Nuclear localization of tetrahydrobiopterin biosynthetic enzymes.

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.

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin.

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.

Plasma tetrahydrobiopterin and its pharmacokinetic following oral administration.

Tetrahydrobiopterin (BH(4)) is widely used as a therapeutic agent in patients with BH(4) deficiencies and mild forms of phenylketonuria (PKU) and there is an increasing need for the measurement of its plasma concentrations in patients with cardiovascular disorders. We measured BH(4) and total biopterin in dithioerythritol (DTE) pretreated plasma from four adults after oral administration of BH(4) (2, 10, and 20mg/kg body weight) using the differential iodine oxidation method. About 80% (range 64.8-92.2% ) of total biopterin was found as BH(4) when analyzed immediately after blood sampling. Compared with ascorbic acid as an antioxidant, DTE was more protective against oxidation of BH(4), particularly in samples stored over a period of 8 months. Without antioxidant (DTE or ascorbic acid) almost no BH(4) was detected. Furthermore, BH(4) and total biopterin were measured at different time intervals (up to 33 h after oral administration) and pharmacokinetic parameters T(max) (1-4h), C(max) (258.7-259.0 nmol/L biopterin at a dosage of 10mg/kg), and area under the curve (AUC=1708-1958 nmol(*)h/L up to T=10h) were estimated. The elimination half-life time was calculated to be 3.3-5.1h. Doubling the BH(4) dosage to 20mg/kg resulted in 60% higher AUC while sublingual BH(4) application (2mg/kg) resulted in 58-76% higher BH(4) plasma concentrations when compared with oral administration. These preliminary data suggest that in patients with BH(4) cofactor defects and BH(4)-responsive phenylalanine hydroxylase deficiency, BH(4) should be given in at least two to three daily doses and that sublingual administration may lower the required BH(4) dosage and subsequently the cost of treatment. Due to inter individual differences in pharmacokinetic properties, in some patients with hyperphenylalaninemia and mild PKU plasma BH(4) levels may be not high enough to fully activate the liver phenylalanine hydroxylase and thus lower blood phenylalanine levels. Assessment of plasma BH(4) or total biopterin concentrations may be a good way to control the efficacy of the loading test.

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase.

Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.