Peng Zeyu, Green Peter G, Arakane Yasuyuki, Kanost Michael R, Gorman Maureen J
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, United States of America.
Department of Civil and Environmental Engineering, University of California Davis, Davis, California, United States of America.
PLoS One. 2014 Oct 20;9(10):e111344. doi: 10.1371/journal.pone.0111344. eCollection 2014.
Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.
典型的多铜氧化酶（MCOs）有10个保守的组氨酸和1个保守的半胱氨酸，它们与4个铜原子配位。这些铜离子是氧化酶活性所必需的。在我们对昆虫MCOs的研究过程中，我们发现了一个基因，我们将其命名为多铜氧化酶相关蛋白（MCORP）。MCORP与MCOs具有序列相似性，但缺少许多铜配位残基。我们在许多昆虫物种中鉴定出了MCORP直系同源物，但在其他无脊椎动物或脊椎动物中未发现。我们预测，由于缺乏铜配位残基，MCORP将缺乏氧化酶活性。为了验证这一预测，我们纯化了重组的赤拟谷盗（红粉甲虫）MCORP，并使用多种底物分析其酶活性。正如预期的那样，未检测到氧化酶活性。为了研究MCORP在体内的功能，我们分析了赤拟谷盗MCORP（TcMCORP）和冈比亚按蚊（非洲疟蚊）MCORP的表达谱，并评估了RNA干扰介导的敲低表型。我们发现，在我们分析的所有组织中，这两种MCORP均以低水平组成性表达。向幼虫注射TcMCORP双链RNA导致在成虫羽化前100%死亡，死亡主要发生在蛹期或晚期成虫期。向蛹期注射TcMCORP双链RNA导致约20%的处理昆虫在蛹到成虫的转变过程中死亡，其余昆虫的寿命大大缩短。此外，雌性中TcMCORP的敲低阻止了卵母细胞成熟，因此，产卵数量大大减少。这些结果表明，TcMCORP是一个必需基因，其功能是繁殖所必需的。了解MCORP在昆虫生理学中的作用可能有助于开发控制害虫的新策略。
