Characterization of Wild-Type and Mutants of Recombinant Human GTP Cyclohydrolase I: Relationship to Etiology of Dopa-Responsive Dystonia Takahiro Suzuki, Tamae Ohye, Hidehito Inagaki, Toshiharu Nagatsu, and Hiroshi Ichinose Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Aichi, Japan Abstract: To explore the molecular etiology of two dis- orders caused by a defect in GTP cyclohydrolase I— he- reditary progressive dystonia with marked diurnal fluctu- ation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency—we purified and analyzed recombinant hu- man wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant-negative effect of chi- meric protein composed of wild-type and mutant sub- units is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD. Key Words: GTP cyclohydro- lase I—Tetrahydrobiopterin—Hereditary progressive dys- tonia with marked diurnal fluctuation—Dopa-responsive dystonia—Hyperphenylalaninemia—Recombinant protein. J. Neurochem. 73, 2510 –2516 (1999). GTP cyclohydrolase I catalyzes the formation of D-erythro-7,8-dihydroneopterin triphosphate (NPH 2 P 3 ) from GTP. This is the first and rate-limiting step for the de novo biosynthesis of tetrahydrobiopterin (BPH 4 ) (Ni- chol et al., 1985). BPH 4 is known as a natural cofactor for three aromatic amino acid monooxygenases, i.e., phenylalanine, tyrosine, and tryptophan hydroxylases, and for nitric oxide synthase (Kaufman, 1993). The ar- omatic amino acid monooxygenases are essential for synthesizing hormones and neurotransmitters such as dopamine, norepinephrine, epinephrine, serotonin, and melatonin. The nitric oxide synthase generates nitric oxide, which is a regulatory factor in various physiolog- ical processes. Besides functioning as a cofactor, BPH 4 has also been suggested to be involved in cell prolifera- tion (Tanaka et al., 1989; Anastasiadis et al., 1997). GTP cyclohydrolase I is induced by cyclic AMP- elevating reagents (Abou-Donia et al., 1986; Zhu et al., 1994) or cytokines such as interferon-, tumor necrosis factor-, and kit ligand (Werner et al., 1993; Ziegler et al., 1993). Recently, in addition to this transcriptional regulation, it has been demonstrated that transient phos- phorylation of GTP cyclohydrolase I correlates with an increase in its activity in receptor- or phorbol ester- stimulated mast cells (Hesslinger et al., 1998) and renal mesangial cells (Lapize et al., 1998). Taken together, the activity of GTP cyclohydrolase I is considered to be strictly regulated in vivo, but the molecular mechanisms that regulate the expression and modification of GTP cyclohydrolase I are still unknown. Hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dys- tonia (DRD), is a disorder typically characterized by childhood onset, concurrent or subsequent development of parkinsonism, and a dramatic therapeutic response to L-dopa (Segawa et al., 1971; Nygaard et al., 1988). We previously reported cloning and the structure of the hu- man and mouse GTP cyclohydrolase I gene (Ichinose et al., 1995) and found the gene to be a causative gene for HPD/DRD (Ichinose et al., 1994). After our first discov- ery, 60 families with patients having HPD/DRD have been reported to date from all over the world. The kinds of mutations found in the coding region of the GTP Received June 1, 1999; revised manuscript received August 11, 1999; accepted August 11, 1999. Address correspondence and reprint requests to Dr. H. Ichinose at Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, 470-1192, Japan. Abbreviations used: BPH 4 , tetrahydrobiopterin; DRD, dopa-respon- sive dystonia; GST, glutathione S-transferase; GST-hGCH, glutathione S-transferase-fusion protein of the wild-type human GTP cyclohydro- lase I; HPD, hereditary progressive dystonia with marked diurnal fluctuation; NP, neopterin; NPH 2 P 3 , D-erythro-7,8-dihydroneopterin triphosphate; PAGE, polyacrylamide gel electrophoresis; PBS, phos- phate-buffered saline; PHA, phytohemagglutinin; rhGCH, recombinant human GTP cyclohydrolase I digested at a designed site; SDS, sodium dodecyl sulfate. 2510 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry