Fuminori Tokunaga

Direct Virus Inactivation of Tachyplesin I and Its Isopeptides from Horseshoe Crab Hemocytes

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity

A glycoprotein (M r = 43 000) from horseshoe crab hemocytes with antimicrobial activity against Gram‐negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti‐human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2‐terminal signal sequence. The COOH‐terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH2‐terminal domain with weak sequence homology with part of the mammalian interleukin‐6 receptor α‐chain. Factor D is likely to have an important role in host defense mechanisms.

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.

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells.

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence ofC9 consists of 261 amino acid residues with a calculated molecular weight of 29496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.

Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells.

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.

cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region

The nucleotide sequence of a cDNA that encodes a new subunit, named RC1, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23,130, which is consistent with the size obtained by electrophoretic analysis of purified RC1. The partial amino acid sequences of several fragments of RC1, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RC1 was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RC1 is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway.

Primary Structures and Functions of Anti-Lipopolysaccharide Factor and Tachyplesin Peptide Found in Horseshoe Crab Hemocytes

The component of lipopolysaccharide (LPS) located in the cell surface of Gram-negative bacteria, has various biological activities, such as pyrogenicity, adjuvant activity, activation of macrophage and B lymphocyte antitumor activity, and so on (19). It is also known that LPS induces the activation and degranulation of horseshoe crab hemocytes and results in the hemolymph coagulation (2, 7). This cellular event is thought to be one of the self-defense mechanisms of horseshoe crab (12, 18, 23). The recent studies on the limulus coagulation system indicate that it consists of the sequential activations of at least three serine protease zymogens and subsequent conversion of coagulogen, an invertebrate fibrinogen-like substance, to coagulin gel (9, 10, 14–16, 21).

Biological Activities of Anti-LPS Factor and LPS Binding Peptide from Horseshoe Crab Amoebocytes

Amoebocytes of horseshoe crabs (such as Limulus polyphemus and Tachypleus tridentatus) contain a coagulation system which is triggered by minute amounts of endotoxic lipopolysaccharide (LPS) and (1–3)-β-D-glucan and result in gelation of hemolymph. Limulus amoebocyte lysate (LAL) is now widely employed as a sensitive assay method for endotoxin. Iwanaga et al. (7), purified reaction components of the gelation cascade and clarified the mechanism of the gelation reaction. In the course of their extensive studies Tanaka et al., (15) discovered a potent anticoagulant, named Anti-LPS Factor (ALF), which specifically inhibits the activation of Factor C by LPS. ALF is a simple basic protein with a molecular weight of 11,600 Da and the unique chemical structure of this protein was described in detail by Iwanaga et al., in this volume and elsewhere (1, 8). In collaborative studies we have found interesting biological activities of ALF such as hemolysis of LPS sensitized erythrocytes (12) and growth inhibition of Gram negative bacteria (10).

Horseshoe Crab Factor G: A New Heterodimeric Serine Protease Zymogen Sensitive to (1→3)-β-D-Glucan

Horseshoe crabs (or limulus), as well as other invertebrate animals, do not have an ordinary immune system like mammals. Although they prefer to live in clean oceans, there are still a number of bacteria in their environment. In order to defend themselves from such microorganisms, they have developed a unique and sophisticated defense system in their hemolymph.