Cuticular proteins of Anopheles gambiae

描述(申请人提供)：冈比亚按蚊是疟疾的主要传播媒介，其编码蛋白质的基因中约有2%用于结构角质层蛋白。这些基因已经被注释，它们的表达模式已经确定，动物中表达许多基因的区域已经确定。其他实验室已经证实，按蚊的角质层蛋白与杀虫剂和干燥抗性、配偶识别以及对血餐的反应有关。我们建议通过验证血餐后角质层组成蛋白转录水平的增加以及角质层蛋白基因的表达是否会随着干燥的反应而改变来确定角质层是否是动态结构。实时定量RT-PCR将验证已经与血餐反应有关的基因是否上调。简单的生理学实验将证明按蚊是否适应干燥挑战；如果是这样，对转录本的全基因组分析将显示是否涉及角质蛋白基因。作为更好地了解角质层组装和结构的第一步，选定蛋白质在角质层中的位置将通过在EM切片上可视化与一次抗体结合的胶体金标记的二次抗体来确定。为了更好地理解组装和结构，下一步将是确定所有角质蛋白对几丁质的亲和力，以及在它们的自然环境中相互之间的亲和力，方法是先用缓冲液提取整个动物粉末，以去除可溶性蛋白质，然后增加干扰几丁质结合的试剂的浓度。不同组分中角质层蛋白的补充量将通过对胰酶降解组分的定量质谱分析来确定。如果代表良好的蛋白质含有未被检测到的多肽，这些缺失的多肽可能是那些参与邻苯二酚交联的多肽。从上述研究中，将选择角质层蛋白的基因，并将使用RNAi降低其转录水平，以确定这种治疗是否会损害蚊子的形态和功能，可能揭示可用于控制疟疾的特征。 与公共卫生相关：按蚊是疟疾的携带者。与所有昆虫一样，蚊子的外部--角质层--既是它的骨骼，也是它的皮肤，含有许多蛋白质，其中大多数已经在我们之前的研究中被发现，并构成了大约2%的蛋白质多样性。我们现在建议将我们的研究扩展到确定这些蛋白质在蚊子生活中的具体作用，希望这一知识将有助于控制这种致命的昆虫。

DESCRIPTION (provided by applicant): Anopheles gambiae, the major vector of malaria, uses ~2% of its protein-coding genes for structural cuticular proteins. These genes have been annotated, their expression patterns determined, and the regions of the animal where many are expressed have been established. Other laboratories have implicated the cuticular proteins of Anopheles in insecticide and desiccation resistance, in mate recognition, and in being synthesized in response to a blood meal. We propose to determine whether cuticle is a dynamic structure by verifying the increased levels of transcripts of its component proteins after a blood meal and whether expression of cuticular protein genes can change in response to desiccation. Quantitative real-time RT-PCR will verify that genes already implicated in responding to a blood meal are upregulated. Simple physiological experiments will demonstrate whether Anopheles adapts to a desiccation challenge; if it does, genome-wide analyses of transcripts will show whether cuticular protein genes are involved. As a first step toward better understanding of cuticle assembly and structure, the location within the cuticle of selected proteins will be determined by visualizing secondary antibodies labeled with colloidal gold that have bound to primary antibodies on EM sections. The next step toward better understanding assembly and structure will be to determine the affinity of all cuticular proteins for chitin and for each other in their natural environment by extracting pulverized whole animals first with buffer to remove soluble proteins and then with increasing concentrations of agents that interfere with chitin binding. The complement of cuticular proteins in the various fractions will be determined by quantitative mass spectrometry on trypsinized fractions. If well represented proteins have peptides that are not detected, these missing peptides may be those that are participating in catechol-based crosslinking. From the studies described above, genes for cuticular proteins will be selected and RNAi will be used to reduce their transcript levels to establish whether this treatment compromises form and function of the mosquito, possibly revealing features that could be exploited in controlling malaria. PUBLIC HEALTH RELEVANCE: The Anopheles mosquito is the carrier of malaria. As with all insects the exterior of the mosquito - the cuticle - is both its skeleton and its skin and contains many proteins, most of which have been identified in our previous study and constitute about 2% of its protein diversity. We now propose to extend our studies into determining the specific role of these proteins in the life of the mosquito, hoping that this knowledge will contribute to the control of this deadly insect.