Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus
Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus
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DOI：
10.1093/chemse/bjx011
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发表时间：
2017-05-01
期刊：
影响因子：
3.5
通讯作者：
Luetje, Charles W.
中科院分区：
文献类型：
--
作者：
Hughes, David T.;Pelletier, Julien;Luetje, Charles W.
在致倦库蚊（Culex quinquefasciatus）中，CquiOR 91是来自冈比亚按蚊（Anopheles gambiae）（Agam\Or40，先前显示响应于几种气味配体，包括广谱驱避剂N，N-二乙基-3-甲基苯甲酰胺（DEET））和埃及伊蚊（Aedes aegypti）（Aaeg\Or40）的2种幼虫特异性气味受体（OR）的直系同源物。当我们从致倦库蚊幼虫头部RNA样品中克隆全长CquiOR 91时，我们发现该OR的2个等位基因，在9个残基处不同。使用爪蟾卵母细胞表达系统和2-电极电压钳电生理学的功能分析显示一个等位基因（CquiOR 91.1）是无功能的，而另一个等位基因（CquiOR 91.2）是功能性的。由CquiOR 91.2和CquiOR 91.2形成的受体对（-）-fenchone、（+）-fenchone和DEET产生响应，类似于对AgamOR 40的报道。我们还鉴定了CquiOR 91.2 + CquiOrco受体的5种新的气味配体：2-异丁基噻唑、藜芦醇、桉叶醇、d-樟脑和番红花醛，其中番红花醛是最有效的。为了探索CquiOR 91.1缺乏功能的可能原因，我们产生了一系列突变体CquiOR 91.2亚基，其中9个多态性残基位置中的每一个的残基从CquiOR 91.2中出现的变为CquiOR 91.1中出现的。CquiOR 91.2的9个突变体中有8个形成了功能性受体，对（-）-小茴香酮有反应。只有CquiOR 91.2 Y183C突变体是无功能的。回复突变（C183Y）赋予CquiOR91.1功能，其变得对（-）-小茴香酮和番红花醛有反应。这些结果表明，CquiOR 91.1中阻止功能的"缺陷"是位置183处的半胱氨酸。
In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.