Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti

In this study we describe the purification and molecular cloning of a dopac hrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti . DCE catalyzes the conversion of L-dopachrome to 5,6-dihydroxyindole in th e melanization pathway. Melanin biosynthesis is involved with crucial prote ctive 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 S DS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7 .5-8.0 and substrate activity for L-dopachrome and aminochromes generated f rom dopa methyl ester, (alpha-methyl dopa and dopamine, Trypsin digestion o f 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 a n 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 clon e containing coding sequence for both internal DCE amino acid sequences, th ereby 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 abun dance above constitutive levels in adult females when a melanotic encapsula tion immune response was initiated by the intrathoracic inoculation of Diro filaria immitis microfilariae. (C) 2001 Elsevier Science Ltd. All rights re served.