Insect Mimetics Research Unit, Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan.
Insect Biochem Mol Biol. 2013 Nov;43(11):1055-67. doi: 10.1016/j.ibmb.2013.08.004. Epub 2013 Aug 24.
LEA proteins are found in anhydrobiotes and are thought to be associated with the acquisition of desiccation tolerance. The sleeping chironomid Polypedilum vanderplanki, which can survive in an almost completely desiccated state throughout the larval stage, accumulates LEA proteins in response to desiccation and high salinity conditions. However, the biochemical functions of these proteins remain unclear. Here, we report the characterization of a novel chironomid LEA protein, PvLEA4, which is the most highly accumulated LEA protein in desiccated larvae. Cytoplasmic-soluble PvLEA4 showed many typical characteristics of group 3 LEA proteins (G3LEAs), such as desiccation-inducible accumulation, high hydrophilicity, folding into α-helices on drying, and the ability to reduce aggregation of dehydration-sensitive proteins. This last property of LEA proteins has been termed molecular shield function. To further investigate the molecular shield activity of PvLEA4, we introduced two distinct methods, turbidity measurement and dynamic light scattering (DLS). Turbidity measurements demonstrated that both PvLEA4, and BSA as a positive control, reduced aggregation in α-casein subjected to desiccation and rehydration. However, DLS experiments showed that a small amount of BSA relative to α-casein increased aggregate particle size, whereas PvLEA4 decreased particle size in a dose-dependent manner. Trehalose, which is the main heamolymph sugar in most insects but also a protectant as a chemical chaperone in the sleeping chironomid, has less effect on the limitation of aggregate formation. This analysis suggests that molecular shield proteins function by limiting the growth of protein aggregates during drying and that PvLEA4 counteracts protein aggregation in the desiccation-tolerant larvae of the sleeping chironomid.
LEA 蛋白存在于脱水生物中，被认为与获得干燥耐受性有关。在幼虫阶段几乎可以完全脱水的休眠摇蚊 Polypedilum vanderplanki 会在受到干燥和高盐条件的刺激时积累 LEA 蛋白。然而，这些蛋白质的生化功能仍不清楚。在这里，我们报告了一种新型摇蚊 LEA 蛋白 PvLEA4 的特征，它是干燥幼虫中积累量最高的 LEA 蛋白。细胞质可溶性 PvLEA4 表现出许多组 3 LEA 蛋白 (G3LEAs) 的典型特征，如干燥诱导积累、高亲水性、干燥时折叠成 α-螺旋以及减少脱水敏感蛋白聚集的能力。LEA 蛋白的最后一个特性被称为分子屏蔽功能。为了进一步研究 PvLEA4 的分子屏蔽活性，我们引入了两种不同的方法，浊度测量和动态光散射 (DLS)。浊度测量表明，PvLEA4 和 BSA（阳性对照）都能减少α-酪蛋白在干燥和复水过程中的聚集。然而，DLS 实验表明，与α-酪蛋白相比，少量 BSA 会增加聚集体颗粒大小，而 PvLEA4 则以剂量依赖的方式降低颗粒大小。海藻糖是大多数昆虫血液中的主要糖，但也是休眠摇蚊中作为化学伴侣的保护剂，它对限制聚集体形成的影响较小。这项分析表明，分子屏蔽蛋白的功能是在干燥过程中限制蛋白质聚集体的生长，而 PvLEA4 则在休眠摇蚊的干燥耐受性幼虫中对抗蛋白质聚集。
