Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification,cloning, expression,assay development,identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse |
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| Authors: | Thomas Ilg Michael Berger Sandra Noack Andreas Rohwer Michael Gaßel |
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| Affiliation: | 1. Dept. of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294, United States;2. Dept. of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States;3. Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States;4. Dept. of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, United States;5. Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States;6. Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233, United States;1. “Grigore Antipa” National Museum of Natural History, Kiseleff Street, No 1, Bucharest 011341, Romania;2. Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Květná 8, 603 65 Brno, Czech Republic;3. Institute of Botany and Zoology, Faculty of Science, Masaryk University, Kotlá?ská 2, 611 37 Brno, Czech Republic;1. Department of Medical Biotechnologies and Translational Medicine, University of Milan, via Vanvitelli, 32, 20129 Milan, Italy;2. Department of Pharmacological and Biomolecular Sciences, University of Milan, via Balzaretti, 9, 20133 Milan, Italy;3. CNR Institute of Neurosciences, Cellular and Molecular Pharmacology Section, University of Milan, via Vanvitelli, 32, 20129 Milan, Italy;1. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA;2. Andlinger Center for Energy and the Environment, Princeton University, Princeton, NJ 08544, USA;3. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA |
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| Abstract: | Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD65 and GAD67 genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD65 and GAD67 resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds. |
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