Coelomic fluid of Eisenia foetida earthworms contains a 42-kDa protein named coelomic cytolytic factor 1 (CCF-1) that was described previously to be involved in cytolytic, opsonizing, and hemolytic properties of the coelomic fluid. Cloning and sequencing of CCF-1 reveal significant homology with the putative catalytic region of ␤-1,3-and ␤-1,3-1,4-glucanases. CCF-1 also displays homology with coagulation factor G from Limulus polyphemus and with Gram-negative bacteria-binding protein of Bombyx mori silkworm, two proteins involved in invertebrate defense mechanisms. We show that CCF-1 efficiently binds both ␤-1,3-glucan and lipopolysaccharide. Moreover, CCF-1 participates in the activation of prophenoloxidase cascade via recognition of yeast and Gram-negative bacteria cell wall components. These results suggest that the 42-kDa CCF-1 protein of E. foetida coelomic fluid likely plays a role in the protection of earthworms against microbes.The prophenoloxidase (pro-PO) 1 -activating system represents an important defense mechanism in a large variety of invertebrates (reviewed in Refs. 1 and 2). This system is based on the recognition of bacterial antigens such as lipopolysaccharide (LPS) or peptidoglycan and ␤-1,3-glucan present as major components of the cell wall of yeast and fungi (3,4). Generally, upon the recognition of such saccharides proteases cleave by limited proteolysis inactive pro-PO to its active state, phenoloxidase (PO). The active enzyme catalyzes the o-hydroxylation of monophenols as well as the oxidation of diphenols to quinones that are subsequently polymerized nonenzymatically to melanin. Melanin and its precursors involved in the pro-POactivating system have cytotoxic and antimicrobial properties and participate in a wide range of other biological activities including phagocytosis/opsonization, encapsulation/nodule formation, degranulation, and wound healing (5-9).The pro-PO-activating system has been detected both in protostomian and deuterostomian species. Although pro-POactivating system is well documented in arthropods, data in other protostomian groups are more scarce. In annelids, melanization reactions and formation of "brown bodies" or nodules have been described in polychaetes and oligochaetes (10 -14). However, biochemical detection of PO activity was so far restricted to a few species with rather controversial results. Whereas Smith and Söderhä ll (15) failed to detect pro-PO system in the polychaete Aphrodite aculeata and Arenicola marina, Fischer (16), Valembois et al. (17), and Porchet-Hennerè and Vernet (13) have documented PO activity in Lumbricus terrestris, Eisenia foetida andrei, and Nereis diversicolor, respectively. More recently using L-DOPA as substrate, a 38-kDa protein responsible for PO activity was identified in the coelomic fluid of E. foetida andrei (18). A report showing that the oxidative activity of the coelomic fluid of earthworms toward L-DOPA in vitro is not affected by trypsin but completely blocked by subtilisin reflects the importance of a correct proteolyt...