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Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana.

ABSTRACT: The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme preferred flavonoid glycosides (e.g. kaempferol-3-glucoside and quercetin-3-glucoside) rather than their aglycone counterparts. Among a series of hydroxyflavones tested, AtSULT202B7 showed the enzymatic activity only for 7-hydroxyflavone. pH-dependency study showed that the optimum pH was relatively low (pH 5.5) compared with those (pH 6.0-8.5) previously reported for other isoforms. Based on the comparison of high performance (pressure) liquid chromatography (HPLC) retention times between sulfated kaempferol and the deglycosylated product of sulfated kaempferol-3-glucoside, the sulfation site in sulfated kaempferol-3-glucoside appeared to be the hydroxyl group of the flavonoid skeleton. In addition, by using direct infusion mass spectrometry, it was found that the sulfated product had one sulfonate group within the molecule. These results indicated that AtSULT202B7 functions as a flavonoid glycoside 7-sulfotransferase.

SUBMITTER: Hashiguchi T

PROVIDER: S-EPMC3905610 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana.

Hashiguchi Takuyu T Sakakibara Yoichi Y Shimohira Takehiko T Kurogi Katsuhisa K Yamasaki Masao M Nishiyama Kazuo K Akashi Ryo R Liu Ming-Cheh MC Suiko Masahito M

Journal of biochemistry 20131106 2

The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, f ...[more]

PMID: 24202284

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Project description:Background and purposeOsteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti-inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms.MethodsThe docking pose of Rob and RANKL was identified by protein-ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor-κB (NF-κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL-induced ROS generation in osteoclasts was detected by H2 DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real-time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen-deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo.ResultsComputational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL-mediated osteoclastogenesis dose-dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL-induced mitochondrial ROS production or enhanced activities of ROS-scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c-Fos, and Mmp9) and reducing Ca2+ oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function.ConclusionsRob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.

Dataset Information

Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana.

Publications

Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana.

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