title: Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs.
abstract: Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta serine proteinase homolog-1 and -2 were present. These proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D004789:Enzyme Activation; D006458:Hemolymph / Q000201:enzymology; D007814:Larva / Q000378:metabolism / Q000382:microbiology; D018402:Manduca / Q000201:enzymology* / Q000382:microbiology; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme; D000581:Amidohydrolases; C019665:amidase