Molecular characterization of a cDNA encoding prophenoloxidase and its expression in Apis mellifera

Insect Biochem Mol Biol. 2005 Jun;35(6):541-52. doi: 10.1016/j.ibmb.2005.01.013. Epub 2005 Feb 24.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Bees / enzymology*
  • Bees / genetics
  • Binding Sites
  • Catechol Oxidase / chemistry
  • Catechol Oxidase / genetics
  • Catechol Oxidase / metabolism*
  • DNA, Complementary / chemistry*
  • Enzyme Precursors / chemistry
  • Enzyme Precursors / genetics
  • Enzyme Precursors / metabolism*
  • Female
  • Gene Expression
  • Insect Proteins / chemistry
  • Insect Proteins / genetics
  • Insect Proteins / metabolism*
  • Male
  • Molecular Sequence Data
  • Phylogeny
  • Sequence Alignment

Substances

  • DNA, Complementary
  • Enzyme Precursors
  • Insect Proteins
  • pro-phenoloxidase
  • Catechol Oxidase