The alpha-2-macroglobulins are plasma proteins that are present in all classes of vertebrates and that function as broad-spectrum protease inhibitors. The proposed research will investigate the structure and function of a form of alpha-2-macroglobulin from the horseshoe crab, Limulus polyphemus. This laboratory has obtained extensive peptide sequence information and antisera to Limulus alpha-2-macroglobulin which will be used to isolate and sequence the full length cDNA from a CDNA library developed to the poly (A)+ RNA of the Limulus amebocyte. In contrast to vertebrates, which possess a large number of plasma protease inhibitors, alpha-2- macroglobulin is the only protease inhibitor in the plasma of Limulus, which should facilitate its functional characterization in vivo. The proposed research will investigate the possibility that alpha-2-macroglobulin participates in a receptor-mediated clearance pathway for proteases. Alpha-2-macroglobulin also participates in a cytolytic system in the plasma of Limulus that may be homologous to the vertebrate complement system. This system will be characterized, the participating molecules identified, and their sequence of action and mechanism of cytolysis characterized. This study, which represents the first molecular characterization of a complement-like cytolytic system in an invertebrate, should yield valuable information concerning this important immune-like defense system. %%% Multicellular organisms have a varied array of defense strategies that limit their susceptibility to damage by potential pathogens. These defense mechanisms generally involve cells and soluble factors of the blood. One of the most ancient of these blood factors is the plasma protein, alpha-2-macroglobulin. This molecule is found in a wide range of animals from invertebrates to humans. Alpha-2-macroglobulin appears to have two general types of function, one as a trap for enzymes in the blood or on cell surfaces that might otherwise do damage by digesting other proteins, and the second is as a part of a specific biochemical chain reaction that functions to kill invading pathogens or foreign cells. The purpose of this research is to characterize the structure and function of the alpha-2-macroglobulin from the horseshoe crab, Limulus, a relatively primitive animal. The results of this research will contribute to understanding of the diverse array of defense mechanisms found in nature, and may also shed light on the functioning of the defense molecules of higher organisms.
