Vol.
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
CLONING
AND PARTIAL GLUTAMIC
David S. Cram, Louise
of Medical Research, 18,
SEQUENCE
AND PANCREATIC
D. Barnett,
Burnet Clinical Research
February
NUCLEOTIDE
ACID DECARBOXYLASE
FROM BRAIN
Received
AND
Joan L. Joseph
1239-l
244
OF HUMAN
cDNA ISLETS
and Leonard
C. Harrison
Unit, Walter and Eliza Hall Institute Parkville, Victoria 3050, Australia
1991
We report partial nucleotide sequences of the human enzyme glutamic acid decarboxylase (GAD) from brain and pancreatic islets which encode the middle 180 amino acids of GAD. The brain and islet GAD sequences display a high degree of sequence homology with the equivalent region of other mammalian brain GAD cDNAs. Alignment of the brain and islet GAD sequences showed that there were 45 nucleotide differences which, at the translational level, would result in seven amino acid substitutions. These results which suggest that different isomeric forms of human GAD exist in brain and pancreas may be relevant to the pathogenesis of stiff man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM), respectively, two distinct but 0 1991Academic associated clinical disorders in which GAD is the target of autoantibodies. Press,Inc. The enzyme glutamic acid decarboxylase
(GAD) catalyzes the conversion of L-
glutamic acid to the inhibitory neurotransmitter y-amino butyric acid (GABA).
GAD is
expressed in the GABA secretory neurons of the central nervous system (l-3) the ficells of the pancreatic islets (4,5) and in spermatozoa (6). Analysis of immunoaffinitypurified animal brain GAD has identified several isomeric forms with M, 54-66,000 (7,8). Antisera raised to purified brain GAD have been used to screen brain cDNA expression libraries and cDNAs encoding the full length rat (9) and feline (10) GAD genes isolated and sequenced.
Comparison of their deduced amino acid sequences shows that rat
and feline GAD are 95% identical and therefore highly conserved during evolution. Autoantibodies reactive with GAD in GABA-ergic neurons are present in 60% of sera from patients with the rare neurological disease stiff man syndrome (SMS) (11 ,12). Almost all patients positive for GAD autoantibodies have associated insulin dependent Abbreviations : GAD, glutamic acid decarboxylase; IDDM, insulin-dependent diabetes mellitus; SMS, stiff man syndrome; PCR, polymerase chain reaction. 0006-291X/91
1239
$1.50
Copwight 0 1991 by Academic Press, Inc. All rights of reproduction in any form resenvd.
Vol.
176,
diabetes
No.
mellitus
recent-onset 64,000
(IDDM).
AND
protein
designated
64K (13).
and the definition the mechanisms
report the cloning
are frequently In a recent
amino acid GAD peptides substitutions.
show
The possible
structure
in SMS and IDDM.
Sequence
with
a 6 cell Mr was
of the human enzyme in
will therefore
that they are identical
implications
against
the 64K autoantigen
of human brain and pancreatic
the middle portion of the protein.
COMMUNICATIONS
detected
report,
of its autoepitopes
of autoimmunity
and sequencing
RESEARCH
phase of IDDM and in patients
identified as GAD (14). The molecular
brain and islets understanding
BIOPHYSICAL
During the pre-clinical
clinical IDDM, autoantibodies
presumptively
encoding
BIOCHEMICAL
3, 1991
analysis except
of these tissue-specific
be important
in
In this study, we islet GAD cDNAs
of the predicted for seven
180
amino acid
differences
in human
GAD are discussed.
MATERIALS
AND METHODS
Human cDNA and RNA. Human pancreatic islet cDNA and poly(A)+ human brain RNA were used as sources of GAD cDNA. A hgt-11 library constructed with poly(A)+ RNA isolated from purified human islets of donor pancreata was kindly donated by Dr. Alan Permutt from the Washington University School of Medicine, St. Louis. cDNA was prepared from phage stocks by a plate lysis method (15). Poly(A)+ normal human brain RNA was a gift of Dr. Claude Bernard from Latrobe University School of Behavioural Sciences, Australia. Polymerase chain reaction (PM). Oligonucleotide primers were synthesized to conserved regions of the rat (9) and feline (10) GAD nucleotide cDNA sequences. First strand synthesis of poly(A)+ RNA (1 OOng) was performed in 10 mM Tris pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 200 PM dNTP’s (PCR buffer) containing 2 pmole of complementary 3’ primer, 40 U of RNAsin and 5 U of mouse moloney leukemia virus reverse transcriptase. hgt-11 cDNA (1 OOng) or 1 Oul of the first strand reaction was amplified for 30 thermal cycles (one cycle: 1.5 min at 95°C; 2.0 min at 37-45°C; 2.0 min at 72°C) in PCR buffer containing 20 pmole of 5’ and 3’ primers and 2.5 U of Taql polymerase. Reaction products were analyzed in low melting agarose gels and products of the expected size purified by phenol extraction (15). Cloning
and DNA sequencing. PCR amplified DNA fragments were cloned into the plasmid expression vector pGEX-3 (16). Nucleotide sequences were determined by the dideoxy chain termination method (17) using plasmid specific primers hybridizing 5’ and 3’ to the cloning site and primers designed from internal GAD sequence. RESULTS
AND DISCUSSION
The nucleotide similarity
(9) which
sequences suggests
of rat and feline
that mammalian
clone human GAD cDNA, oligonucleotide conserved
between
brain GAD cDNAs
exhibit
95%
GAD genes are highly conserved.
To
primer pairs overlapping
rat and feline sequences 1240
were
synthesized
nucleotide and used
stretches in PCR
Vol.
176,
No.
BIOCHEMICAL
3, 1991
reactions
to amplify
expression
library.
oligonucleotide generated
AND
RNA extracted
PCR reactions
and temperatures
5’ ACTGCCAATACCAATATGTTCACATATGA
respectively, cDNA
3’ (complementary, to nucleotide
COMMUNICATIONS
isolated
various
from the islet
combinations
a product
of 600 bp was
with the oligonucleotide
an artificial &IRI
!&RI
and ml
transformants
were
digested
and transformed identified
primer pair
739-768
site) which correspond,
and 1312-l 330 of the published
(lo), and span the middle portion of the GAD open reading frame.
bp PCR products
of
3’ and 5’ CCGAATTCTGTGAGGGTT
contains
positions
using
of annealing,
from both brain and islet cDNA templates
CCAGGTGAC
RESEARCH
from brain and cDNA
In extensive
primers
BIOPHYSICAL
with BRI, into E.coli.
ligated with pGEX-3 Restriction
analysis
a human brain GAD clone (HBGAD)
feline
The two 600
DNA cleaved
with
of plasmid DNA from
and an islet GAD clone
(HIGAD). The 540 nucleotide excluding display sequence HBGAD
the oligonucleotide a high degree
determined
primers,
of homology
(10) and therefore sequence
sequences
are shown
for both
HBGAD
in Fig. 1. These
with the equivalent
region
sequence
showed
two sequences
of feline brain GAD
confirm the identity of the human clones.
with the HIGAD
and HIGAD,
that there were
Alignment
of the
45 nucleotide
HBGAD HIGAD
1 30 ATTGCACCAGTGTTTGTCCTCATGGAACAAATAACACTTAAGAAGATGAGAGAGATAGTT ATTGCACCCGTGTTTGTTCTCATGGAACAGATTACTCTTAAGARGRTGAAGATGAG~GATCGTT 90 GGATGGTCAAGTAAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCC~CATG GGATGGTCAAATAAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCCAATATG
120
HBGAD HIGAD
150 TACAGCATCATGGCTGCTCGCTACAAGTACTTCCCGGAAGTTAAGACAAAGGGCATGGCG TACAGCATCATGGCTGCTCGTTACTTCCCAGAAGGCATGGCG
180
HBGAD HIGAD
210 GCTGTGCCTAAACTGGTCCTCTTCACCTCAGAACAGAGTCACTATTCCATAAAGAAAGCT GCTGTGCCCAAACTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATARAGAAAGCC
240
HBGAD HIGAD
210 GGGGCTGCACTTGGCTTTGGAACTGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG GGGGCTGCGCTTGGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG
300
HBGAD HIGAD
330 ARAATAATTCCAGCTGATTTTGAGGCAAAAATTCTTGAAGCCAAACAGAAGGGATATGTT AAGATAATTCCGGCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTT
360
HBGAD HIGAD
390 CCCTTTTATGTCAATGCAACTGCTGGCACGACTGTTTATGGAGCTTTTGATCCGATACAA CCCCTTTATGTCAATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCGATCC~TCCAG
420
HBGAD HIGAD
450 GAGATTGCAGATATATGTGAGAAATATAT~CCTTTGGTTGCATGTCGATGCTGCCTGGGGA GFAATTGCGGACATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGT
480
HBGAD HIGAD
510 GGTGGGCTGCTCATGTCCAGGAAGCACCGCACCGCCAT~ACTC~CGGCATAG~GGGCC~C GGTGGACTGCTCATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAG~GGGCC~T
540
HBGAD HIGAD
60
Figure 1. Alignment of human brain (HB) and islet (HI) GAD nucleotide sequences. The partial human GAD sequences shown correspond to nucleotide positions 760-l 308 of the feline brain GAD cDNA (10). Nucleotide differences in the HIGAD sequence are indicated by bold lettering. 1241
BIOCHEMICAL
Vol. 176, No. 3, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HBGAD HIGAD FBGAD
218 IleAlaProValPheValLeuMetGluGlnIleThrLeuLysLysMetArgGluIleVal ---------------------------I--
237
HBGAD HIGAD FBGAD
GlyTrpSerSe+LysAspG1yAspGlyIlePheSerProGlyGlyAlaIleSerAsnMet -Asn_ -----------
HBGAD HIGAD FBGAD
TyrSerIleMetAlaAlaArgTyrLysTyrPheProGluValLysThrLysGlyMetAla -----_-_-__ - phe _
HBGAD HIGAD FBGAD
AlaValProLysLeuValLeuPheThrSerGluGlnSerHisTyrSerIleLysLysAla --_-_-_ -His-
HBGAD HIGAD FBGAD
GlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCysAs~GluArgGly ------------------_-
HBGAD HIGAD FBGAD
LysIleIleProAlaAspPheGluAlaLysIleLeuGluAlaLysGlnLysGlyTyrVal - Leu -
HBGAD HIGAD FBGAD
ProPheTyrValAsnAlaThrAlaGlyThrThrValTyrGlyAlaPheAspProIleGln -J&u-&xu-
HBGAD HIGAD FBGAD HBGAII HIGAD FBGAD
-
---
_
_ ---
-Lys-
251
_
-
-
-
---
-
-
-
-
-
-_-
-
-
-
_
_
-
-
_
_
-
_
_
_
_
-
-
-
---
211
-
297
_
-HiS-
317
-
-----
-
337
_
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
GluIleAlaAspIleCysGluLysTyrAsnLeuT+pLeuHisValAspAlaAlaTrpGly ------------
-
---
-
-
-
GlyGlyLeuLeuMetSerArgLysHisArgHisLysLeuAsnGlyIleGluArgAlaAsn ------------------
-Sex - Se1
-
-
-
-
-
-
-
-
-Jpu-
-
-
-
-Asp-
-
-
-
-
-
357
377
397
-
-
-
Figure 2. Partial amino acid structure of human and feline GAD. Human brain (HB), human islet (HI) and feline brain (FB) GAD amino acid sequences (180 amino acids)
are shown. Numbers indicate the amino acid position in the FBGAD protein. Dashes denote the same aminoacids as in HBGAD.
differences between the two sequences giving an overall homology of 92%. As PCR amplification has been reported to generate sequence variants, particularly those with A.T+G.C
transitions (l&19), the authenticity of these nucleotide differences was cross-
checked by determining the sequence of the 600 bp fragment derived from independent clones. Of a further five brain and islet sequences analyzed, all were identical to their counterpart HBGAD and HIGAD sequences. Figure 2 shows the deduced amino acid sequences of HBGAD and HIGAD and their alignment with the equivalent region in the feline GAD protein (amino acids 218397). The 45 nucleotide differences between HBGAD and HIGAD would result in six conservative amino acid changes at residues 241 (Ser + Asn), 289 (Gln + His), 330 (Glu + Asp), 324 (Phe + teu), 339 (Phe + Leu) and 391 (Asn j
Ser) and one non-
conservative change at residue 235 (Glu -+ Lys); the remaining 38 nucleotide changes occur predominantly at the third position of each codon and are silent. These amino 1242
Vol.
176,
No.
3, 1991
acid differences the existence
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
Infiltration
isomeric
of the pancreatic
islets
of the insulin-producing
the ability of several
with
mononuclear
cells culminates
8 cells and clinical IDDM (20).
IDDM sera to co-precipitate
we have shown
that peripheral
and clinical IDDM can be activated GAD (21).
The presence
might explain autoantigen
the selective
respectively,
T cells in subjects screened
between
destruction
the former being recognized
with IDDM.
in IDDM, based on
blood T cells from subjects
with pre-clinical that contain
islet GAD and brain GAD
of 8 cells in IDDM.
in both IDDM and SMS it could contain
epitope(s),
If GAD is an
islet- and brain-specific only by GAD-specific
T cell
autoreactive
Human brain and islet cDNA libraries are currently
with the 600 bp PCR product
GAD
the 64K p cell protein and GAD (14).
differences
autoimmune
in the
The enzyme
by human islets and foetal pig proislets
of sequence
with
forms of human GAD.
has recently been identified as a putative primary 8 cell autoantigen
Recently,
COMMUNICATIONS
in the mid region of the brain and islet GAD proteins are consistent of tissue-specific
destruction
AND
in order to clone and express
being
the full length
GAD proteins for use in detailed T cell studies.
ACKNOWLEDGMENTS: Senior Principal of Australia. Thompson
Research We thank
This work
was supported
Fellow of the National Dr. Ross
Coppel
by AMKAID
Pty Ltd.
Health and Medical Research
for helpful
discussions
LCH is a Council
and Margaret
for typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Roberts, E., Chase, T.N., and Tower, D.B. (1976) Kroc Foundation Series, Vol 5: GABA in Nervous System Function. Raven Press, New York. Mugaini, E., and Oertel, W.H. (1985) In Handbook of Chemical Neuroanatomy (A. Bjorklund and T. Hokfelt, Eds.) Vol 4, pp. 436-608, Elsevier, New York. Blessing, W.W. (1990) Neuroscience 37, 171-l 85. Okada, Y., Taniguchi. H., and Shimada, C. (1976) Science 194, 620-622. Garry, D.J., Appel, N.M., Garry, M.G., and Sorensen, R.L. (1988) J. Histochem. Cytochem. 36,573-580. Persson, H., Pelto-Huikko, M., Metsis, M., Seder O., Brene, S., Skog, S., Hokfelt, T., and Ritzen, EM. (1990) Mol. Cell. Biol. 19, 4701-4711. Gottlieb, D.I., Chang, Y-C., and Schwab., J.E. (1986) Proc. Natl. Acad. Sci. USA. 83, 8808-8812. Chang, Y-C., and Gottlieb, D.I. (1988) J. Neuroscience 8, 2123-2130. Julien, J-F., Samana, P., and Mallet, J. (1990) J. Neurochemistry 54, 703-705. Kobayashi, Y., Kaufman, D.L., and Tobin, A.J. (1987) J. Neuroscience 7, 27682772. Sofimena, M., Folli, F., Denis-Donini, S., Comi, G.C., Pozza, G., DeCamilli, P., and Vicari, A.M. (1988) N. Engl. J. Med. 318, 1012-1020. 1243
“-I VOI.
176,
No.
3, 1991
BIOCHEMICAL
AND
12.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Solimena, M., Folk, F., Aparisi, R., Pozza, G., and DeCamilli, P. (1990) N. Engl. J. Med. 322, 1555-l 560. S., Nielson, J.H., Marner, B., Bilde, T., Ludvigsson, J., and 13. Baekkeskov, Lernmark, A. (1982) Nature 298, 167-l 69. 14. Baekkeskov, S., Aanstoot, H-J., Christgau, S., Reetz, A., Solimena, S., Cascalho, M., Folli, F., Richter-Olesen, H., and DeCamilli, P. (1990) Nature 347, 151-156. 15. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In Molecular Cloning. A Laboratory Manual. Vol l-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 16. Smith, D.B., and Johnson, K.S. (1988) Gene 67, 31-40. 17. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA. 74, 5463-5467. 18. Keohavong, P., and Thilly, W.G. (1989) Proc. Natl. Acad. Sci. USA. 86, 92539257. 19. Belyavsky, A., Vinogradova, T., and Rajewsky, K. (1989) Nucleic Acids Res. 8, 2919-2932. 20. Harrison, L.C., Colman, P.G., Chosich, N., Kay, T.W.H., Tait, B.D., Bartholomeusz, R.K., De Aizpurua, H.J., Joseph, J.L., Chu, S., and Kielczynski, W.E. (1990) Adv. Endocrinol. Metab. 1,35-94. 21. Harrison L.C., De Aizpurua, H., Loudovaris, T., Campbell, I.L., Cebon, J.S. and Colman, P.G. Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with pre-clinical and clinical insulin-dependent diabetes (submitted).
1244
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
CLONING
AND PARTIAL GLUTAMIC
David S. Cram, Louise
of Medical Research, 18,
SEQUENCE
AND PANCREATIC
D. Barnett,
Burnet Clinical Research
February
NUCLEOTIDE
ACID DECARBOXYLASE
FROM BRAIN
Received
AND
Joan L. Joseph
1239-l
244
OF HUMAN
cDNA ISLETS
and Leonard
C. Harrison
Unit, Walter and Eliza Hall Institute Parkville, Victoria 3050, Australia
1991
We report partial nucleotide sequences of the human enzyme glutamic acid decarboxylase (GAD) from brain and pancreatic islets which encode the middle 180 amino acids of GAD. The brain and islet GAD sequences display a high degree of sequence homology with the equivalent region of other mammalian brain GAD cDNAs. Alignment of the brain and islet GAD sequences showed that there were 45 nucleotide differences which, at the translational level, would result in seven amino acid substitutions. These results which suggest that different isomeric forms of human GAD exist in brain and pancreas may be relevant to the pathogenesis of stiff man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM), respectively, two distinct but 0 1991Academic associated clinical disorders in which GAD is the target of autoantibodies. Press,Inc. The enzyme glutamic acid decarboxylase
(GAD) catalyzes the conversion of L-
glutamic acid to the inhibitory neurotransmitter y-amino butyric acid (GABA).
GAD is
expressed in the GABA secretory neurons of the central nervous system (l-3) the ficells of the pancreatic islets (4,5) and in spermatozoa (6). Analysis of immunoaffinitypurified animal brain GAD has identified several isomeric forms with M, 54-66,000 (7,8). Antisera raised to purified brain GAD have been used to screen brain cDNA expression libraries and cDNAs encoding the full length rat (9) and feline (10) GAD genes isolated and sequenced.
Comparison of their deduced amino acid sequences shows that rat
and feline GAD are 95% identical and therefore highly conserved during evolution. Autoantibodies reactive with GAD in GABA-ergic neurons are present in 60% of sera from patients with the rare neurological disease stiff man syndrome (SMS) (11 ,12). Almost all patients positive for GAD autoantibodies have associated insulin dependent Abbreviations : GAD, glutamic acid decarboxylase; IDDM, insulin-dependent diabetes mellitus; SMS, stiff man syndrome; PCR, polymerase chain reaction. 0006-291X/91
1239
$1.50
Copwight 0 1991 by Academic Press, Inc. All rights of reproduction in any form resenvd.
Vol.
176,
diabetes
No.
mellitus
recent-onset 64,000
(IDDM).
AND
protein
designated
64K (13).
and the definition the mechanisms
report the cloning
are frequently In a recent
amino acid GAD peptides substitutions.
show
The possible
structure
in SMS and IDDM.
Sequence
with
a 6 cell Mr was
of the human enzyme in
will therefore
that they are identical
implications
against
the 64K autoantigen
of human brain and pancreatic
the middle portion of the protein.
COMMUNICATIONS
detected
report,
of its autoepitopes
of autoimmunity
and sequencing
RESEARCH
phase of IDDM and in patients
identified as GAD (14). The molecular
brain and islets understanding
BIOPHYSICAL
During the pre-clinical
clinical IDDM, autoantibodies
presumptively
encoding
BIOCHEMICAL
3, 1991
analysis except
of these tissue-specific
be important
in
In this study, we islet GAD cDNAs
of the predicted for seven
180
amino acid
differences
in human
GAD are discussed.
MATERIALS
AND METHODS
Human cDNA and RNA. Human pancreatic islet cDNA and poly(A)+ human brain RNA were used as sources of GAD cDNA. A hgt-11 library constructed with poly(A)+ RNA isolated from purified human islets of donor pancreata was kindly donated by Dr. Alan Permutt from the Washington University School of Medicine, St. Louis. cDNA was prepared from phage stocks by a plate lysis method (15). Poly(A)+ normal human brain RNA was a gift of Dr. Claude Bernard from Latrobe University School of Behavioural Sciences, Australia. Polymerase chain reaction (PM). Oligonucleotide primers were synthesized to conserved regions of the rat (9) and feline (10) GAD nucleotide cDNA sequences. First strand synthesis of poly(A)+ RNA (1 OOng) was performed in 10 mM Tris pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 200 PM dNTP’s (PCR buffer) containing 2 pmole of complementary 3’ primer, 40 U of RNAsin and 5 U of mouse moloney leukemia virus reverse transcriptase. hgt-11 cDNA (1 OOng) or 1 Oul of the first strand reaction was amplified for 30 thermal cycles (one cycle: 1.5 min at 95°C; 2.0 min at 37-45°C; 2.0 min at 72°C) in PCR buffer containing 20 pmole of 5’ and 3’ primers and 2.5 U of Taql polymerase. Reaction products were analyzed in low melting agarose gels and products of the expected size purified by phenol extraction (15). Cloning
and DNA sequencing. PCR amplified DNA fragments were cloned into the plasmid expression vector pGEX-3 (16). Nucleotide sequences were determined by the dideoxy chain termination method (17) using plasmid specific primers hybridizing 5’ and 3’ to the cloning site and primers designed from internal GAD sequence. RESULTS
AND DISCUSSION
The nucleotide similarity
(9) which
sequences suggests
of rat and feline
that mammalian
clone human GAD cDNA, oligonucleotide conserved
between
brain GAD cDNAs
exhibit
95%
GAD genes are highly conserved.
To
primer pairs overlapping
rat and feline sequences 1240
were
synthesized
nucleotide and used
stretches in PCR
Vol.
176,
No.
BIOCHEMICAL
3, 1991
reactions
to amplify
expression
library.
oligonucleotide generated
AND
RNA extracted
PCR reactions
and temperatures
5’ ACTGCCAATACCAATATGTTCACATATGA
respectively, cDNA
3’ (complementary, to nucleotide
COMMUNICATIONS
isolated
various
from the islet
combinations
a product
of 600 bp was
with the oligonucleotide
an artificial &IRI
!&RI
and ml
transformants
were
digested
and transformed identified
primer pair
739-768
site) which correspond,
and 1312-l 330 of the published
(lo), and span the middle portion of the GAD open reading frame.
bp PCR products
of
3’ and 5’ CCGAATTCTGTGAGGGTT
contains
positions
using
of annealing,
from both brain and islet cDNA templates
CCAGGTGAC
RESEARCH
from brain and cDNA
In extensive
primers
BIOPHYSICAL
with BRI, into E.coli.
ligated with pGEX-3 Restriction
analysis
a human brain GAD clone (HBGAD)
feline
The two 600
DNA cleaved
with
of plasmid DNA from
and an islet GAD clone
(HIGAD). The 540 nucleotide excluding display sequence HBGAD
the oligonucleotide a high degree
determined
primers,
of homology
(10) and therefore sequence
sequences
are shown
for both
HBGAD
in Fig. 1. These
with the equivalent
region
sequence
showed
two sequences
of feline brain GAD
confirm the identity of the human clones.
with the HIGAD
and HIGAD,
that there were
Alignment
of the
45 nucleotide
HBGAD HIGAD
1 30 ATTGCACCAGTGTTTGTCCTCATGGAACAAATAACACTTAAGAAGATGAGAGAGATAGTT ATTGCACCCGTGTTTGTTCTCATGGAACAGATTACTCTTAAGARGRTGAAGATGAG~GATCGTT 90 GGATGGTCAAGTAAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCC~CATG GGATGGTCAAATAAAGATGGTGATGGGATATTTTCTCCTGGGGGAGCCATATCCAATATG
120
HBGAD HIGAD
150 TACAGCATCATGGCTGCTCGCTACAAGTACTTCCCGGAAGTTAAGACAAAGGGCATGGCG TACAGCATCATGGCTGCTCGTTACTTCCCAGAAGGCATGGCG
180
HBGAD HIGAD
210 GCTGTGCCTAAACTGGTCCTCTTCACCTCAGAACAGAGTCACTATTCCATAAAGAAAGCT GCTGTGCCCAAACTGGTCCTCTTCACCTCAGAACACAGTCACTATTCCATARAGAAAGCC
240
HBGAD HIGAD
210 GGGGCTGCACTTGGCTTTGGAACTGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG GGGGCTGCGCTTGGCTTTGGAACCGACAATGTGATTTTGATAAAGTGCAATGAAAGGGGG
300
HBGAD HIGAD
330 ARAATAATTCCAGCTGATTTTGAGGCAAAAATTCTTGAAGCCAAACAGAAGGGATATGTT AAGATAATTCCGGCTGATTTAGAGGCAAAAATTCTTGATGCCAAACAAAAGGGCTATGTT
360
HBGAD HIGAD
390 CCCTTTTATGTCAATGCAACTGCTGGCACGACTGTTTATGGAGCTTTTGATCCGATACAA CCCCTTTATGTCAATGCAACCGCAGGCACGACTGTTTACGGAGCATTCGATCGATCC~TCCAG
420
HBGAD HIGAD
450 GAGATTGCAGATATATGTGAGAAATATAT~CCTTTGGTTGCATGTCGATGCTGCCTGGGGA GFAATTGCGGACATATGTGAGAAATACAACCTTTGGCTGCATGTGGATGCTGCCTGGGGT
480
HBGAD HIGAD
510 GGTGGGCTGCTCATGTCCAGGAAGCACCGCACCGCCAT~ACTC~CGGCATAG~GGGCC~C GGTGGACTGCTCATGTCCCGGAAGCACCGCCACAAACTCAGCGGCATAG~GGGCC~T
540
HBGAD HIGAD
60
Figure 1. Alignment of human brain (HB) and islet (HI) GAD nucleotide sequences. The partial human GAD sequences shown correspond to nucleotide positions 760-l 308 of the feline brain GAD cDNA (10). Nucleotide differences in the HIGAD sequence are indicated by bold lettering. 1241
BIOCHEMICAL
Vol. 176, No. 3, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HBGAD HIGAD FBGAD
218 IleAlaProValPheValLeuMetGluGlnIleThrLeuLysLysMetArgGluIleVal ---------------------------I--
237
HBGAD HIGAD FBGAD
GlyTrpSerSe+LysAspG1yAspGlyIlePheSerProGlyGlyAlaIleSerAsnMet -Asn_ -----------
HBGAD HIGAD FBGAD
TyrSerIleMetAlaAlaArgTyrLysTyrPheProGluValLysThrLysGlyMetAla -----_-_-__ - phe _
HBGAD HIGAD FBGAD
AlaValProLysLeuValLeuPheThrSerGluGlnSerHisTyrSerIleLysLysAla --_-_-_ -His-
HBGAD HIGAD FBGAD
GlyAlaAlaLeuGlyPheGlyThrAspAsnValIleLeuIleLysCysAs~GluArgGly ------------------_-
HBGAD HIGAD FBGAD
LysIleIleProAlaAspPheGluAlaLysIleLeuGluAlaLysGlnLysGlyTyrVal - Leu -
HBGAD HIGAD FBGAD
ProPheTyrValAsnAlaThrAlaGlyThrThrValTyrGlyAlaPheAspProIleGln -J&u-&xu-
HBGAD HIGAD FBGAD HBGAII HIGAD FBGAD
-
---
_
_ ---
-Lys-
251
_
-
-
-
---
-
-
-
-
-
-_-
-
-
-
_
_
-
-
_
_
-
_
_
_
_
-
-
-
---
211
-
297
_
-HiS-
317
-
-----
-
337
_
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
GluIleAlaAspIleCysGluLysTyrAsnLeuT+pLeuHisValAspAlaAlaTrpGly ------------
-
---
-
-
-
GlyGlyLeuLeuMetSerArgLysHisArgHisLysLeuAsnGlyIleGluArgAlaAsn ------------------
-Sex - Se1
-
-
-
-
-
-
-
-
-Jpu-
-
-
-
-Asp-
-
-
-
-
-
357
377
397
-
-
-
Figure 2. Partial amino acid structure of human and feline GAD. Human brain (HB), human islet (HI) and feline brain (FB) GAD amino acid sequences (180 amino acids)
are shown. Numbers indicate the amino acid position in the FBGAD protein. Dashes denote the same aminoacids as in HBGAD.
differences between the two sequences giving an overall homology of 92%. As PCR amplification has been reported to generate sequence variants, particularly those with A.T+G.C
transitions (l&19), the authenticity of these nucleotide differences was cross-
checked by determining the sequence of the 600 bp fragment derived from independent clones. Of a further five brain and islet sequences analyzed, all were identical to their counterpart HBGAD and HIGAD sequences. Figure 2 shows the deduced amino acid sequences of HBGAD and HIGAD and their alignment with the equivalent region in the feline GAD protein (amino acids 218397). The 45 nucleotide differences between HBGAD and HIGAD would result in six conservative amino acid changes at residues 241 (Ser + Asn), 289 (Gln + His), 330 (Glu + Asp), 324 (Phe + teu), 339 (Phe + Leu) and 391 (Asn j
Ser) and one non-
conservative change at residue 235 (Glu -+ Lys); the remaining 38 nucleotide changes occur predominantly at the third position of each codon and are silent. These amino 1242
Vol.
176,
No.
3, 1991
acid differences the existence
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
Infiltration
isomeric
of the pancreatic
islets
of the insulin-producing
the ability of several
with
mononuclear
cells culminates
8 cells and clinical IDDM (20).
IDDM sera to co-precipitate
we have shown
that peripheral
and clinical IDDM can be activated GAD (21).
The presence
might explain autoantigen
the selective
respectively,
T cells in subjects screened
between
destruction
the former being recognized
with IDDM.
in IDDM, based on
blood T cells from subjects
with pre-clinical that contain
islet GAD and brain GAD
of 8 cells in IDDM.
in both IDDM and SMS it could contain
epitope(s),
If GAD is an
islet- and brain-specific only by GAD-specific
T cell
autoreactive
Human brain and islet cDNA libraries are currently
with the 600 bp PCR product
GAD
the 64K p cell protein and GAD (14).
differences
autoimmune
in the
The enzyme
by human islets and foetal pig proislets
of sequence
with
forms of human GAD.
has recently been identified as a putative primary 8 cell autoantigen
Recently,
COMMUNICATIONS
in the mid region of the brain and islet GAD proteins are consistent of tissue-specific
destruction
AND
in order to clone and express
being
the full length
GAD proteins for use in detailed T cell studies.
ACKNOWLEDGMENTS: Senior Principal of Australia. Thompson
Research We thank
This work
was supported
Fellow of the National Dr. Ross
Coppel
by AMKAID
Pty Ltd.
Health and Medical Research
for helpful
discussions
LCH is a Council
and Margaret
for typing of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Roberts, E., Chase, T.N., and Tower, D.B. (1976) Kroc Foundation Series, Vol 5: GABA in Nervous System Function. Raven Press, New York. Mugaini, E., and Oertel, W.H. (1985) In Handbook of Chemical Neuroanatomy (A. Bjorklund and T. Hokfelt, Eds.) Vol 4, pp. 436-608, Elsevier, New York. Blessing, W.W. (1990) Neuroscience 37, 171-l 85. Okada, Y., Taniguchi. H., and Shimada, C. (1976) Science 194, 620-622. Garry, D.J., Appel, N.M., Garry, M.G., and Sorensen, R.L. (1988) J. Histochem. Cytochem. 36,573-580. Persson, H., Pelto-Huikko, M., Metsis, M., Seder O., Brene, S., Skog, S., Hokfelt, T., and Ritzen, EM. (1990) Mol. Cell. Biol. 19, 4701-4711. Gottlieb, D.I., Chang, Y-C., and Schwab., J.E. (1986) Proc. Natl. Acad. Sci. USA. 83, 8808-8812. Chang, Y-C., and Gottlieb, D.I. (1988) J. Neuroscience 8, 2123-2130. Julien, J-F., Samana, P., and Mallet, J. (1990) J. Neurochemistry 54, 703-705. Kobayashi, Y., Kaufman, D.L., and Tobin, A.J. (1987) J. Neuroscience 7, 27682772. Sofimena, M., Folli, F., Denis-Donini, S., Comi, G.C., Pozza, G., DeCamilli, P., and Vicari, A.M. (1988) N. Engl. J. Med. 318, 1012-1020. 1243
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