Hirotoshi Fuda

SITE-SELECTED MUTAGENESIS OF A CONSERVED NUCLEOTIDE BINDING HXGH MOTIF LOCATED IN THE ATP SULFURYLASE DOMAIN OF HUMAN BIFUNCTIONAL 3'-PHOSPHOADENOSINE 5'-PHOSPHOSULFATE SYNTHASE

3′-Phosphoadenosine-5′-phosphosulfate (PAPS) synthase is a bifunctional protein consisting of an NH2-terminal APS kinase and a COOH-terminal ATP sulfurylase. Both catalytic activities require ATP; the APS kinase domain involves cleavage of the β-γ phosphodiester bond of ATP, whereas the ATP sulfurylase domain involves cleavage of the α-β phosphodiester bond of ATP. Previous mutational studies have suggested that β-γ phosphodiesterase activity involves a highly conserved NTP-binding P-loop motif located in the adenosine-5′-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in the ATP sulfurylase domain of PAPS synthases, a motif implicated in the α-β phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP sulfurylase domain of human PAPS synthase (amino acids 425–428) was performed to examine this possibility. Either H425A or H428A mutation produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an important role for the HXGH histidines in the ATP sulfurylase activity of bifunctional PAPS synthase and support the hypothesis that the highly conserved HXGH motif found in the ATP sulfurylase domain of PAPS synthases is involved in ATP binding and α-β phosphodiesterase activity.

Expression of Cholesterol Sulfotransferase (SULT2B1b) in Human Platelets

Background—Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. Methods and Results—We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4°C but substantially diminished over a period of 4 hours at 37°C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. Conclusions—Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37°C is subject to regulation by the apoA-I component of HDL.

Cholesterol sulfate induces expression of the skin barrier protein filaggrin in normal human epidermal keratinocytes through induction of RORα.

Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased RORα expression. Further studies are required to fully elucidate the mechanisms involved.

Regulation of SULT2B1a (pregnenolone sulfotransferase) expression in rat C6 glioma cells: Relevance of AMPA receptor-mediated NO signaling

The neurosteroid pregnenolone sulfate (PREGS), which is synthesized in glial cells, plays a significant role in learning and memory performance. The aim of this study was to investigate the regulation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes the conversion of pregnenolone to PREGS, using the rat C6 glioma cell line. Rat C6 glioma cells expressed the SULT2B1a isoform, which sulfonates pregnenolone, but, neither the SULT2B1b isoform, which catalyzes cholesterol, nor the prototypical steroid sulfotransferase SULT2A1 were expressed in these cells. Increasing concentrations of l-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression; however, neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide also inhibited SULT2B1a expression. Attenuation of SULT2B1a expression by L-glutamic acid was reversed by the selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and partially reversed by the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI). Induction of inducible NOS by TNF-alpha in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression; this was partially reversed by the specific inducible NOS inhibitor N(6)-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Furthermore, exposure to exogenous NO donors inhibited SULT2B1a mRNA expression, and exposure to sodium nitroprusside, LPS/TNF-alpha and L-glutamic acid in combination with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.