Potentiation of Limulin-Mediated Cytolysis by Thiol Ester-Reacted Forms of Limulus α2-Macroglobulin: Identification of Functionally Important Domains.

Abstract

The hemolytic activity of Limulus plasma is mediated by the sialic acid-binding plasma lectin, limulin (1). We have recently demonstrated that the hemolytic activity of purified limulin can be potentiated by thiol ester-reacted Limulus Lu>-macroglobulin (LAM)(Z). Proteins of the cyz-m~roglobulinfamily are unique for the presence of an internal thiol ester bond, which in LAM links Cys-999 with Gin-1002 (Ikawi et al., unpub. data). Thiol ester activation hydrolyses this bond, releasing the y-carbonyl ofGIn, which in LAM establishes isopeptide bonds with the t-amino group of Lys-254 (3) and the thiol of Cys. Thiol ester activation is initiated by reaction with proteases and is a key event in protease binding by a2-macroglobulin. Activation can also be initiated by small primary amines, such as methylamine. LAM is a large protein with several different functional domains. We have been interested in identifying the domains that are important in its ability to potentiate limulin-mediated hemolysis. Hemolysis of sheep erythrocytes and purification of limulin and LAM were carried out as described previously ( 1,4,5). The binding of limulin to LAM was quantified with a solid phase assay. Limulin immobilized to microtiter wells was exposed to LAM, and the amount of bound LAM was determined by its subsequent reaction with affinity-purified anti-LAM antibodies and a horseradish peroxidase-conjugated second antibody. Native, un-reacted LAM lacks free thiols (6). The new thiol exposed by reaction of LAM with proteases or methylamine is necessary for the potentiation of hemolysis because its acylation by treatment with iodoacetamide (0.1 M iodoacetamide in 0. I M Tris buffer, pH 8.0, 4 h, room temp.) eliminates the potentiating activity of methylamine-activated LAM (Fig. I). Under the conditions used here, the binding of iodoacetamide was limited to the thiol of Cys-999, because the only [‘“Cl-labeled tryptic peptides of [‘4C]-iodoacetamide-reacted MALAM had the peptide sequence of the thiol ester domain, PTGCGIQNMIK (3). Human and Limulus forms of az-macroglobulin share significant sequence identity at key functional domains but show no homology in the stretch delineated by residues 24-105. Instead, this stretch of LAM shows modest sequence homology with human complement factor-& (C8,). C8, is part of the membrane attack complex generated by the activation of the mammalian complement pathway that inserts in the plasma membrane of targeted foreign cells and that mediates cytolysis. The possibility that this domain of LAM contributes to the potentiation of limulin-mediated hemolysis appears unlikely however, because thiol-reacted, but not native, human N2-mac-