Tatsushi Muta

THE ROLE OF HEMOLYMPH COAGULATION IN INNATE IMMUNITY

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1-->3)-beta-D-glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-κB in lipopolysaccharide-stimulated macrophages

Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor‐κB (NF‐κB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor‐β activated kinase 1 (TGF‐TAK1), a mitogen‐activated protein kinase kinase kinase (MAPKKK), in the LPS‐induced signaling cascade. A kinase‐negative mutant of TAK1 inhibited the LPS‐induced NF‐κB activation both in a macrophage‐like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll‐like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS‐induced signaling pathway.

Molecular mechanism of hemolymph clotting system in Limulus

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains (Sushi or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif (cliplike domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen (easter) in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Stimulus-specific induction of a novel NF-κB regulator, IκB- , via toll/interleukin-1 receptor is mediated by mRNA stabilization

We have recently identified an inducible nuclear factor-κB (NF-κB) regulator, IκB-ζ, which is induced by microbial ligands for Toll-like receptors such as lipopolysaccharide and the proinflammatory cytokine interleukin (IL)-1β but not by tumor necrosis factor (TNF)-α. In the present study, we examined mechanisms for stimulus-specific induction of IκB-ζ. The analysis of the IκB-ζ promoter revealed an essential role for an NF-κB binding sequence in transcriptional activation. The activation, however, did not account for the Toll-like receptor/IL-1 receptor-specific induction of IκB-ζ, because the promoter analysis and nuclear run-on analysis indicated that its transcription was similarly induced by TNF-α. To examine post-transcriptional regulation, we analyzed the decay of IκB-ζ mRNA, and we found that it was specifically stabilized by lipopolysaccharide or IL-1β but not by TNF-α. Furthermore, we found that costimulation with TNF-α and another proinflammatory cytokine, IL-17, elicited the IκB-ζ induction. Stimulation with IL-17 alone did not induce IκB-ζ but stabilized its mRNA. Therefore, IκB-ζ induction requires both NF-κB activation and stimulus-specific stabilization of its mRNA. Because IκB-ζ is essential for expression of a subset of NF-κB target genes, the stimulus-specific induction of IκB-ζ may be of great significance in regulation of inflammatory reactions.

CDNA AND DEDUCED AMINO ACID SEQUENCE OF HUMAN PK-120, A PLASMA KALLIKREIN-SENSITIVE GLYCOPROTEIN

PK‐120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK‐120 mRNA. The deduced amino sequence of PK‐120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK‐120 may be a precursor of a bioactive peptide. Most interestingly, PK‐120 showed significant sequence identities to heavy chains (HCs) of the inter‐α‐trypsin inhibitor (ITI) superfamily.PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-alpha-trypsin inhibitor (ITI) superfamily.

Crystal structure of coagulogen, the clotting protein from horseshoe crab - a structural homologue of nerve growth factor

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C‐terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

SummaryThe structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 μm in diameter) and less electron-dense than the D-granules (less than 0.6 μm in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.

Clotting and immune defense in Limulidae.

The blue blood of the horseshoe crab contains a sophisticated defense system very sensitive to pathogens or foreign materials. The hemocytes circulating in the hemolymph detect trace amounts of LPS molecules on the invading microorganisms and respond quickly to release the granular components into the external milieu. The coagulation system composed of three serine protease zymogens, factor C, factor B, and proclotting enzyme, and a clottable protein, coagulogen, is activated by LPS to form insoluble coagulin gel. The coagulation system also responds to beta-(1,3) glucan through the activation of unique heterodimeric serine protease zymogen, factor G. The pathogens are, thus, engulfed in the gel and subsequently killed by antimicrobial substances with various specificities, which are also released from cells. The horseshoe crab has developed two kinds of serine protease zymogens as biological sensors, factor C and factor G, which are responsive to LPS and beta-(1,3) glucan on the surface of Gram-negative bacteria and fungi, respectively. These are possible invaders for horseshoe crabs and also for most animals including humans. This novel heterodimeric serine protease zymogen, factor G, may open a new way to develop an innovative assay system to quantitate beta-(1,3) glucans. Furthermore, these LPS and beta-(1,3) glucan sensitive factors could be utilized as a unique tool to analyze other biological reactions caused by LPS or the glucan. Although the coagulation reaction in horseshoe crabs is famous, it is not the only defense mechanism of this animal. Many agglutinins are present either in hemolymph plasma or in the cell. The hemolymph plasma also has cytolytic activity against foreign cells. These cellular and humoral defense systems, in concert, defend themselves from invading foreign organisms. Such a sophisticated defense system has allowed the horseshoe crab to survive for more than 200 million years on the earth. Horseshoe crabs are often called ¿living fossils. However, they are not fossils. They are living.

Role of Hemocyte-Derived Granular Components in Invertebrate Defensea

Figure 2 illustrates an outline of the cellular and humoral defense systems in limulus. On the basis of the knowledge described above, it is suggested that granular components present in L and S granules in the hemocytes play a decisive role in the biological defense for this animal. The isolated L granules contain at least three clotting factors plus coagulogen as the major component. The known anti-LPS factor and a number of additional unknown protein components are also present in the L granules. On the other hand, the isolated S granules contain antimicrobial tachyplesins as the major component, in addition to six unidentified proteins. We speculate that the L-granule-derived protein components, which probably contain all the factors essential for the Limulus clotting system participate, in immobilizing invading microbes, and that the S-granule-derived tachyplesins contribute to a self-defense system against invaders. Although we have not mentioned hemolymph plasma components, there are many humoral factors, such as proteinase inhibitors, alpha 2-macroglobulin, various lectins, C-reactive protein, and polyphemin, all of which are important for antimicrobial defense. Furthermore, Liu and colleagues have reported several endotoxin-binding proteins and a cell-adhesion protein found in the Limulus hemocytes. Although the exact functions of these substances are unknown, they may act in concert with other components to provide biological defense for the animal. Nevertheless, compared to our knowledge of mammalian blood cells, much less remains to be learned of biological/physiological events in horseshoe crab hemocytes.

HORSESHOE CRAB COAGULOGEN IS AN INVERTEBRATE PROTEIN WITH A NERVE GROWTH FACTOR-LIKE DOMAIN

The rapid clotting of the horseshoe crab hemolymph is essential for both its host defense and hemostasis. It is mediated by the clotting cascade system which consists of four serine proteinase zymogens and the clottable protein coagulogen. Coagulogen, the target protein of the cascade, is converted to an insoluble gel upon activation of the cascade, giving rise to clot formation. Thus this cascade is reminiscent of the mammalian blood coagulation leading to fibrin clot. The structural analysis of coagulogen revealed a polypeptide fold and disulfide bridge pattern in the C-terminal half of the molecule very similar to nerve growth factor (NGF). This finding assigns coagulogen as the first structurally characterized invertebrate protein which belongs to the cystine knot superfamily. The putative structural similarity of coagulogen and the Drosophila morphogen Spaetzle as well as the homology of its processing proteinases suggests a common origin of the two functionally different cascades. This would exemplify a divergent evolution of two proteinase cascades having totally different functions from common ancestors in a long history of evolution.