Comp. Biochem. Physiol. Vol. 105B, No. I, pp. 79-85, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd AGGLUTINATION ACTIVITY OF LIMULUS POLYPHEMUS COAGULOGEN FOLLOWING LIMITED PROTEOLYSIS CONSUELO L. FORTES-DIAs,* CONCEI~.~O A. S. A. MINETTI, YUAN Ll~q and TEH-YuNG LIU~" Division of Biochemistry and Biophysics, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, U.S.A. (Received 14 October 1992; accepted 18 November 1992) Abstract--l. A 14 kDa protein with cell agglutination properties has been purified from endotoxin- activated L. polyphemus amebocyte lysate. Amino terminal sequence analysis indicates that this protein corresponds to a proteolytically cleaved product (coagulin) of coagulogen. 2. Similar cell agglutination activity can be generated, in vitro, by proteolytic cleavage of the coagulogen with either trypsin, endogenous protease or an a2M/enzyme complex isolated from amebocytes. 3. Studies with [t2~I]-labeledcoagulogen showed that only coagulin, not the intact coagulogen, binds to rabbit erythrocytes and formalin-fixed amebocytes. 4. The cell agglutination activity of coagulin towards erythrocytes was not inhibited by various sugars tested, and was not Ca~ +-dependent. 5. These findings suggest that coagulogen and coagulin are reminiscent of their mammalian counter- parts, fibrinogen and fibrin, in their clotting and relative adhesive properties. INTRODUCTION Coagulogen is a major protein in the hemolymph of many invertebrates and its role as a substrate in clot formation has been studied extensively (Nakamura et al., 1976; Tai et al., 1977; Moseson et al., 1979; Takagl et al., 1984; Cheng et al., 1986). The complete amino acid sequences of coagulogens have been reported for four species of horseshoe crabs (Tai et al., 1977; Takagi et al., 1984; Cheng et al., 1986; Miyata et al., 1984a,b; Srimai et al., 1985b). In Limulus polyphemus, coagulogen is a 175 amino acid protein containing eight disulfide bonds. During the last step of the clotting process, the endotoxin- induced clotting enzyme cleaves coagulogen be- tween amino acids 18-19 and 46-47 to release to peptide fragment of 28 amino acids. The 18 amino acid-peptide at the N-terminus remains linked to the rest of the molecule through a disulfide bond and the resultant cleaved product, coagulin, polymedzes to form an insoluble clot (Miyata et al., 1984b). Previous studies have shown the presence of a transglutaminase-catalyzed covalent cross-linking re- action of polypeptide chains during clot formation (Chung et al., 1977). However, there is no substantial evidence to demonstrate that the coagulin generated Abbreviations--Me2SO: dimethylsulfoxide; PMSF: phenyl- methylsulfonylfluoride; HPLC: high-performance liquid chromatography; EDTA: ethylenediaminetetraacetic acid. *Visiting Fellow from Fundae~o Ezequiel Dias, Belo Hori- zonte, MG, Brazil. Supported by Conselho National de Pesquisa Cientifica e Technologica (CNPq proc. 201124/0). tTo whom correspondence should be addressed. following cleavage of coagulogen is a substrate for such an enzyme. In contrast to the fibrin clot pro- duced in the mammalian system, the coagulin clot can be dissociated either by simple mechanical disruption or dilution (Roth et al., 1989). Among a variety of biologically active components, a 55 kDa protein from L. polyphemus amebocytes with cell adhesion- promoting properties has been isolated and charac- terized at both the protein and eDNA levels (Liu et al., 1991). A 24 kDa agglutinin has been isolated, but not fully characterized from the amebocytes of an Indian horseshoe crab, Carcinoscorpius rotundicauda (Srimal et al., 1985a). Agglutination activities have also bccn observed in the hemolymph of other species of horseshoe crabs (Marchalonis and Edelman, 1968; Pistole, 1976; Shishikura and Sekiguchi, 1983). These iectin-like agglutination activities in invertebrates bear some similarities to the vertebrate immune sys- tem and could assume a crucial role in the recognition of foreign substances, resulting in their ultimate removal from the circulation (Vasta, 1990). During the course of our studies of proteins with agglutinin/lectin activities from L. polyphemus ame- bocytes, we have observed that the ability of total amebocyte lysate to promote cell agglutination of rabbit erythrocytes or human leukocytes increases upon activation by endotoxin of the clotting cascade. Several other studies also suggest that agglutination activities observed either in the amebocytes (Srimal et al., 1985a) or in the hemolymph (Shishikura and Sekiguchi, 1983) could arise from activation of the clotting cascade and further granular extrusion via exocytosis. In the present study, we investigated the course of the proteolytic reactions which occur during activation of the clotting cascade with the cero IO~/~--F 79