The recognition of broadly conserved microorganism components known as pathogen-associated molecular patterns is an essential step in initiating the innate immune response. In the horseshoe crab, stimulation of hemocytes with lipopolysaccharide (LPS) causes the activation of its innate immune response, and Factor C, a serine protease zymogen, plays an important role in this event. Here, we report that Factor C associates with LPS on the hemocyte surface and directly recognizes Gram-negative bacteria. Structure-function analyses reveal that the LPS binding site is present in the N-terminal cysteine-rich (Cys-rich) region of the molecule and that it contains a tripeptide sequence consisting of an aromatic residue flanked by two basic residues that is conserved in other mammalian LPS-recognizing proteins. Moreover, we have demonstrated that the Cys-rich region specifically binds to LPS on Gram-negative bacteria and that mutations in the tripeptide motif abrogate its association with both LPS and Gram-negative bacteria, underscoring the importance of the tripeptide in LPS interaction. Although the innate immune response to LPS in the horseshoe crab is distinct from that of mammals, it appears to rely on structural features that are conserved among LPS-recognizing proteins from diverse species.

Hemolymph coagulation in arthropods plays key roles in host defense, including sealing wounds to staunch bleeding and immobilizing invading microorganisms. We have previously reported that horseshoe crab transglutaminase (TGase) promotes cross-linking of a clotting protein (coagulin) with hemocyte-derived proteins (proxins), resulting in the formation of stable coagulin fibrils. Here we show that TGase also cross-links proxins to another hemocyte-derived protein named stablin. Stablin is a cysteine-rich protein of 131 residues. Surface plasmon resonance analysis revealed the specific interaction of stablin with proxin-1 at K d ‫؍‬ 4.0 ؋ 10 ؊9 M. Stablin was predominantly localized in the large granules of hemocytes and secreted by lipopolysaccharide-induced exocytosis. Interestingly, stablin bound to chitin at K d ‫؍‬ 1.5 ؋ 10 ؊8 M, as determined by using a quartz-crystal microbalance. Stablin also interacted with lipopolysaccharides and lipoteichoic acids and exhibited bacterial agglutinating activity against Gram-positive and -negative bacteria. Immunostaining showed that stablin is co-localized with coagulin in the clotting fibrils that effectively trap bacteria. Moreover, an anti-stablin antibody strongly inhibited the proper formation of the clotting fibrils. These data suggest that stablin promotes the formation of the clotting mesh and the immobilization of invading microbes at injury sites. In arthropods, the TGase-mediated cross-linking may play an important role in the initial stage of host defense, wound closure, and healing, as in the case of mammals.