The role of lysozyme in the prophenoloxidase activation system of Manduca sexta: an in vitro approach.
Activation of the prophenoloxidase (proPO) system and synthesis of antimicrobial peptides (including lysozyme) are two key defense mechanisms in arthropods. Activation of proPO involves a cascade of serine proteinases that eventually converts proPO to active phenoloxidase (PO). However, a trade-off between lysozyme/antibacterial activity and PO activity has been observed in some insects, and a mosquito lysozyme can inhibit melanization. It is not clear whether lysozyme can inhibit PO activity and/or proPO activation. In this study, we used in vitro assays to investigate the role of lysozyme in proPO activation in the tobacco hornworm Manduca sexta. We showed that lysozymes from M. sexta, human milk and hen egg white did not inhibit PO activity in the pre-activated naïve plasma of M. sexta larvae, but significantly inhibited proPO activation in the naïve plasma. Western blot analysis showed that direct incubation of M. sexta lysozyme with the naïve plasma prevented conversion of proPO to PO, but stimulated degradation of precursor proteins for serine proteinase homolog-2 (SPH2) and proPO-activating proteinase-1 (PAP1), two key components required for proPO activation. Far-western blot analysis showed that M. sexta lysozyme and proPO interacted with each other. Altogether, our results suggest that lysozymes may inhibit the proPO activation system by preventing conversion of proPO to PO via direct protein interaction with proPO.
Rao
Xiang-Jun
XJ
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Ling
Erjun
E
Yu
Xiao-Qiang
XQ
D000818:Animals; D015153:Blotting, Western; D004156:Catechol Oxidase / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology*; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D018402:Manduca / Q000276:immunology / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism*; D000073718:Pancreatitis-Associated Proteins; D012697:Serine Endopeptidases / Q000378:metabolism
D004792:Enzyme Precursors; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D009113:Muramidase; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme