Recombinant Tyrosine Hydroxylase Research Articles

The Effect of Recombinant Tyrosine Hydroxylase Expression on the Neurogenic Differentiation Potency of Mesenchymal Stem Cells

ObjectiveTyrosine hydroxylase (TH) is a rate-limiting enzyme in dopamine synthesis, making the enhancement of its activity a target for ensuring sufficient dopamine levels. Rat bone marrow mesenchymal stem cells (rBM-MSCs) are known to synthesize TH after differentiating into neuronal cells through chemical induction, but the effect of its ectopic expression on these cells has not yet been determined. This study investigated the effects of ectopic recombinant TH expression on the stemness characteristics of rBM-MSCs. MethodsAfter cloning, a cell line with stable TH expression was maintained, and the proliferation, the gene expression profile, and differentiation potential of rBM-MSCs were analyzed. Analysis of the cells showed an increment in the proliferation rate that could be reversed by the neutralization of TH. ResultsThe constitutive expression of TH in rBM-MSCs was successfully implemented, without significantly affecting their osteogenic and adipogenic differentiation potential. TH expression improved the expression of other neuronal markers, such as glial fibrillary acidic protein, β-tubulin, nestin, and c-Fos, confirming the neurogenic differentiation capacity of the stem cells. The expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) significantly increased after the chemical induction of neurogenic differentiation. ConclusionIn this study, the expression of recombinant TH improved the neuroprotective effect of MSCs by upregulating the expression of BDNF and CNTF. Although the neuronal markers were upregulated, the expression of recombinant TH alone in rBM-MSCs was not sufficient for MSCs to differentiate into neurogenic cell lines.

Effect of tyrosine hydroxylase overexpression in lymphocytes on the differentiation and function of T helper cells.

The aim of the present study was to examine the effect of the overexpression of tyrosine hydroxylase(TH), a rate-limiting enzyme for the synthesis of catecholamines(CAs), in lymphocytes on the differentiation and function of Thelper(Th) cells. A recombinant TH overexpression plasmid (pEGFP-N1-TH) was constructed and transfected into mesenteric lymphocytes using nucleofection technology. These cells were stimulated with concanavalinA(ConA) for 48h and then examined for THexpression and CAcontent, as well as for the percentage of Th1 and Th2cells, cytokine concentrations and for the levels of signaling molecules. The lymphocytes overexpressing TH also expressed higher mRNA and protein levels of TH, and synthesized more CAs, including norepinephrine(NE), epinephrine(E) and dopamine(DA) than the mock-transfected control cells. THgene overexpression in the lymphocytes reduced the percentage of interferon-γ(IFN-γ)-producing CD4+cells and the ratio of CD4+IFN-γ+/CD4+IL-4+cells, as well as the percentages of CD4+CD26+ and CD4+CD30+cells and the ratio of CD4+CD26+/CD4+CD30+cells. THoverexpression also reduced the secretion of IFN-γ and tumor necrosis factor(TNF) from lymphocytes. Moreover, NE inhibited the ConA-induced lymphocyte proliferation and decreased both cyclic adenosine monophosphate(cAMP) levels and p38mitogen-activated protein kinase(MAPK) expression in the lymphocytes. Our findings thus indicate that THgene overexpression promotes the polarization and differentiation of CD4+cells towards Th2cells, and this effect is mediated by the cAMP and p38MAPK signaling pathways.

Characterization of tyrosine hydroxylase from Manduca sexta

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.

Assessment of recovery in the hemiparkinson rat: Drug-induced rotation is inadequate

Recovery from apomorphine-induced rotational behavior was compared to sensorimotor and motor function in hemiparkinsonian rats receiving intrastriatal grafts of astrocytes expressing recombinant tyrosine hydroxylase (TH) or control β-galactosidase (β-gal). Rats received unilateral intranigral infusions of 6-hydroxydopamine (6-OHDA). Animals with large lesions, as determined by apomorphine-induced rotation, received grafts of astrocytes into the denervated striatum. Behavioral recovery was assessed on days 14–16 post-transplantation using apomorphine-induced rotation, somatosensory neglect, and reaching for pellets using the Montoya staircase method. Rats that received transplants of TH-transfected astrocytes showed a 34% decrease in rotational behavior, but no consistent recovery of somatosensory neglect or skilled reaching. Post-mortem histological analyses revealed survival of grafted astrocytes in host striatum and expression of TH at 17 days post-transplantation. We suggest that TH-expressing astrocytes may reverse post-synaptic dopamine (DA) receptor supersensitivity; however, sensorimotor and motor abilities are not restored due to a failure by TH-expressing astrocytes to reestablish dopaminergic circuitry. The present results demonstrate the need to utilize a variety of sensory and motor behavioral tests that cohesively provide greater interpretability than a single behavioral measure used in isolation, such as drug-induced rotational behavior, to assess the efficacy of experimental gene therapies.

Production of functional recombinant tyrosine hydroxylase by the BPV-1 expression plasmids in the cell cultures

Bovine papilloma virus type-1 (BPV-1)-based expression plasmids TkBPVTH and CGalBPVTH encoding the rat tyrosine hydroxylase (TH) enzyme have been designed for the development of gene therapy for experimental Parkinson’s disease. The aim of the present work was to examine the transfection of BPVTH plasmids to express a dopaminergic transgene in the monkey CV1-P fibroblast, rat C6 glioma and human NHA astrocyte cell cultures. The biological function of the transgene was estimated by analyzing the production of recombinant TH mRNA and protein, and the synthesis of l-dopa and dopamine. The highest transfection efficiency was obtained using TkBPVTH plasmids (5 μg). Furthermore, the expression of TkBPVTH plasmids was associated with significant synthesis of TH enzyme and l-dopa in the C6 and NHA cell cultures.

Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain

In this study, we developed an immuno-autoradiographic method to obtain quantitative estimates of tyrosine hydroxylase (TH) protein in tissue sections from post-mortem human brain. Protein from tissue sections containing the locus coeruleus (LC) was directly transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoreactive TH on PVDF membranes was identified with optimized concentrations of TH antibody followed by application of [ 125I]labeled secondary antibody. Quantities of TH on autoradiograms were estimated by comparing optical densities of transferred immunoblots to a calibrated standard curve produced with purified recombinant TH dotted onto the same PVDF membranes. Amounts of TH-immunoreactivity in the LC were proportional to the thickness of tissue sections up to 15 μm. However, the amounts of total protein, as measured by Ponceau S staining, were linearly related to section thicknesses up to 30 μm. Comparisons of quantities of immunoreactive TH in the LC using this method to amounts determined using traditional Western blotting, in which LC tissue was punched from adjacent sections from the same subject, showed a positive correlation ( r 2=0.99, P<0.01). Using the transfer immunoblot method, an uneven distribution of TH protein was observed along the rostrocaudal axis of the human LC ( P<0.01). This method may provide a sensitive and useful tool for the study of the role of human TH expression in the pathophysiology of psychiatric disease.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.

The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in L-DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser19 (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser40 phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser40, as opposed to Ser19, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser40 but not Ser19. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser19 phosphorylation had been eliminated (by substitution of Leu for Ser19). Thus, depolarization-induced increases in the stoichiometry of Ser19 phosphorylation do not appear to influence directly the activity of TH in situ.

P-205 - Dopamine synthesis and release in AtT20 cells expressing recombinant tyrosine hydroxylase (TH)

P-205 - Dopamine synthesis and release in AtT20 cells expressing recombinant tyrosine hydroxylase (TH)

Regulation of L-DOPA biosynthesis by site-specific phosphorylation of tyrosine hydroxylase in AtT-20 cells expressing wild-type and serine 40-substituted enzyme.

De novo L-DOPA biosynthesis was studied in stably transfected AtT-20 cells expressing wild-type- or [Leu40]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser4O (S40L-rTH) than by those expressing wild-type rTH (WT-rTH). Treatment of WT-rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT-rTH phospho-Ser40 content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of 32Pi-prelabeled cells, WT-rTH was phosphorylated at Ser8, Ser19, Ser31, and Ser40, whereas 32P incorporation into S40L-rTH was restricted to Ser8, Ser19, and Ser31. Relatively prolonged treatment of AtT-20 cells expressing WT-rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser40; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH. Thus, the present studies demonstrate that Ser40 phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser40 phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.

Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis. This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain. In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH. We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria. Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (NΔ40, NΔ155, NΔ165, NΔ175 and NΔ200; NΔ denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (CΔ19 and CΔ50). The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479. The amino-terminal deletion mutants, NΔ40, NΔ155 and NΔ165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (CΔ19) results in a 70% reduction in enzyme activity. Removal of additional sequences (ten more residues from the N-terminus [NΔ175]; or an additional 31 amino acids from the C-terminus [CΔ50]) results in protein that is totally without enzyme activity. As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA. We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme. These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment.

Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR

Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4). No effects of hTH1-Zn2+ on 1/T1 or 1/T2 are found. From paramagnetic effects of hTH1-Co2+ on 1/T1 of phenylalanine protons at 250 and 600 MHz, in the presence of BH2, a correlation time (tau c) of 1.8 +/- 0.1 ps was found. Using this tau c value, and assuming that only one proton of the pairs H3,H5, and H2,H6 is experiencing the total paramagnetic effect (asymmetric limiting case), distances from enzyme-bound Co2+ to phenylalanine (+/- 1.2 A) of 6.1 A (H3 or H5), 6.3 A (H2 or H6), 7.0 A (H4), 7.3 A (H alpha), > or = 7.4 A (H beta-pro-S), and > or = 7.6 A (H beta-pro-R) were calculated. The distances to H3 or H5 and to H2 or H6 are slightly increased to 6.8 and 7.0 A, respectively, if each proton of both degenerate pairs equally experiences the paramagnetic effect of Co2+ (symmetric limiting case). These distances place the aromatic ring of phenylalanine in the second coordination sphere of the metal, which would permit an Fe-bound oxy or peroxy species to approach molecular contact with C3/C4, suggesting a direct role of Fe2+ in the hydroxylation reaction. The same correlation time and similar distances were found in the absence of BH2 with H4 of phenylalanine slightly closer to the metal. In the ternary hTH1-Zn(2+).BH2.phenylalanine complex, eight interproton distances in the enzyme-bound phenylalanine were determined by NOESY spectra at 600 MHz at 35-, 50-, and 75-ms mixing times. The conformation of enzyme-bound phenylalanine, consistent with the six Co(2+)-proton distances and the eight interproton distances, is partially extended with torsional angles chi 1 = 97 degrees +/- 3 degrees and chi 2 = -78 degrees +/- 2 degrees.

Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing.

The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics. The polypeptides have molecular weights similar to those of TH expressed in Xenopus oocytes and react with antibodies to TH. The two isoforms were purified with a purity of 90% using a three-step procedure. The phosphorylation sites have been determined in the two isoforms after labeling with [gamma-32P]ATP in the presence of cAMP-dependent protein kinase (PKA) or calmodulin-dependent protein kinase II (CaM-PK II). In both isoforms, Ser-40 was found to be phosphorylated by PKA, and Ser-19 and Ser-40 were found to be phosphorylated by CaM-PK II. The putative phosphorylation site generated by alternative splicing (Ser-31) was phosphorylated specifically by CaM-PK II in TH-2 only. The kinetic properties of the two isoforms in the presence of various concentrations of the substrate (tyrosine) and of the natural cofactor [6R)-tetrahydrobiopterin) were also analyzed. TH produced in E. coli is unphosphorylated but nevertheless active. At 50 microM tyrosine and 300 microM (6R)-tetrahydrobiopterin, the specific activities of TH-1 and TH-2 are 1300 and 620 nmol of dihydroxyphenylalanine/min/mg, respectively. Phosphorylation of TH-1 and TH-2 by PKA activates both isoenzymes as shown by the increase in the affinity for the cofactor. No changes in kinetic parameters of the isoenzymes were observed after phosphorylation by CaM-PK II. Dopamine was found to inhibit both TH isoenzymes to the same extent as shown by their similar Ki values for dopamine. These values were increased after phosphorylation of each enzyme by PKA. Unlike TH-1, phosphorylation of TH-2 by CaM-PK II resulted in an increase of the Ki value for dopamine. This property may be related to the presence of the additional phosphorylated residue in TH-2 isoform.

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-BH 4) cofactor were determined to be 7.2 μM (420 μM 6R-BH 4), 19μM ([6R-BH 4] < 55 μM, 20 μM tyrosine) and 54 μM ([6R-BH 4] > 55 μM, 20 μM tyrosine), respectively. These were in good agreement with previously reported values for this enzyme.