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

加拿大树突甲虫抗冻蛋白及其增强剂的结构/功能研究

• 批准号: 0212907
• 负责人: John Duman
• 金额: $ 34万
• 依托单位: University of Notre Dame
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-10-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212907&HistoricalAwards=false
• 关键词: Structure; Function; Studies; Antifreeze; Proteins

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.

在某些昆虫中发现了最活跃的抗冻蛋白（AFP），如加拿大石斛甲虫的幼虫，它们的功能是防止由外部冰穿过角质层引发的接种冻结，并通过抑制血淋巴和肠液中潜在的冰成核剂来促进过冷。 越冬D. canadensis产生12种AFP，其由12-和13- mer重复序列组成，所述重复序列在序列和重复数目上略有不同。 这些AFP中的某些在脂肪体（类似于脊椎动物的肝脏）中产生并分泌到血淋巴中，而其他的在中肠上皮中产生并分泌到肠液中。 单层表皮细胞位于角质层下，有助于防止接种冷冻，也会产生独特的AFP组合，产生尿液的马尔皮基管上皮也是如此。 后一种AFP可能分泌到小管腔中以促进尿液的过冷。 不同DAFP在这些不同位点的独特组合表明，某些D。加拿大AFP（DAFP）已经进化到专门在这些组织中发挥作用。 本研究将继续早期的工作，试图更好地了解功能和机制的DAFP与这些网站。 此外，这项工作将澄清抗冻蛋白活性季节性和年度变化的原因。最近的研究表明，为了达到最佳水平的DAFP活性（凝固点降低、抑制冰成核剂、防止接种冻结），需要存在某些增强剂、蛋白质增强剂和/或低分子量增强剂如甘油。 虽然甘油和某些蛋白质增强子已在血淋巴中被鉴定，但其他未鉴定的蛋白质增强子也存在于那里，并且其他部位（中肠液等）中的增强子的性质是未知的。不明 这项研究将确定这些增强子。由于其高比活，昆虫AFP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。 已经产生了表达降低植物冷冻温度的DAFP的转基因拟南芥植物。 提高了对D. canadensis幼虫具有极大地改善昆虫AFP在这些应用中的性能的潜力。