Novel silkworm (Bombyx mori) sulfotransferase swSULT ST3 is involved in metabolism of polyphenols from mulberry leaves

Abstract

Polyphenols in plants are important for defense responses against microorganisms, insect herbivory, and control of feeding. Owing to their antioxidant, anti-cancer, and anti-inflammatory activities, their importance in human nutrition has been acknowledged. However, metabolism of polyphenols derived from mulberry leaves in silkworms (Bombyx mori) remains unclear. Sulfotransferases (SULT) are involved in the metabolism of xenobiotics and endogenous compounds. The purpose of this study is to investigate the metabolic mechanism of polyphenols mediated by B. mori SULT. Here, we identified a novel SULT in silkworms (herein, swSULT ST3). Recombinant swSULT ST3 overexpressed in Escherichia coli effectively sulfated polyphenols present in mulberry leaves. swSULT ST3 showed high specific activity toward genistein among the polyphenols. Genistein-7-sulfate was produced by the activity of swSULT ST3. Higher expression of swSULT ST3 mRNA was observed in the midgut and fat body than in the hemocytes, testis, ovary, and silk gland. Polyphenols inhibited the aldo-keto reductase detoxification of reactive aldehydes from mulberry leaves, and the most noticeable inhibition was observed with genistein. Our results suggest that swSULT ST3 plays a role in the detoxification of polyphenols, including genistein, and contributes to the effects of aldo-keto reductase in the midgut of silkworms. This study provides new insight into the functions of SULTs and the molecular mechanism responsible for host plant selection in lepidopteran insects.

Fig 1. SULT mRNA relative expression.

(A) swSULT ST3 mRNA was detected using qPCR in different tissues of Bombyx mori. The corresponding values are relative to that of reference gene rp49. (B) Time-dependent expression of swSULT ST3 mRNA. Error bars indicate the standard deviation of three separate experiments.

Fig 2. SDS-PAGE analysis of purified B. mori swSULT ST3.

Proteins were run on a 15% polyacrylamide gel and stained with Coomassie Blue. Lanes 1 and 2 correspond to molecular size markers and swSULT ST3 after Superdex 200 column, respectively. Molecular sizes are shown on the right.

Fig 3. Thin-layer chromatography (TLC) of [³⁵S] sulfated derivatives produced by swSULT ST3 with various substrates.

Representative autoradiographs derived from TLC plates after elution are shown here. Lane numbers for (A) and (B) indicate 1) DMSO, 2) p-nitrophenol, 3) 1-naphthol, 4) naphthylamine, 5) dopamine, 6) serotonin, 7) triiodothyronine, 8) daidzein, 9) resveratrol, 10) xanthurenic acid, 11) 17β-estradiol, 12) dehydroepiandrosterone, 13) corticosterone, 14) chlorogenic acid, 15) caffeic acid, 16) rutin, 17) genistein, 18) catechin, 19) apigenin, 20) DMSO, and 21) quercetin. Cellulose (A) and silica gel (B) TLC plates were used. The arrows and square brackets indicate the sulfated products of each substrate.

Fig 4. Analysis of reaction products of swSULT ST3 using liquid-chromatography-tandem mass spectrometry (LC-MS/MS).

Mass chromatograms are shown on selective mass traces at m/z 269.0450 (blue), m/z 349.0018 (magenta) and m/z 428.9256 (brown). The reaction mixture after incubation of genistein with swSULT ST3 and PAPS (A) and genistein-7-sulfate standard (B) were loaded onto LC-MS/MS. Chromatographic conditions are described in the Methods section. Genistein, the reaction product, and genistein-7-sulfate are indicated by an arrow.

Fig 5. Negative ion mass-spectrometry (MS) spectra and tandem MS product ions spectra of genistein metabolite and genistein-7-sulfate of swSULT ST3.

Analysis of the genistein metabolite (A) and genistein-7-sulfate (B) was carried out. Each panel includes the result of MS full scan and production ion scan at m/z 349.0018.

Fig 6. Phylogenetic analysis of sulfotransferase (SULT) amino acid sequences.

Primary amino acid sequence comparisons were performed by the neighbor-joining method using various SULT sequences retrieved from the Swiss-Prot database (http://web.expasy.org/docs/swiss-prot_guideline.html). The bootstrap value for each node is shown.