title: Expression of a serine proteinase homolog prophenoloxidase-activating factor from the blue crab, Callinectes sapidus.
abstract: Tanning, or sclerotization, of crustacean cuticle provides initial reinforcement by cross linking cuticular proteins attached to the cuticle chitin-fiber matrix. This process is catalyzed in part by phenoloxidase, which is under the control of a serine protease activation cascade. The cDNA of a prophenoloxidase-activating factor (PPAF) was cloned and sequenced from the hypodermal tissue of the blue crab, Callinectes sapidus. It codes for a serine proteinase homolog containing a single clip domain. If it is involved in sclerotization, its transcription might be expected to be molt-cycle related. Expression patterns were determined by quantitative PCR and Northern blotting in hypodermis underlying both arthrodial and dorsal (calcifying) cuticles. Transcript levels in pre-molt RNA from both hypodermis types were high, suggesting that the PPAF produced may be incorporated into the pre-ecdysial cuticle layers and then activated at ecdysis to regulate tanning. After a decrease at ecdysis, a second increase in PPAF mRNA occurred at three to four hours post-molt in arthrodial membrane hypodermis but not dorsal hypodermis. This suggests that cuticle deposited after ecdysis may tan in the non-calcifying regions but may not tan where calcification occurs. The PPAF gene is also transcribed at low levels in the hemocytes of intermolt crabs, but not in the hepatopancreas.
authors.affiliation: Department of Biological Sciences, University of North Carolina at Wilmington, 601 S. College Rd., Wilmington, NC 28403, USA.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Buda
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Shafer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003386:Brachyura / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme