Cuticular proteins of Anopheles gambiae

描述（由申请人提供）：冈比亚按蚊是疟疾的主要传播媒介，其约2%的蛋白质编码基因用于结构表皮蛋白。对这些基因进行了注释，确定了它们的表达模式，并确定了许多基因表达的动物区域。其他实验室认为，按蚊的表皮蛋白与杀虫剂和干燥抗性、配偶识别以及对血食的反应有关。我们建议通过验证血食后角质层组成蛋白转录本水平的增加，以及角质层蛋白基因的表达是否会因干燥而改变，来确定角质层是否是一个动态结构。定量实时RT-PCR将证实已经涉及到对血餐反应的基因被上调。简单的生理实验将证明按蚊是否适应干燥的挑战；如果确实如此，对转录本的全基因组分析将显示角质层蛋白基因是否参与其中。作为更好地了解角质层组装和结构的第一步，选择的蛋白质在角质层内的位置将通过可视化标记胶体金的二抗来确定，这些二抗体在EM切片上与一抗结合。为了更好地理解组装和结构，下一步将是确定所有表皮蛋白对几丁质的亲和力，以及它们在自然环境中相互之间的亲和力，首先用缓冲液提取粉碎的整个动物，以去除可溶性蛋白，然后增加干扰几丁质结合的药物浓度。角质层蛋白在不同组分中的补体将通过胰蛋白酶化组分的定量质谱测定。如果有代表性的蛋白质有未被检测到的肽，这些缺失的肽可能是那些参与基于儿茶酚的交联的肽。从上述研究中，将选择表皮蛋白基因，并使用RNAi来降低其转录水平，以确定这种治疗是否会损害蚊子的形态和功能，从而可能揭示可用于控制疟疾的特征。

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.