Expression of human tyrosine hydroxylase cDNA in invertebrate cells using a baculovirus vector

A human cDNA containing the complete coding sequence for a human tyrosine hydroxylase (EC 1.14.16.2, form 2) was introduced into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) downstream to the polyhedrin promoter. Infection of Spodoptera frugiperda cells (SF9) with recombinant virus resulted in the expression of human tyrosine hydroxylase in these invertebrate cells. Characterization of tyrosine hydroxylase activity in infected SF9 cells demonstrated both substrate and cofactor kinetics that were characteristic of those previously reported for the native human enzyme. Both 3-iodotyrosine and alpha-methyl-p-tyrosine competitively inhibited the recombinantly produced tyrosine hydroxylase with Ki values of 1.2 and 16 microM, respectively, similar to those previously reported for the rat and human enzymes. Western blot analysis of extracts of SF9 cells infected with the recombinant baculovirus containing human tyrosine hydroxylase cDNA revealed a major immunoreactive band with an apparent Mr of 60 kDa, identical to the size of the immunoreactive protein from rat adrenal and caudate nucleus. The use of the baculovirus expression system to produce abundant quantities of each of the multiple forms of active human tyrosine hydroxylase in eukaryotic cells should facilitate structural analysis and help clarify the physiological significance of each of the isoenzymes.     

Expression of tyrosine hydroxylase is epigenetically regulated in neural stem cells

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis of catecholamines, and its expression is regulated in a developmental stage- and cell type-specific manner. Our previous work suggested that the genetic elements responsible for cell type-specific expression of TH were in the repressor region of the TH promoter between −2187 and −1232 bp. To investigate the molecular mechanisms underlying the specificity of TH expression, the DNA methylation patterns of the CpG islands in the repressor region of the TH promoter were examined in human neural stem cells (NSCs) and dopaminergic neuron-like cells. Using a bisulfite sequencing method, we found that the cytosine residues of CpG islands within the NRSE-R site were specifically methylated in NSCs, but not in SH-SY5Y neuroblastoma cells. In NSCs, CpG methylation correlated with reduced TH gene expression, and inhibition of DNA methylation with 5-azacytidine restored TH expression. Furthermore, methyl-CpG binding domain proteins (MBDs) bound to the highly methylated X-1 and X-2 regions of the TH gene in NSCs. Taken together, these results suggest that region-specific methylation and MBDs play important roles in TH gene regulation in NSCs.     

Expression of functional human C1 inhibitor in COS cells

Full length human C1 inhibitor cDNA was cloned into a vector suitable for transient expression in COS-1 cells. Transfected COS cells secreted an immunoreactive protein of Mr approximately 110,000 that appeared to be functionally equivalent to the plasma-derived protein as established by the following criteria: 1) ability to form sodium dodecyl sulfate-stable complexes with C1s, factor XIIa, and kallikrein; 2) inhibition of C1s-mediated C4 consumption; and 3) susceptibility to inactivation by the nontarget proteinase elastase. Quantitation of secreted recombinant C1 inhibitor by radioimmunoassay indicated that 72 h after transfection the level was approximately 2.2 micrograms/ml. Treatment of transfected cells with tunicamycin resulted in secretion of a protein of Mr approximately 90,000 that was also capable of complex formation with C1s.     

The human tyrosine hydroxylase gene

1. Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis, and it is a pterin-requiring monooxygenase. Both cDNAs and genomic DNA of human TH have been cloned and the nucleotide sequence has been determined. 2. Four similar but distinct mRNAs encode human TH. The results of Southern blot analysis and the nucleotide sequence of the human TH genomic DAN indicate that the four types of human TH mRNA are produced through alternative splicing from a single gene. 3. The human TH gene was split into 4 exons and 13 introns. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon and the inclusion/exclusion of the second exon. 4. The four types (type 1-4) were expressed in COS cells, and all had enzymatic activities. The type 1 enzyme had the highest homospecific activity (activity per enzyme protein), the values for the other enzymes ranging from 30 to 40%. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were similar.     

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.     

酪氨酸羟化酶与视网膜疾病

酪氨酸羟化酶(tyrosine hydroxylase,TH)是儿茶酚胺(catecholamines,CAs)合成过程中的限速酶,提高或抑制该酶的活性可大幅度影响CAs的合成。近年来对于TH的关注越来越多,大量的研究表明,TH含量及活性的异常改变能通过影响CAs的含量而导致相应生理功能的异常,从而导致疾病。本文将TH的结构功能、分布、表达变化及其与视网膜疾病关系做一综述。     

酪氨酸羟化酶与视网膜疾病

酪氨酸羟化酶（tyrosine hydroxylase,TH）是儿茶酚胺（catech01amines,CAs）合成过程中的限速酶,提高或抑制该酶的活性可大幅度影响CAs的合成。近年来对于TH的关注越来越多,大量的研究表明,TH含量及活性的异常改变能通过影响CAs的含量而导致相应生理功能的异常,从而导致疾病。本文将TH的结构功能、分布、表达变化及其与视网膜疾病关系做一综述。     

Expression of mouse tyrosine hydroxylase in Escherichia coli.

Enzymatically active mouse tyrosine hydroxylase (TH) was successfully expressed at a high level in Escherichia coli using a T7 RNA polymerase directed expression system. The specific activity of mouse TH in E. coli cell lysate was 7.5 nmol/mg protein/min. Kinetic characteristics of recombinant TH were examined. Km for tyrosine and (6R)-tetrahydrobiopterin (6R-BH4) cofactor were determined to be 7.2 microM (420 microM 6R-BH4), 19 microM [( 6R-BH4] less than 55 microM, 20 microM tyrosine) and 54 microM [( 6R-BH4] greater than 55 microM, 20 microM tyrosine), respectively. These were in good agreement with previously reported values for this enzyme.     

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.     

Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase

As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339], tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated at an identical site (site A) by cyclic AMP-dependent protein kinase, the calmodulin-dependent multiprotein kinase and protein kinase C, while the calmodulin-dependent multiprotein kinase also phosphorylates another unique site (site C). Preparations of tyrosine hydroxylase purified from this source are also contaminated with traces of a fourth protein kinase which phosphorylates another unique site (site E). We have isolated tryptic peptides containing each of these sites and determined their amino acid sequences. By comparison of these data with the known cDNA sequence for rat tyrosine hydroxylase, we have been able to identify these sites as Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also phosphorylated a secondary site which was identified as ser-153. All of these phosphorylation sites are in the amino-terminal region, where there is no significant homology with the closely related enzyme, phenylalanine hydroxylase. Our data also establish that the initiator methionine is removed by post-translational processing to leave pro-2 as the amino-terminus of the mature protein. The significance of these results for the mechanism of action of extracellular signals on catecholamine biosynthesis is discussed.     

Distribution of tyrosine hydroxylase in human and animal brain

The activity of tyrosine hydroxylase (EC 1.10.3.1) when assayed under ideal conditions in young human brains, was comparable to that in brains of other species in level of activity and distribution. The highest levels of activity were in the putamen, caudate nucleus and substantia nigra, in keeping with data on other species. The caudate activity in human brain appeared to decrease substantially with increasing age. In both humans and baboons, the enzyme in the neostriatum was particle-bound and inhibited by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. In the substantia nigra it was soluble and stimulated by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. The data suggest that tyrosine hydroxylase may be produced in a soluble form in the cell bodies of the substantia nigra but become bound as it moves toward the nerve endings in the putamen and caudate nucleus. The bound form of the enzyme was unstable but the soluble form exhibited considerable stability.     

Frequent sequence variant in the human tyrosine hydroxylase gene

A polymorphism of human tyrosine hydroxylase changing the amino acid ⁸¹Val to ⁸¹Met is located in exon 2 of the human tyrosine hydroxylase gene.     

Subcellular distribution of human tyrosine hydroxylase isoforms 1 and 4 in SH-SY5Y cells

Tyrosine hydroxylase (TH) is the key enzyme that controls the rate of synthesis of the catecholamines. SH-SY5Y cells with stable transfections of either human tyrosine hydroxylase isoform 1 (hTH1) or human tyrosine hydroxylase isoform 4 (hTH4) were used to determined the subcellular distribution of TH protein and phosphorylated TH, under basal conditions and after muscarine stimulation. Muscarine was previously shown to increase the phosphorylation of only serine 19 and serine 40 in hTH1 cells. Under basal conditions, the hTH1 and hTH4 proteins, their serine 19 phosphorylated forms and hTH1 phosphorylated at serine 40 were all similarly distributed; with ~80% in the cytosolic fraction, ~20% in the membrane fraction, and less than 1%, or not detectable, in the nuclear fraction. However, hTH4 phosphorylated at serine 71 had a significantly different distribution with ~65% cytosolic and ~35% membrane associated. Muscarine stimulation led to hTH1 being redistributed from the cytosol and nuclear fractions to the membrane fraction and hTH4 being redistributed from the cytosol to the nuclear fraction. These muscarine stimulated redistributions were not due to TH phosphorylation at serine 19, serine 40, or serine 71 and were most likely due to TH binding to proteins whose phosphorylation was increased by muscarine. This is the first study to show a difference in subcellular distribution between two human TH isoforms under basal and stimulated conditions.     

Purification and immunochemical properties of tyrosine hydroxylase in human brain

Tyrosine hydroxylase (TH) was isolated from human brain (caudate nucleus + putamen). The major form of the active enzyme in the cytoplasmic fraction was purified to apparent homogeneity. The molecular weight of the purified enzyme was estimated to be 280 kdalton by gel filtration. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) of the purified enzyme gave a single subunit with mol. wt 60 kdalton, which is similar to the subunit of human adrenal TH. Using a sandwich enzyme immunoassay (EIA), the presence of inactive form(s) of TH in human brain was demonstrated, and the total content of this immunoinactive form(s) was approx. 8 times higher than that of the active form. By the Western blot technique after two-dimensional (2-D) electrophoresis, TH in the crude fraction of the human brain was found to consist of multiple forms with different pI-values and with the same molecular weight. The pl of the major spots ranged from 5.3 to 5.8, and that of the minor spot was 6.0. Because the pl of the purified enzyme preparation was 6.0, this protein with pI at 6.0 may be the active form of TH.     

Purification and immunochemical characterization of human adrenal tyrosine hydroxylase

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M_r of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M_r of 60,000 similar to the M_r of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the “Western Blot” method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.