Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.
Protein J. 2014 Feb;33(1):75-84. doi: 10.1007/s10930-013-9539-5.
Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.
许多芸薹科植物都具有不可光转化的水溶性叶绿素结合蛋白（Class II WSCPs），在创伤、害虫/病原体攻击和/或环境胁迫导致细胞破坏时，这些蛋白作为叶绿素的清除剂发挥作用。Class II WSCPs 具有两个延伸肽，一个位于 N 端，一个位于 C 端。N 端肽作为信号肽，将蛋白靶向内质体体，内质体体是仅在芸薹科植物中发现的独特防御细胞器。然而，C 端延伸肽的生理和生化功能以前尚未得到表征。为了研究 C 端延伸肽的功能，我们构建了表达带有或不带有 C 端延伸肽的重组 WSCPs 的表达构建体。所用的 WSCP 来自抱子甘蓝（Brassica oleracea）、日本野生萝卜（Raphanus sativus）和维吉尼亚辣椒（Lepidium virginicum）。所有带有 C 端延伸肽的 WSCP 的溶解度都明显低于没有 C 端延伸肽的重组 WSCP。此外，与没有 C 端延伸肽的蛋白相比，带有 C 端延伸肽的 WSCP 复体的稳定性发生了改变。这些发现表明，C 端延伸肽不仅影响蛋白的溶解度，还影响 Class II WSCP 的稳定性。此外，我们在 40%甲醇溶液中对来自日本野生萝卜的重组 WSCP（RshWSCP-His）的叶绿素结合特性进行了表征。电泳迁移率变动分析显示，RshWSCP-His 形成四聚体需要半摩尔比的 Chls。
