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

ABSTRACT

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 bp 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.