCTGT'KTC'IGACCTlGGTGG~~MACTATGTG 2610
T C A T G A ~ M ' I G A ~ T T C m A A T T C T A G T C M T G W T A ~ C l T A ~ A T A ~ A G A G A T G T A C C A ' I G l T ~ A G G c G2T6 9C7 m G T A T T T T C ' I T C C C A T T P C T M T G T A T C ' I T A ~ A T A T A T G A A G T A A G T T C T G A A A A C T G ~ A T G O T A ~ G l C C A T T I C T G2~7 0 4 C W G A G ~ A ~ T A ~ U A ~ T A G ~ A G A T T ~ C T A ~ A T A ~ T M A G T G C C C ~ T W T M T G A ~ ~ A ~ A C T G T C G ~ T ~ M A2 8 7 1
A T T C T M G A T C G T A C A T A A A G ~ A T A T A T A T G O W T C C T G T T A C l T W T A G C A T C m T C ~ l T A C ~ T C T C T G T C W T2 G 958 TACGTCTCCTGTGCTCAATCC~TCTACCAACTCTTGGATMTMCTAGATCTCCTGTM~TAGTAGT'KATGACCM~TC 3045 ~ A A C A ~ C G A A G A C C T C C T G A A G A T C T C A G A T W G T G A C C A G G ~ T C A C A A 3 1C 32 T G ~ ~ " TGMGMOCGAAATTCACAC'IGTGCG~AGAGTATGCAAGAAGAATAT~~~~TA'ITCTCCATGOAGM~MCA
Fig. 2. Nudeotide sequence and deduced amino acid sequence of human GAD,,. Initiation codon, termination cwdon, polyadenylation signal, pyridoxal phosphate binding site and primers synthesized for chromosomal allocation are boxed.
3219
260
C. D. KELLYAND
OTHERS
59 59 60 119 119 120 179 179 18 0 239 239 240 299 299 300 359 3 59 360 4 19 4 19 420 479 479 480 539 539 54 0
Fig. 3 . Deduced amino-acid sequence of human GAL),, compared with published rat and rat sequrnc'e (Julien et 01. 1987, Wyborski et al. 1990)- a single residue deletion in the human sequence is indicated k>y A . Residues in the cat and rat sequence which are identical to human are indicated by -.
in Table 1. Human DNA was specifically amplified to give the expected 703 bp fragment. X o band of this size was seen in the mouse, rat or hamster parent cell lines. The results show that the presence of the human GAD,, gene in the hybrids correlates with the presence of chromosome 2 . There are at least 2 cases of discordance with every human chromosome except chromosome 2 .
Immunohistochemistry and in situ hybridization The incubation of rat brain sections with polyclonal antibody raised to the fusion protein of human GAD,, cDNA clone, HGSblb, demonstrates the localization of GAD,, protein to neurones in the cerebral cortex (interneurones), in the striatum, in the Purkinje cells (Fig. 5a.) and in many other smaller neurones throughout the brain stem (Fig. 5 b ) . Parallel experiments were carried out using cDNA probes, hybridizing [32P]dCTPand [35S]dCTPlabelled HG3cla cDNA and feline GAD cDNA to adjacent sections and these preliminary experiments indicated a similar distribution for GAD,, mRNA (not shown).
Sequence and assignment of CAD
26 1
(D
u3
W
c cv c1
M
t
a L
t
(D
r
cv
C cd
-E
z
Fig. 4.W R analysis. iising oligonucleotides specific for human CAD6,. of human, rodent and various rodent/hunian hyhritl wll lines. The amplified fragment is 703 bp long. M, markers; A. ladder. DISCUSSION
The human CAI),, cDNA nucleotide sequence given here extends the sequence information previously reported for this isoform. While the cDNA appears t o be full length therc is some discrepancy between the size 3219 bp and an estimation of 3700 bp determined as transcript sizr by Nort hcrn analysis. This may simply reflect t,he inaccuracies associated with sizing mRKA after clcctrophoretic separation or may imply that additional untranslated sequences remain to be isolated. Identification of the transcription/initiation site of GAD,, mRNA or isolation of further GAD,, cDNA clones may throw light on this observation. The GAD,, gene has been assigned to chromosome 10 (Karlsen etal. 1991). In this paper we make a firm assignment for the gene for human GAD,, to chromosome 2 using PCR methods. The GAL),, homologue in the mouse has been mapped to mouse chromosome 2. The area of homology between the human and mouse chromosomes includes genes for interleukin-I a and p, sodium-channel type I1 a-polypeptide, cholinergic receptor, nicotinic a-polypeptide, nebulin and horncobox region 4 arid extends from 2 q l l to 2q37. It seems reasonable to suggest that the human GAL),, gene also lies within this distal region of the long arm of chromosome 2, since other regions of human chromosome 2 are homologous to mouse chromosomes 1, 6 and 12. Comparison of human GAD,, nucleotide sequence with that from other species reveals an extremely high degree of homology indicating remarkable sequence conservation. The
Table 1. Segregation of human glutamic acid decarboxylase 67 (GAD6q i n rodentlhumn hybrids
FGlOE8EP2.2 TWIN19F6 FGlOE8EP2 MOG2E5 FST917 MOG2C2 FG lOE8EP2.6 DUR4.3 TWIN19D12
1 2 2 4
+
5
3 6 0 0
1 1 2 5
2 3 1 3
-
+
+ + +-
- + + + -
0 3 3 3
4
+ + ++
3
+ + +- - + + - +
2
+ + + - +- --
-
1
-
-
-
+
2 3 0 3
0 3 3 2
3 1 0 5
0 3 1 1
0 2 3 4
+ - + - -
-
+
-
-
/
-
10
-
/
9
-
+
8
- + - - + / + + / + - + / + + + + +
-
7
+ + + +
I
6
-
-
+
+ + I +
-
+ + + +
-
1
2 2 4
3 3 2
0
2 1 3
1
1 2 5
1 1 4
2
+ + + + + / -
I - + / + -
14 15
+ - +
0
-
-
+ + + / + +
-
-
-
11 12 13
Human chromosome
+ - - - + - + + + - - - + - + + + + + - - + - +
+
-
/
0 3 3 3
1 2 2 4
3 0 0 6
0 5 3 1
1 3 2 2
+ + + - +
-
-
18 19 20
- + + - +
-
16 17
+ + + + I +
+ + + + + / +
/
+
+ + + + +
X
0 2
5
4
1 I 0
3
4
1 1
2
+ + -
22
21
+- _+ +-+
GAD,, chromosome
-
(Note. All of these hybrids are referenced in Wong et al. (1987)except for the FGlOE8EP hybrids which are described in Purdue et al. (1991). The + and - indicate definite scoring of the presence or absence of a human chromosome; / not tested, fragment of chromosome present or equivocal result.)
0
E 5U
I 5
wM
p
0
Sequence and assignment of GAD
263
Fig. 5. Immunohistochemical localization of GAD,,, in rat brain sections. (a)Specific staining in Purkinje cells (arrowed)(magnificationx 250). ( b ) Specific staining of GABAergic neurones (arrowed) in the nucleus of the spinal tract of the trigeminal nerve (N).Also shown are the parts of the medulla oblongata (MO) and the spinal tract of the trigeminal nerve (TN) (magnification x 120).
conservation of the GAD co-factor pyridoxal phosphate binding site was expected but the extensive homology exhibited across all regions of the GAD cDNA sequence suggests the existence of intense selective pressure during phylogeny. There is also a moderate degree of structural homology between the human GAD,, and GAD,, sequences (Karlsen et al. 1991), and it has been shown that the DrosophiZa GAD sequence resembles both forms at different positions (Jackson etal. 1990), suggesting a common parental gene for the two isoforms. Gene duplication, followed by chromosomal translocation and sequence divergence seems a likely evolutionary history for the two human GAD genes. In this paper, we demonstrate localized expression of GAD,, protein in rat brain Purkinje cells (Fig. 5 a ) and in other GABAergic cell types (Fig. 5 b ) . This confirms an earlier study which also demonstrated GAD,, in rat Purkinje cells (Lindefors et al. 1989). The presence of GAD autoantibodies is the earliest predictor of IDDM and is also diagnostic of SMS. The isolation of cDNAs for both human GAD isoforms will surely facilitate the production of pure GAD antigen for the routine detection of GAD autoantibodies.
18
HCR5ti
264
C. D. KELLYAND
OTHERS
Note During the preparation of this manuscript a paper has been published reporting the cloning and deduced protein sequence of both human GAD,, and GAD,, and their assignment to chromosomes lOpll.23 and 2q31, respectively, by in situ hybridization (Bu et al. 1992). The authors would like to thank Dr A. J. Tobin for his kind gift of the feline GAD cDNA clone. This work was partly funded by St George’s Hospital Special Trustees and South West Thames Locally Organised Research Scheme. REFERENCES
BAEKKESKOW, S.,LANDIN, M., KRISTENSEN, J. K., SRIKANTA, S., BRUININQ, G. J., MANDRUP-POULSEN, T., D E BEAUFORT, C., SOELDNER, J. S., EISENBARTH, G., LINDQREN, F., SUNDQUIST, G. & LERNMARK, A. (1987). Antibodies to 64OOO M,human islet cell antigen precede the clinical onset of insulin dependent diabetes. J . Clin. Invest. 79, 926-934. BAEKKESKOV, S., AANSTOOT,H-J., CHRISTQAU, S., REETZ,A., SOLIMENA, M., CASCALHO, M., FOLLI, F., RICHTER-OLESEN, H. & DE CAMILLI,P. (1990). Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesising enzyme glutamic acid decarboxylase. Nature 347, 151-156. BOSSA,F., MARTINI,F., BARRA, D., BOLTATTORNI, C. B., MINELLI,A. & TORANO, C. (1977). The chymotryptic phosphopyridoxyl peptide of DOPA decarboxylase from pig kidney. Biochem. Biophys. Res. Comm. 78, 177-1 84.
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glutamic acid decarboxylase mRKA expression by dopamine neurons in rat brain. Exp. Brain. Res. 77, 61 1-620. DELUCA, V., MARINEAN,C. & BRISSON,K. (1989). Molecular cloning and analysis of cDKA encoding a plant tryptophan decarboxylase : comparison with animal decarboxylases. Proc. Natl Acad. Sci. lJSA 86, 2582-2586. MANIATIS,T., FRITSCH, E.F. & SAMnRooK, J . (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory. MICHELSON,B. K., PETERSON, ,J. S., BOEL,E., MOLDRUP,A., DYRBERG, T. & MADSEN,0. D. (1991). Cloning, characterisation and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus. Proc. Nut1 Acad. Sci. USA 88, 8754-8758. H. & SHIMADA, C. (1976). High concentration of GABA and high glutamate OKADA,Y., TANIOUCHI, decarboxylase activity in rat pancreatic islets and human insulinoma. Science 194, 62s622. PERSSON, H., PELTO-HIJIKKO, M., METSIS, M., SODER, O., BRENE,S., SKOG, S., HOKFELT, T. & RITZEN,E. M. (1990). Expression of the neurotransmitter-synthesising enzyme glutamic acid decarboxylase in male germ cells. Mol. Cell. Biol. 10, 47014711. PURDUE, P. E., LUMB,M. J., Fox, M., GRIFFO,G., HAMON-BENAIS, C., POVEY, S. & DANPURE, C. J . (1991). Characterisation and chromosomal mapping of a genomic clone encoding human alanine : glyoxylate aminotransferase. Genomics 10, 34-42. REETZ,A,, SOLIMENA, M., MATTEOLI,M.. FOLLI, F., TAKEI,K. & DECAMILLI, P. (1991). GABA and pancreatic 8-cells : colocalisation of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J . 10, 1275-1284. 6.& COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. SANGER, F., NICKLEN, Natl. Acad. Sei. USA 74, 5463-5467. M., FOLLI,F., APARISI,R., POZZA, G. & DECAMILLI, P. (1990). Autoantibodies to GABAergic SOLIMENA, neurons and pancreatic 8-cells in stiff man syndrome. New Eng. J . Med. 322, 1555-1560. I., POVEY, S. & JEFFREYS, A. J . (1987). Characterization of a panel of highly WONIJ, Z., WILSON,V., PATEL, variable minisatellites cloned from human DKA. Ann. Hum. Gen. 51, 26S288. D. I. (1990). Characterisation of a cDNA coding for rat glutamic WYnoRsKr, R. J., BOND,R. W. & GOTTLIEB, acid decarboxylase. Mol. Brain Res. 8, 193-198.
