Cloning and expression of large isoform of glutamic acid decarboxylase from human pancreatic islet.
Glutamic acid decarboxylase (GAD) catalyzes formation of gamma-aminobutyric acid from glutamic acid and is a major autoantigen in insulin-dependent diabetes mellitus. Its two isoforms, GAD65 and GAD67, are encoded by two separate genes. We prepared human islet cDNA library and screened it with cDNA probes of rat brain GAD67. We cloned the cDNA for GAD67, the large isoform of glutamic acid decarboxylase, and determined its nucleotide sequence. Sequencing of the resulting clone identified a 1,785 residue open-reading frame encoded a 594 amino acid polypeptide that showed a 99.4% similarity with GAD67 from human brain. The bacterially expressed human islet GAD67 protein was enzymatically active and immunoreactive. The isolation of cDNA for this additional islet GAD isoforms will be important in studying the etiology and pathogenesis of IDDM.
Kawasaki
E
E
First Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
Moriuchi
R
R
Watanabe
M
M
Saitoh
K
K
Brunicardi
F C
FC
Watt
P C
PC
Yamaguchi
T
T
Mullen
Y
Y
Akazawa
S
S
Miyamoto
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D015342:DNA Probes; D015870:Gene Expression; D015723:Gene Library; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D015342:DNA Probes; D007527:Isoenzymes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase