马氏珠母贝磺基转移酶基因的克隆及功能

硫酸角质素具有丰富的携带大量负电荷的磺酸基团,参与生物矿化的成核过程。磺基转移酶催化磺酸基团的转移,对硫酸角质素的生物合成起决定性作用。本研究利用RACE技术克隆马氏珠母贝磺基转移酶PmCHST1a全长,并通过RNA干扰技术检测PmCHST1a对硫酸角质素合成及贝壳珍珠层形成的影响。结果显示,PmCHST1a基因全长1385 bp,编码366个氨基酸;含有磺基转移酶结构域,具有跨膜结构和信号肽,定位于高尔基体上。组织差异表达分析发现,PmCHST1a在中央膜显著高表达。注射PmCHST1a的RNAi探针后,PmCHST1a在中央膜的表达量显著下调,并且外套膜外液中硫酸角质素的浓度显著降低;SEM检测发现珍珠层结构紊乱。综上所述,PmCHST1a可能通过影响外套膜外液中硫酸角质素的合成,参与珍珠层的形成。本研究为进一步探讨磺基转移酶及其参与合成的糖胺聚糖硫酸角质素在马氏珠母贝生物矿化中的作用提供依据。     

Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae)

ABSTRACT: A novel sulfotransferase (O-ST), which transferred the sulfate group of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to O-22 of 11-α,β-hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500-fold and the yield was 4%. On affinity chromatography with a PAP-agarose column, O-ST was observed in the bound fraction, and N-ST specific to N-21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O-ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O-ST did not require metal cations for its activity. O-ST required PAPS as the sole source of sulfate. O-ST transferred a sulfate group from PAPS to only O-22 of 11-α,β-hydroxy STX and not to N-21 of these toxins. These observations suggested that two ST, N-ST and O-ST, participate in the sulfation of PSP toxins.     

The sex-specific sulfation of the major metabolite of the novel fungicide cyprodinil in the rat

The metabolism of cyprodinil, a novel broad-spectrum fungicide, was investigated in rats. After single oral administration of 0.5 or 100 mg kg⁻¹ body weight, [phenyl-U-¹⁴C]cyprodinil was rapidly eliminated, principally in the urine. The metabolite pattern in urine exhibited a significant sex-related difference with respect to the major metabolite. Males and females both produced a dihydroxy metabolite, N-4-(hydroxyphenyl)-4-cyclopropyl-5-hydroxy-6-methylpyrimidin-2-ylamine. Female rats conjugated this metabolite with sulfate exclusively at the 5-hydroxypyrimidinyl moiety, while males formed equal amounts of the monosulfate and a disulfate conjugate. The sex dimorphism in the conjugation reaction indicates the involvement of a sex-specific sulfotransferase that catalyzed the transfer of the second sulfate group.     

Preliminary study of the gene expression of sulfation and degradation enzymes for chondroitin sulfate in glycerol-treated C2C12 myoblast cells

In this study, we induced chemical damage of C2C12 myoblasts that had differentiated into myotubes with glycerol, and four sulfation enzymes for chondroitin sulfate (CS) [carbohydrate sulfotransferase (Chst) 12, Chst15 and Chst3 and uronyl 2-O-sulfotransferase (UST)] and two CS degradation enzymes [hyaluronidase (Hyal) 1 and Hyal2] were examined for changes in gene expression. Treatment of myoblasts with 5% glycerol significantly increased the expression levels of the sulfation enzymes Chst12 and Chst15 and the degradation enzymes Hyal1 and Hyal2. However, the expression levels of the other two genes (Chst3 and Ust) showed no change. Differences in the expression levels of these enzymes may help to understand the difference in responsiveness of myoblasts to glycerol after muscle injury in vivo or in vitro.