Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Dev Comp Immunol. 2010 Mar;34(3):264-71. doi: 10.1016/j.dci.2009.10.004. Epub 2009 Oct 24.
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.
酚氧化酶原（proPO）系统的激活和抗菌肽（包括溶菌酶）的合成是节肢动物的两种主要防御机制。proPO 的激活涉及一系列丝氨酸蛋白酶，最终将 proPO 转化为活性酚氧化酶（PO）。然而，在一些昆虫中观察到溶菌酶/抗菌活性和 PO 活性之间存在权衡，并且一种蚊子溶菌酶可以抑制黑化。目前尚不清楚溶菌酶是否可以抑制 PO 活性和/或 proPO 激活。在这项研究中，我们使用体外测定法研究了溶菌酶在烟粉虱 Manduca sexta 中 proPO 激活中的作用。我们表明，来自 M. sexta、人乳和鸡卵清的溶菌酶不会抑制 M. sexta 幼虫预激活的幼稚血浆中的 PO 活性，但显着抑制了幼稚血浆中的 proPO 激活。Western blot 分析表明，M. sexta 溶菌酶与幼稚血浆直接孵育可防止 proPO 转化为 PO，但可刺激丝氨酸蛋白酶同源物-2（SPH2）和 proPO 激活蛋白酶-1（PAP1）前体蛋白的降解，这是 proPO 激活所必需的两个关键成分。远 Western blot 分析表明，M. sexta 溶菌酶和 proPO 相互作用。总之，我们的结果表明，溶菌酶可能通过与 proPO 的直接蛋白相互作用来防止 proPO 转化为 PO，从而抑制 proPO 激活系统。
