Structure/Function Studies of Antifreeze Proteins and Their Enhancers from the Beetle Dendroides canadensis

最活跃的抗冻蛋白（AFPs）存在于某些昆虫中，如加拿大石蜡甲虫（Dendroides canadensis）的幼虫，它们的功能是防止外部冰穿过角质层引起的接种性冻结，并通过抑制血淋巴和肠液中潜在的冰核来促进过冷。越冬加拿大杜鹃产生12个AFPs，由12- mer和13- mer重复序列组成，在序列和重复次数上有所不同。其中一些afp在脂肪体中产生（类似于脊椎动物的肝脏）并分泌到血淋巴中，而另一些在中肠上皮中产生并分泌到肠液中。位于角质层下的单层表皮细胞有助于防止接种冻结，也产生独特的afp组合，就像产生尿液的马尔比氏小管上皮一样。后一种磷酸腺苷可能被分泌到小管腔中，以促进尿液的过冷。存在于这些不同位点的不同DAFPs的独特组合表明，某些加拿大丹的AFPs （DAFPs）已经进化到专门在这些组织中起作用。这项研究将继续早期的工作，试图更好地了解与这些位点相关的DAFPs的功能和机制。此外，这项工作将澄清抗冻蛋白活性的季节性和年度变化的原因。最近的研究表明，要达到最佳水平的DAFP活性（冰点降低，抑制冰核，防止接种冻结）需要某些增强剂，蛋白质增强剂和/或低分子质量增强剂，如甘油。虽然已经在血淋巴中发现了甘油和某些蛋白质增强剂，但其他未知的蛋白质增强剂也存在于血淋巴中，而其他部位（中肠液等）的增强剂的性质尚不清楚。这项研究将确定这些增强剂。由于其高比活性，昆虫蛋白在农业和生物医学材料的低温保存等方面具有巨大的应用潜力。转基因拟南芥植株已经产生，表达DAFPs，降低植物的冷冻温度。进一步了解控制加拿大夜蛾幼虫AFP活性的因素有可能大大提高昆虫AFP在这些应用中的性能。

The most active antifreeze proteins (AFPs) are found in certain insects, such as larvae of the beetle Dendroides canadensis where they function to prevent inoculative freezing initiated by external ice across the cuticle and to promote supercooling by inhibiting potential ice nucleators in hemolymph and gut fluid. Overwintering D. canadensis produce 12 AFPs which consist of 12- and 13- mer repeat sequences which differ somewhat in sequence and number of repeats. Certain of these AFPs are produced in the fat body (analogous to the vertebrate liver) and secreted into the hemolymph, while others are produced in the midgut epithelium and secreted into the gut fluid. The single layer of epidermal cells, which lie under the cuticle and help to prevent inoculative freezing, also produces a unique combination of AFPs, as does the Malpighian tubule epithelium which produces the urine. The latter AFPs are presumable secreted into the tubule lumen to promote supercooling of the urine. The unique combinations of the different DAFPs present in these different sites suggest that certain D. canadensis AFPs (DAFPs) have evolved specifically to function in these tissues. This study will continue earlier work attempting to better understand the functions and mechanisms of the DAFPs in relation to those sites. In addition, this work will clarify the reasons for seasonal and yearly variations in antifreeze protein activity. Recent studies have shown that to achieve optimal levels of DAFP activity (freezing point depression, inhibition of ice nucleators, prevention of inoculative freezing) requires the presence of certain enhancers, protein enhancers and/or low molecular mass enhancers such as glycerol. While glycerol and certain protein enhancers have been identified in hemolymph, other unidentified protein enhancers are also present there, and the nature of the enhancers in other sites (midgut fluid, etc.) is unknown. This study will identify these enhancers. Because of their high specific activities, insect AFPs have tremendous potential for applications, such as agriculture and the cryopreservation of biomedical materials. Transgenic Arabidopsis plants have been produced which express DAFPs that lower the freezing temperatures of the plants. Improved understanding of the factors which control AFP activity in D. canadensis larvae has the potential to greatly improve the performance of the insect AFPs in these applications.