Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade.
Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.
Gorman
Maureen J
MJ
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Wang
Yang
Y
Jiang
Haobo
H
Kanost
Michael R
MR
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D006458:Hemolymph / Q000378:metabolism*; D007107:Immune System; D019476:Insect Proteins / Q000378:metabolism / Q000502:physiology*; D007313:Insecta; D018402:Manduca; D008954:Models, Biological; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D019476:Insect Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D011994:Recombinant Proteins; D010450:Endopeptidases; C490363:hemolymph proteinase 14, Manduca sexta; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme