Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae
Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae
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DOI：
10.1016/j.ibmb.2003.08.003
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发表时间：
2004-01-01
影响因子：
3.8
通讯作者：
Kanost, MR
中科院分区：
文献类型：
--
作者：
Dittmer, NT;Suderman, RJ;Kanost, MR
漆酶（EC 1.10.3.2）是具有对二酚氧化酶活性的酶，其是统称为多铜或蓝铜氧化酶的一组蛋白质的成员。漆酶在昆虫角质层硬化过程中起重要作用，它将角质层中的儿茶酚氧化成相应的醌类化合物，然后催化蛋白质交联反应。为了研究昆虫漆酶的结构、功能和调控机制，我们克隆了烟草天蛾（Manducasexta）和冈比亚按蚊（Anophelesgambiae）的漆酶基因（MsLac 1和2，AgLac 1）。MsLac 1和2 cDNA分别编码801个氨基酸和760个氨基酸的蛋白质，而AgLac 1 cDNA编码1009个氨基酸的蛋白质。所有三个cDNA都含有推定的分泌信号序列，以及形成铜结合中心的10个组氨酸和一个半胱氨酸，以及T1铜中心的甲硫氨酸。相对于真菌和植物漆酶，昆虫漆酶的新的是更长的氨基末端序列，其特征在于由几个保守的半胱氨酸、芳香族和带电残基组成的独特结构域。北方印迹分析鉴定了MsLac 1、MsLac 2和AgLac 1分别约3.6、3.5和4.4 kb的单个转录物，并且还显示AgLac 1在蚊子的所有生命阶段中表达。RT-PCR显示MsLac 1转录本在中肠、马尔皮基管和表皮中最丰富，而MsLac 2转录本在表皮中最丰富。MsLac 2在蛹中表现出强表达，而在早期蛹表皮中表达减少，这与漆酶在角质层硬化中的假定作用一致。(C)2003 Elsevier Ltd.保留所有权利。
Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization. (C) 2003 Elsevier Ltd. All rights reserved.