Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti
Introduction
Melanization is a biochemical event that plays important physiological roles in insects. There are numerous studies that discuss the role of melanin biosynthesis in cuticle and egg chorion hardening, wound healing, and immune responses against parasites in insects. Phenoloxidase (PO) is the key enzyme responsible for catalyzing melanization reactions in insects. Tyrosine is the initial precursor, and the overall melanization pathway involves the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (dopa), oxidation of dopa to dopaquinone and then to dopachrome (DC), structural rearrangement of DC to 5,6-dihydroxyindole (DI), and oxidation of DI to DI-quinone that polymerizes to form melanin. PO can catalyze the hydroxylation of tyrosine and the oxidation of dopa and DI. The rate of tyrosine hydroxylation by PO is about one order of magnitude lower than the rate of dopa and DI oxidation by the same enzyme (Korner and Pawelek, 1982). Therefore, hydroxylation of tyrosine to dopa is considered to be one of the rate-limiting steps in the melanization pathway. Oxidation of dopa by tyrosinase or PO results in the accumulation of a relatively stable red-colored DC. In the melanization pathway, DC has to be converted to DI. It had generally been considered that the production of DI from DC was a nonenzymatic process. Under neutral conditions, DC is converted to DI most likely by base-catalyzed reactions, because the rate of DC conversion to DI shows a linear relationship to the buffer concentrations (Li and Christensen, 1994). The rate of DC conversion under physiological conditions is fairly slow; therefore, conversion of DC to DI is another rate-limiting step in the melanization pathway. An enzyme that catalyzes the conversion of DC to DI has been found in several insect species (Andersson et al., 1989, Aso et al., 1984, Aso et al., 1989, Aso et al., 1990, Aso et al., 1995, Cherqui et al., 1998, Li and Nappi, 1991, Sugumaran and Semensi, 1991) and this enzyme enhances greatly the melanization process. However, no information is available concerning the molecular structure of the insect dopachrome conversion enzyme (DCE). In this study, we purified DCE to homogeneity from A. aegypti larvae, elucidated pH optima of the enzyme as well as its substrate specificities, and isolated a DCE cDNA clone from an A. aegypti pupal cDNA library by screening with a PCR product generated from primers based on partial amino acid sequences obtained by the Edman degradation of the purified DCE. These data provide requisite tools for the elucidation of the molecular regulation of DCE, as well as, contribute an important basis for studying DCE from other insects.
Section snippets
Chemicals and reagents
Ammonium sulfate (NH2SO4), ascorbic acid, l-dopa, dopa methyl ester, α-methyl dopa, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), dl-threo-3,4-dihydroxyphenylserine, 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), 2-[N-morpholino] ethanesulfonic acid (MES), 3-[N-morpholino]propanesulfonic acid (MOPS), sodium periodate, sodium phosphate, phenylmethylsulfonyl fluoride (PMFS), β-mercaptoethanol (ME), phenyl thiourea (PTU), and trifluroacetic acid were supplied by Sigma (St. Louis, MO).
Mosquito rearing and maintenance
The
Purification of DCE
Purification of DCE from 30 to 60% (NH4)2SO2 precipitated proteins was achieved by phenyl sepharose, QA-cellulose, hydroxyapatite, UNO-Q and reverse-phase column chromatographies. Fig. 1 illustrates the results during hydroxyapatite, UNO-Q and reverse-phase chromatographies. Under the applied conditions of hydroxyapatite chromatography, DCE activity was eluted from 140 to 210 mM phosphate (Fig. 1A). The DCE peak was separated from other protein peaks during the first UNO-Q column chromatography (
Discussion
Results from recent studies demonstrate the presence of a specific enzyme, DCE, that accelerates the melanization pathway by catalyzing the conversion of DC to DI in insects. DCE plays an important physiological role in insects because critical to their survival is the ability to rapidly tan their exoskeleton following molts, as well as, egg chorion following oviposition, and parasites following their entry into the hemocoel environment. Studies by Li and Nappi (1991) demonstrated a
Acknowledgements
The authors thank L. Christensen, C. Lowenberger, and C. Smartt for technical assistance, and A.A. James for providing the A. aegypti pupal cDNA library. This study was supported by NIH grants AI 19769 to B.M.C. and AI 37789 to J.L. The pAaDce1 clone has been given the GenBank accession number AF288384.
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