Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: Molecular phylogeny and evidence for a new GAD paralog

The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD(65) and GAD(67). These are the products of separat e genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a compreh ensive phylogenetic analysis, molecular information of GAD forms in other v ertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from bra ins of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldf ish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (C oryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebra l ganglion plus neural gland of Ciona intestinalis, a protochordate. Wherea s GAD(65) and GAD(67) homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD form s was found in the protochordate. This indicates that the duplication of th e vertebrate GAD gene occurred between 400 and 560 million years ago. For b oth GAD(65) and GAD(67), the generated phylogenetic tree followed the gener al tree topology for the major vertebrate classes. In turtle, an alternativ e spliced form of GAD(65), putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from b oth GAD(65) and GAD(67), is expressed in C. (N.) armatus. This third form m ight have originated from an ancient genome duplication specific to modern ray-finned fishes.