The Plasma-Based Cytolytic System of the American Horseshoe Crab, Limulus polyphemus: Cooperative Interaction of the Sialic Acid-Binding Lectin Limulin and Thiol Ester-Reacted α2-Macroglobulin

Abstract

The cytolytic destruction of invading pathogens by proteins of the plasma is an important immune defense strategy of higher animals. The principal protein effector of the plasmabased cytolytic system of Limulus is limulin, a sialic acid-binding lectin present in the plasma. In assays with sheep red cells as the model foreign cell, purified limulin is hemolytic at 4-6 nM, and removal of the limulin from plasma eliminates hemolytic activity (1). Hemolysis depends on the sialic acid-binding activity of limulin, because sialylated glycoconjugates, such as fetuin, and the sialic acids N-acetyl neuraminic acid and colominic acid inhibit hemolysis. The third most abundant protein in Limulus plasma (after hemocyanin and C-reactive protein) is Limulus az-macroglobulin (LAM), which functions as the principal mediator of protease clearance from the plasma (2). The reaction of LAM with proteases activates a unique internal @-cysteinyl-y-glutamyl thiol ester bond, generating a reactive glutamyl residue and a free cysteinyl thiol. Activation of the thiol ester initiates a substantial molecular compaction (3) exposing a previously cryptic domain that is recognized by cell surface receptors. Recognition results in the binding, internalization, and degradation of LAM with its cargo ofbound protease (2, lkawi et al., unpub. data). Small primary amines such as methylamine (MA) react with the thiol-esterified glutamine and induce a nearly identical conformational change in LAM, with a concomitant molecular compaction and activation of receptor recognizability. In this report, we identify a new function for LAM: thiol ester-reacted LAM binds to limulin and potentiates its hemolytic activity. Native, unreacted LAM has no effect on the hemolytic activity of limulin. Limulin (1) and LAM (4) were purified as described previously. Hemolysis was conducted as described in (5). The action of the proteaseand MA-reacted forms of LAM is complex: at high concentrations of limulin and LAM, we see a modest depression of hemolysis (6) but at subhemolytic concentrations of limulin, we see a marked potentiation. Potentiation requires a 20-40-fold molar excess of proteaseor MAreacted LAM. This approximates the ratio of these proteins in the plasma: Limulus plasma contains l-5 ~.LM LAM (7) and about 30-50 nM limulin. Native, unreacted LAM failed to potentiate limulin-mediated hemolysis. The selective action of reacted forms of LAM on the hemo-