RESUMO
Phenoloxidase (PO), a melanin-synthesizing enzyme known to play an important role in insect defense, is found as a zymogen (ProPO) in hemolymph and cuticle, where it is activated by proteolysis. We characterized the first proPO cDNA in an eusocial insect, the Apis mellifera honey bee. The AmproPO cDNA contains an ORF of 2079 bp encoding 693 amino acids, and is composed of 9 exons and 8 introns. Southern blot of digested genomic DNA suggested that only one copy of the proPO gene is present in A. mellifera. The molecular mass of the deduced ProPO and the active enzyme was predicted to be 80.1 and 74.4 kDa, respectively. The calculated pI was 6.28. BLASTp search of the deduced amino acid sequence, and neighbor-joining analysis, showed similarity with ProPOs from other insects, ranging from 47% to 63%. Protein signature analyses revealed four conserved regions, including the two copper binding sites characteristic of arthropod ProPOs. RT-PCR and Southern blot showed the highest amount of AmproPO transcripts in workers whole body, followed by queens and drones. Expression was also detected in hemocytes and integument. Real time RT-PCR showed higher amounts of AmproPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmproPO in adult exoskeleton melanization and differentiation.
Assuntos
Abelhas/enzimologia , Catecol Oxidase/metabolismo , DNA Complementar/química , Precursores Enzimáticos/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Abelhas/genética , Sítios de Ligação , Catecol Oxidase/química , Catecol Oxidase/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Feminino , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Masculino , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.