Invertebrate animals, which lack adaptive immune systems, have developed other systems of biological host defense, so called innate immunity, that respond to common antigens on the cell surfaces of potential pathogens. During the past two decades, the molecular structures and functions of various defense components that participated in innate immune systems have been established in Arthropoda, such as, insects, the horseshoe crab, freshwater crayfish, and the protochordata ascidian. These defense molecules include phenoloxidases, clotting factors, complement factors, lectins, protease inhibitors, antimicrobial peptides, Toll receptors, and other humoral factors found mainly in hemolymph plasma and hemocytes. These components, which together compose the innate immune system, defend invertebrate from invading bacterial, fungal, and viral pathogens. This review describes the present status of our knowledge concerning such defensive molecules in invertebrates.

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.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems, so-called innate immunity, that respond to common antigens on the surface of potential pathogens. One such defense system is involved in the cellular responses of horseshoe crab hemocytes to invaders. Hemocytes contain two types, large (L) and small (S), of secretory granules, and the contents of these granules are released in response to invading microbes via exocytosis. Recent biochemical and immunological studies on the granular components of L- and S-granules demonstrated that the two types of granules selectively store granule-specific proteins participating in the host defense systems. L-Granules contain all the clotting factors essential for hemolymph coagulation, protease inhibitors including serpins and cystatin, and anti-lipopolysaccharide (LPS) factor and several tachylectins with LPS binding and bacterial agglutinating activities. On the other hand, S-granules contain various new cysteine-rich basic proteins with antimicrobial or bacterial agglutinating activities, such as tachyplesins, big defensin, tachycitin, and tachystatins. The co-localization of these proteins in the granules and their release into the hemolymph suggest that they serve synergistically to construct an effective host defense system against invaders. Here, the structures and functions of these new types of defense molecules found in the Japanese horseshoe crab (Tachypleus tridentatus) are reviewed.

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol. Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows: (Table: see text). The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine alpha-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine alpha-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.

The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three F-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.

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We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of ''bouquet arrangement'' of ficolins and collectins. Electron microscopy revealed that TLs-5 form two-to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonselfrecognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

Hemocytes of the horseshoe crab (limulus) contain a family of arthropodous peptide antibiotics, termed the tachyplesin family, and antibacterial protein, called anti-LPS factor, of which the former is located in the small (S) granules and the latter in the large (L) granules of the hemocytes. In our ongoing studies on granular components, we have identified here a novel defensin-like substance present in both L- and S-granules. This substance strongly inhibits the growth of Gram-negative and -positive bacteria, and fungi, such as Candida albicans. The isolated substance, tentatively termed "big defensin," consists of 79 amino acid residues, of which the COOH-terminal 37 residues have a sequence similar to those of mammalian neutrophil-derived defensins, especially rat defensin. Characterization of the disulfide motif in big defensin indicated that the disulfide array is identical to that of beta-defensins from bovine neutrophils. One clear structural difference is that the limulus hemocyte-derived big defensin has an extension of the NH2-terminal hydrophobic sequence with 35 amino acid residues followed by the COOH-terminal cationic defensin portion. This amphipathic nature of big defensin seems likely to be associated with its potent antibacterial activity. Furthermore, antibacterial activities of the NH2-terminal hydrophobic region and the COOH-terminal defensin portion separated by tryptic digestion are significantly different: the former displays a more potent activity against Gram-positive bacteria, whereas the latter is more potent against Gram-negative bacteria. Big defensin, therefore, may prove to represent a new class of defensin family possessing two functional domains with different antimicrobial activities.