α2-macroglobulin does not function as a C3 homologue in the plasma hemolytic system of the American horseshoe crab, Limulus

A major problem of comparative immunology is the characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens that have gained entry into the body. The plasma cytolytic system of the American horseshoe crab, Limulus polyphemus, is sensitive to treatment with methylamine, which inactivates the abundant plasma defense protein alpha(2)-macroglobulin. This has been interpreted to mean that alpha(2)-macroglobulin plays an important role in hemolysis, analogous to the role of complement component C3 of the mammalian complement system (Enghild et al., 1990). Sensitivity to methylamine has been suggested to reflect an evolutionary homology with the plasma cytolytic system of mammals, in which the complement system is inactivated by the reaction of methylamine with complement components C3 and C4. C3, C4 and alpha(2)-macroglobulin contain an internal thiol ester bond linking cysteinyl and glutamic acid residues and methylamine inactivates all three proteins by reaction with the thiol-esterified glutamic acid. However, we have recently shown that the principal effector of hemolysis in Limulus is the plasma lectin, limulin (Armstrong et al., 1996). In this article we show that native, unreacted alpha(2)-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted alpha(2)-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins alpha(2)-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus alpha(2)-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis. (C) 1998 Elsevier Science Ltd. All rights reserved.