In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.

Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the horseshoe crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000)

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to -agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescencelabeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-␤-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-␤-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-␤-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-␤-D-glucan affinity column. The purified protein specifically bound to 1,3-␤-Dglucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-␤-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-␤-Dglucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-␤-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.

Bacterial lipopolysaccharide (LPS)-induced exocytosis of granular hemocytes is a key component of the horseshoe crab's innate immunity to infectious microorganisms; stimulation by LPS induces the secretion of various defense molecules from the granular hemocytes. Using a previously uncharacterized assay for exocytosis, we clearly show that hemocytes respond only to LPS and not to other pathogen-associated molecular patterns, such as ␤-1,3-glucans and peptidoglycans. Furthermore, we show that a granular protein called factor C, an LPS-recognizing serine protease zymogen that initiates the hemolymph coagulation cascade, also exists on the hemocyte surface as a biosensor for LPS. Our data demonstrate that the proteolytic activity of factor C is both necessary and sufficient to trigger exocytosis through a heterotrimeric GTPbinding protein-mediating signaling pathway. Exocytosis of hemocytes was not induced by thrombin, but it was induced by hexapeptides corresponding to the tethered ligands of proteaseactivated G protein-coupled receptors (PARs). This finding suggested the presence of a PAR-like receptor on the hemocyte surface. We conclude that the serine protease zymogen on the hemocyte surface functions as a pattern-recognition protein for LPS.

Background:The B subunit of factor XIII (FXIII-B) was previously thought to inhibit fibrin cross-linking by preventing thrombin-mediated activation of the A subunit (FXIII-A). Results: FXIII-B accelerated FXIII-A activation and subsequent fibrin cross-linking by formation of an FXIII-A, fibrinogen, and thrombin ternary complex. Conclusion: FXIII-B accelerated fibrin cross-linking. Significance: FXIII-B deficiency leads to impaired fibrin stabilization.

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Limulin, a sialic‐acid‐binding and phosphorylethanolamine‐binding hemagglutinin in the hemolymph plasma of the American horseshoe crab (Limulus polyphemus), is a hemolytic C‐reactive protein [Armstrong, P.B., Swarnakar, S., Srimal, S., Misquith, S., Hahn, E.A., Aimes, R.T. & Quigley, J.P. (1996) J. Biol. Chem. 271, 14717–14721]. We have now identified three types of C‐reactive protein in the plasma of the Japanese horseshoe crab (Tachypleus tridentatus), based on different affinities against fetuin–agarose and phosphorylethanolamine–agarose determined by quantitative precipitin assays using fetuin and an artificial phosphorylethanolamine–protein conjugate. Partial amino acid sequences of the isolated C‐reactive proteins identified homologous proteins which were named Tachypleus tridentatus CRP‐1 (tCRP‐1), tCRP‐2 and tCRP‐3, each of which possibly constitute isoprotein mixtures. tCRP‐2 and tCRP‐3, but not tCRP‐1, agglutinated mammalian erythrocytes. tCRP‐1, the most abundant C‐reative protein in the plasma, exhibited the highest affinity to the phosphorylethanolamine–protein conjugate but lacked both sialic‐acid‐binding and hemolytic activities. tCRP‐2 bound to both fetuin–agarose and phosphorylethanolamine‐agarose, and exhibited Ca2+‐dependent hemolytic and sialic‐acid‐binding activities, suggestive of limulin‐like properties. Furthermore, tCRP‐2 exhibited a higher affinity to colominic acid, a bacterial polysialic acid. By contrast, tCRP‐3 shows stronger hemolytic, sialic‐acid‐binding and hemagglutinating activities than tCRP‐2. tCRP‐3 has no affinity to phosphorylethanolamine–agarose, phosphorylethanolamine–protein conjugate and colominic acid. This suggests tCRP‐3 is a novel hemolytic C‐reactive protein lacking a common characteristic of phosphorylethanolamine–agarose binding affinity. Twenty‐two clones of tCRPs with different deduced amino acid sequences were obtained by PCR using oligonucleotide primers based on the N‐terminal and C‐terminal sequences of tCRPs and with templates including genomic DNA and cDNA of hemocytes or hepatopancreas derived from one individual. The translation products of the tCRP clones possess high molecular diversity which falls into three related groups, consistent with classification based on their biological activities. Only tCRP‐3 contained a unique hydrophobic nonapeptide sequence that appears in the transmembrane domain of a major histocompatibility complex class I heavy chain of rainbow trout, suggesting the importance of the hydrophobic patch to the hemolytic activity of tCRP‐3. The structural and functional diversities of tCRPs provide a good model for studying the properties of innate immunity in invertebrates, which survive without the benefit of acquired immunity.