Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica
Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica
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DOI：
10.1016/j.algal.2016.03.030
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发表时间：
2016-06-01
影响因子：
5.1
通讯作者：
Ojima, Takao
中科院分区：
文献类型：
--
作者：
Inoue, Akira;Satoh, Aya;Ojima, Takao
与细菌产生的藻酸盐相比，褐藻产生的藻酸盐含有不同比例和不同序列的两种糖醛酸-β-D-甘露糖酸和α-L-古糖醛酸。褐藻中藻酸盐的这种多样性是由甘露聚糖C5-差向异构酶(MC5Es)引起的，该酶催化β-D-甘露糖酸转化为α-L-古糖醛酸。尽管一些细菌的MC5E酶已经得到了很好的鉴定，但到目前为止，还没有关于真核生物MC5E的生化特性的信息。本研究通过免疫印迹分析检测了MC5E在棕藻Saccharina Japan onica孢子体中的表达。我们还利用RT-PCR法对日本血吸虫的MC5E基因进行了搜索，发现了8个氨基酸的部分序列，SjC5-I to-。我们以出现频率最高的克隆SjC5-VI为研究对象，并对其全长cDNA和可能的基因结构进行了研究。翻译的SjC5-VI蛋白由499个氨基酸组成，N端21个氨基酸预测为分泌信号序列。利用昆虫细胞表达系统成功地表达了功能性重组蛋白SjC5-VI(rSjC5-VI)，并用钙离子诱导凝胶实验确定了其最适温度为35℃，最适pH为7.0-8.2，最适氯化钠浓度为300 mM。此外，在rSjC5-VI活性之后，Ca~(2+)对凝胶的促进作用提高了1.7倍。此外，经rSjC5-VI处理的聚合物的H-1-核磁共振谱显示，β-D-甘露糖酸交替异构化为α-L-古糖醛酸。据我们所知，这是关于真核生物中MC5E活性的第一个报告。(C)2016爱思唯尔B.V.保留所有权利。
Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, beta-D-mannuronic acid and alpha-L-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to -VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35 degrees C, 7.0-8.2, and 300 mM, respectively, using the Ca2+-induced gel-formation assay. In addition, Ca2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, H-1-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes. (C) 2016 Elsevier B.V. All rights reserved.