Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti
Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti
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DOI：
10.1016/s0965-1748(01)00072-8
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发表时间：
2001-10-01
影响因子：
3.8
通讯作者：
Christensen, BM
中科院分区：
文献类型：
--
作者：
Johnson, JK;Li, J;Christensen, BM
在这项研究中，我们描述了从黄热病蚊子，埃及伊蚊多巴色素转换酶（DCE）的纯化和分子克隆。DCE在黑化途径中催化L-多巴色素转化为5，6-二羟基吲哚。黑色素生物合成与蚊子的重要保护现象有关，包括卵绒毛膜和表皮晒黑、伤口愈合和黑色素包裹免疫反应。用各种层析技术从A. SDS-PAGE分析表明，其相对分子质量为51 kDa。DCE的理化分析表明，最适pH为7.5-8.0，并对多巴甲酯、α-甲基多巴和多巴胺产生的L-多巴色素和氨基色素具有底物活性。分离的DCE经胰蛋白酶消化和随后的反相分离导致分离出几个多肽片段，通过Edman降解从其中获得两个部分内部氨基酸序列。使用基于一个内部氨基酸序列的简并引物和oligo-dT引物进行PCR扩增，产生650 bp的DNA片段。随后对A.埃及蛹cDNA文库导致分离含有两个内部DCE氨基酸序列的编码序列的1.6kb克隆，从而证实分离的基因产物（pAaDce 1）为DCE。北方分析显示，DCE信息在发育阶段和成年中的组成型表达，大部分转录本定位于成年雌性的脂肪体和卵巢。AaDce 1 mRNA的丰度增加，在成年女性的组成水平以上时，黑色素包囊免疫反应开始胸内接种犬心丝虫微丝蚴。(C)2001爱思唯尔科技有限公司版权所有。
In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of L-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5-8.0 and substrate activity for L-dopachrome and aminochromes generated from dopa methyl ester, (alpha-methyl dopa and dopamine, Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 by DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae. (C) 2001 Elsevier Science Ltd. All rights reserved.