title: Nuclear localization of tetrahydrobiopterin biosynthetic enzymes.
abstract: Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.
authors.affiliation: Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland.
authors.forename: Lina
authors.initials: L
authors.lastname: Elzaouk
authors.forename: Stephanie
authors.initials: S
authors.lastname: Laufs
authors.forename: Dirk
authors.initials: D
authors.lastname: Heerklotz
authors.forename: Walter
authors.initials: W
authors.lastname: Leimbacher
authors.forename: Nenad
authors.initials: N
authors.lastname: Blau
authors.forename: Annette
authors.initials: A
authors.lastname: Résibois
authors.forename: Beat
authors.initials: B
authors.lastname: Thöny
mesh_terms: D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D001921:Brain / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D007684:Kidney Tubules / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D001708:Biopterins; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D006136:GTP Cyclohydrolase; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin