Toshiyuki Miyata

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.

Purification and amino acid sequence of basic protein I, a lysine-49-phospholipase A2 with low activity, from the venom of Trimeresurus flavoviridis (Habu snake)

A basic protein (pI 10.2), named basic protein I, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides produced by chemical (cyanogen bromide and formic acid) and enzymatic (chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid and viperid snakes. A most striking feature of this protein is that aspartic acid at the 49th position common in phospholipases A2 is replaced by lysine. When the protein acted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, oleic acid was preferentially released, indicating that the protein has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was 1.5% that of T. flavoviridis phospholipase A2 isolated previously. The fact that both affinity to Ca2+ and reactivity to p-bromophenacyl bromide of basic protein I are approximately one order of magnitude lower than those of T. flavoviridis phospholipase A2 might explain the low activity of basic protein I.

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.

Demonstration of arginyl-bradykinin moiety in rat HMW kininogen: Direct evidence for liberation of bradykinin by rat glandular kallikreins

The amino acid sequence around kinin moiety in rat High-Molecular-Weight (HMW) kininogen was determined by isolating a peptide containing bradykinin after cyanogen bromide treatment of the purified kininogen as follows; NH2-Thr-Ser-Val-Ile-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ala-Pro-Arg- Val-Lys-Lys-. The data indicated that rat HMW kininogen contains the arginyl-bradykinin moiety, instead of lysyl-bradykinin. Kinins liberated from rat HMW kininogen by rat urinary and submaxillary kallikreins were identified to be bradykinin, not arginyl-bradykinin.

The hemolymph coagulation system in invertebrate animals

A hemocyte lysate from horseshoe crab produced a gel, when exposed to Gram-negative bacterial endotoxins. This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1 → 3)-β-d-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, factor G and anti-LPS factor, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. Among these components, the complete amino acid sequences of coagulogens isolated from one American and three Asian species of horseshoe crabs have been established. Moreover, the reconstitution experiment using the isolated clotting factors, C, B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin get. Based on these results, we propose here a mechanism for the Limulus coagulation cascade.

Factor IX Kawachinagano: impaired function of the Gla-domain caused by attached propeptide region due to substitution of arginine by glutamine at position −4

Summary. Factor IX Kawachinagano (KWC) is a mutant factor IX protein initially recognized in a patient with severe haemophilia B, who had 46% of normal factor IX antigen and no detectable clotting activity. Previous studies indicated that factor IX KWC was not activated by factor XIa in the presence of Ca ions. In the present study, we purified and analysed factor IX KWC at a structural level in an attempt to clarify the nature of the impaired reaction with factor XIa. Kinetic studies showed that activation of factor IX KWC by factor X activator from Russells viper venom (RVV‐X) was normal, whereas activation by factor XIa was defective. Amino acid sequence analysis of tryptic peptides and direct analysis of the NH2‐terminal sequence of factor IX KWC demonstrated that this mutant factor IX retained the propeptide region of 18 amino acids due to a substitution of arginine ‐(‐4) by glutamine. These data suggested that the Gla‐domain of factor IX KWC was dysfunctional, although the total γ‐Gla content, measured by alkaline‐hydrolysis, was normal. We assumed that this attached propeptide region of the molecule directly interferes with the adjacent NH2‐terminus and prevents the metal‐induced conformational changes which are essential for biological activity of normal factor IX.

Isolation of cDNA clones of human argininosuccinate lyase and corrected amino acid sequence

In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by OBrien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211–7215] had one‐base deletions at three independent positions in the coding regions near the COOH‐terminus, which caused frame‐shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.

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).

Identification of rat urinary kinin as bradykinin.

Bradykinin (BK)-like activity, which was detected by BK-enzyme-immunoassay, was purified from 80 ml of ureter urine of Sprague-Dawley rats by Sephadex G 25 chromatography, FPLC, and reversed phase HPLC. The purified kinin fraction showed the same retention time as authentic BK on HPLC and produced contraction of isolated rat uterus, the contraction being suppressed by a B2-antagonist Hoe140. There was no other kinin detected on the HPLC at the corresponding retention time to kallidin, arginyl-BK or T-kinin. The peptide showed an amino acid sequence identical to that of BK by amino acid sequence analysis.

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).