ALPHA-MACROGLOBULIN FROM LIMULUS-POLYPHEMUS EXHIBITS PROTEINASE INHIBITORY ACTIVITY AND PARTICIPATES IN A HEMOLYTIC SYSTEM

Significant primary sequence homology between the α-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus α-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus α-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus α-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general “trapping” mechanism shared by most α-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus α-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus α-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the α-macroglobulin family. Because both properties are contained in one molecule, we propose the name “limac” to describe this Limulus α-macroglobulin complement-like protein. © 1990, American Chemical Society. All rights reserved.