Primary structure of limulus anticoagulant anti-lipopolysaccharide factor

A potent anticoagulant, anti-lipopolysaccharide (LPS) factor, found in limulus hemocytes inhibits the LPS-mediated activation of limulus coagulation cascade and shows an antibacterial action against R-types of Gram-negative bacteria (Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M. (1985) J. Biochem. (Tokyo) 97, 1611-1620). The complete amino acid sequence of this substance was determined by sequencing the peptides obtained by selective proteolytic cleavage. The NH2-terminal end of anti-LPS factor was pyroglutamic acid. Anti-LPS factor had two variant residues at position 36 and the COOH-terminal end, respectively. The following sequence was assigned to anti-LPS factor, and it was also confirmed by fast atom bombardment mass spectrometry. less than EGGIWTQLALALVKNLATLWQSGDFQFLGHE (formula; see text) Limulus anti-LPS factor consisted of a single chain of 102 residues with 2 half-cystines in disulfide linkage. Its NH2-terminal region up to 20 residues was highly hydrophobic, and positively charged residues were clustered mainly within the disulfide loop. By searching the homologous sequence in known protein sequences with that of anti-LPS factor, we found a structural homology between anti-LPS factor and alpha-lactalbumin/lysozyme family.     

Interaction between lipopolysaccharide and intracellular serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes

The interaction between lipopolysaccharide (LPS) and an LPS-sensitive serine protease zymogen, factor C, purified from horseshoe crab (Tachypleus tridentatus) hemocytes, was investigated to elucidate the LPS-mediated activation of factor C. The rate of activation of the zymogen factor C was highly dependent on the concentration of LPS and on temperature, and the curve of amount of LPS versus activation showed saturation at 37 degrees C. Moreover, a high-molecular-mass complex formed between factor C and LPS was found in a gel-filtration experiment on a Sepharose 4B column. This complex formation was also confirmed by double diffusion analysis on agarose plates. Triton X-100, which destroys LPS micelles, strongly inhibited the LPS-mediated activation of factor C but not activated factor C. These results indicate that the binding of factor C with LPS is required for its activation and that only LPS-associated factor C generates the active factor C. On the other hand, the LPS-mediated activation of factor C was strongly inhibited by the S-alkylated heavy chain derived from factor C. In contrast, the S-alkylated factor C-light chain did not show any inhibitory effect on the activation of factor C, suggesting that the heavy chain located in the NH2-terminal portion of factor C contains an LPS-binding region.     

Limulus anti-LPS factor: An anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system

Exposure of $\text{Limulus}$ amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of $\text{Tachypleus}\text{tridentatus}$ and $\text{Limulus}\text{polyphemus}$. The principle purified partially from $\text{Tachypleus}$ amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the $\text{Limulus}$ coagulation system was discussed.     

Anti-LPS factor in the horseshoe crab, Tachypleus tridentatus. Its hemolytic activity on the red blood cell sensitized with lipopolysaccharide

Anti-LPS factor, which inhibits the endotoxin mediated coagulation system in the horseshoe crab, Tachypleus tridentatus, was found to lyse red blood cells sensitized with gram-negative bacterial LPS, but not to lyse unsensitized cells. This hemolysis occurred even at 0 degree C and was completed within 1 min. The binding of anti-LPS factor to LPS must be essential for the hemolysis, because free LPS inhibited the hemolytic action of anti-LPS factor.     

Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.     

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Isolation and characterization

Bacterial endotoxin (lipopolysaccharide, LPS) induces coagulation of horseshoe crab hemolymph. Our previous studies had demonstrated that a hemolymph factor, designated factor B, was associated with the LPS-mediated activation of the Limulus clotting system [Ohki et al. (1980) FEBS Lett. 120, 318-321]. On further purification of factor B we found that an additional component, designated factor C, was required to generate factor B activity in the presence of LPS in order to activate the proclotting enzyme. To elucidate the role of factor C in the LPS-mediated reaction, factor C was isolated and characterized from the hemocyte lysate under sterile conditions. The preparation exhibited a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while two protein bands on SDS-PAGE were observed after reduction. Thus, factor C had a Mr of 123 000 consisting of a heavy chain of Mr = 80 000 and a light chain of Mr = 43 000. Factor C was converted to an activated form in the presence of LPS with a Mr = 123 000, designated factor C. Upon activation, cleavage of the light chain occurred resulting in the accumulation of two new fragments of Mr = 34000 and 8500 on reduced SDS-PAGE. A diisopropylfluorophosphate-sensitive active site was localized in the light chain (Mr = 34000) of factor C. The reconstitution experiments, using factor C, factor B, proclotting enzyme and LPS, demonstrated that all of these proteins are essential for the endotoxin-mediated coagulation system. On the basis of these results we propose that a cascade pathway of LPS-induced activation of the Limulus clotting system consists of three sequential activations of hemolymph serine protease zymogens.     

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)--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.     

Cloning of anti-LPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro

In this article we report the cloning and expression of a cDNA encoding Tachypleus anti-lipopolysaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gramnegative bacterial endotoxins. First, two degenerate primers were designed based on the sequence homology of anti-LPS factors purified from different species of horseshoe crab. The total RNA was extracted from amebocytes of Tachypleus tridentatus. The cDNA was then obtained by using the RT-PCR methods. Second, the cDNA of Tachypleus anti-LPS factor (TALF) was expressed in Bombyx mori larvae using baculovirus expression system, which showed a yield of up to 600 mg/L. Last, we determined the biological activity of the recombinant proteins by LPS neutralization assay and bacteriostatic assay in vitro.     

Tachyplesins isolated from hemocytes of Southeast Asian horseshoe crabs (Carcinoscorpius rotundicauda and Tachypleus gigas): identification of a new tachyplesin, tachyplesin III, and a processing intermediate of its precursor

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.     

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

Biochemical properties and cDNa cloning of two new lectins from the plasma of Tachypleus tridentatus: Tachypleus plasma lectin 1 and 2+

A Sepharose CL-4B-binding protein, Tachypleus plasma lectin 1 (TPL-1), and a lipopolysaccharide (LPS)-binding protein, Tachypleus plasma lectin-2 (TPL-2), have been isolated from the plasma of Tachypleus tridentatus and biochemically characterized. Each protein is coded by a homologous family of multigenes. TPL-1 binds to Sepharose CL-4B and was eluted with buffer containing 0.4 m GlcNAc. The deduced amino acid sequence of TPL-1 consisted of 232 amino acids with an N-glycosylation site, Asn-Gly-Ser at residues 74-76. It shares a 65% sequence identity and similar internal repeats of about 20 amino acid motifs with tachylectin-1. Tachylectin-1 was identified as a lipopolysaccharide-agarose binding nonglycosylated protein from the amebocytes of T. tridentatus. TPL-2 was eluted from the LPS-Sepharose CL-4B affinity column in buffer containing 0.4 m GlcNAc and 2 m KCl. The deduced amino acid sequence of TPL-2 consisted of 128 amino acids with an N-glycosylation site, Asn-Cys-Thr, at positions 3-5. It shares an 80% sequence identity with tachylectin-3, isolated from the amebocytes of T. tridentatus. TPL-2 purified by LPS-affinity column from the plasma predominantly exists as a dimer of a glycoprotein with an apparent molecular mass of 36 kDa. Tachylectin-3 is an intracellular nonglycosylated protein that also exists as a dimer in solution with an apparent molecular mass of 29 kDa. It recognizes Gram-negative bacteria through the 0-antigen of LPS. Western blot analyses showed that, in the plasma, TPL-1 and TPL-2 exist predominantly as oligomers with molecular masses above 60 kDa. They both bind to Gram-positive and Gram-negative bacteria, and this binding is inhibited by GlcNAc. Possible binding site of TPL-1 and TPL-2 to the bacteria could be at the NAc moiety of GlcNAc-MurNAc of the peptidoglycan. The physiological function of TPL-1 and TPL-2 is most likely related to their ability to form a cluster of interlocking molecules to immobilize and entrap invading organisms.     

Effect of extracellular products of Pseudoalteromonas atlantica on the edible crab Cancer pagurus

Previous studies have shown that injection of extracellular products (ECP) of Pseudoalteromononas atlantica isolated from shell disease-infected edible crabs (Cancer pagurus) into healthy crabs causes rapid death. In this study we examined the nature of the active lethal factor(s) in ECP. Injection of ECP into crabs caused a rapid decline in the total number of circulating hemocytes (blood cells), and the crabs died within 60 to 90 min. The individuals that died showed eyestalk retraction, limb paralysis, and lack of antennal sensitivity, suggesting that the active factor(s) targeted the nervous system. Histopathological investigations showed that affected crabs had large aggregates of hemocytes in the gills, and there was destruction of the tubules in the hepatopancreas. The active factor in ECP was not sensitive to heat treatment (100 degrees C for 30 min) and proteinase K digestion. As lipopolysaccharide (LPS) was a potential candidate for the lethal factor, it was purified from whole P. atlantica bacteria or ECP and subsequently injected into crabs. These crabs had all of the external symptoms observed previously with ECP, such as limb paralysis and eyestalk retraction, and they died within 90 min after challenge, although no significant decline in the number of circulating hemocytes was observed. Similarly, in vitro incubation of hemocytes with purified LPS (1 to 20 microg) from P. atlantica did not result in the clumping reaction observed with ECP but did result in a degranulation reaction and eventual cell lysis. Injection of crabs with Escherichia coli or Pseudomonas aeruginosa LPS (1 microg g of body weight(-1)) did not cause any of the characteristic symptoms observed following exposure to P. atlantica LPS. No mortality of crabs followed the injection of E. coli LPS, but P. aeruginosa LPS caused ca. 80% mortality at 2 h after injection. Overall, these results show that the main virulence factor of P. atlantica for edible crabs is LPS either alone or in combination with other heat-stable factors.     

Calcineurin mediates the immune response of hemocytes through NF-κB signaling pathway in pearl oyster (Pinctada fucata)

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IκBα, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IκBα degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-κB signaling pathway.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

Detection of lipopolysaccharides in polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: "LPS blotting"

Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria.     

Activation of human meningeal cells is modulated by lipopolysaccharide (LPS) and non-LPS components of Neisseria meningitidis and is independent of Toll-like receptor (TLR)4 and TLR2 signalling

The interactions of Neisseria meningitidis with cells of the meninges are critical to progression of the acute, compartmentalized intracranial inflammatory response that is characteristic of meningococcal meningitis. An important virulence mechanism of the bacteria is the ability to shed outer membrane (OM) blebs containing lipopolysaccharide (LPS), which has been assumed to be the major pro-inflammatory molecule produced during meningitis. Comparison of cytokine induction by human meningeal cells following infection with wild-type meningococci, LPS-deficient meningococci or after treatment with OM isolated from both organisms, demonstrated the involvement of non-LPS bacterial components in cell activation. Significantly, recognition of LPS-replete OM did not depend on host cell expression of Toll-like receptor (TLR)4, the accessory protein MD-2 or CD14, or the recruitment of LPS-accessory surface proteins heat shock protein (HSP)70, HSP90alpha, chemokine receptor CXCR4 and growth differentiation factor (GDF)5. In addition, recognition of LPS-deficient OM was not associated with the expression of TLR2 or any of these other molecules. These data suggest that during meningococcal meningitis innate recognition of both LPS and non-LPS modulins is dependent on the expression of as yet uncharacterized pattern recognition receptors on cells of the meninges. Moreover, the biological consequences of cellular activation by non-LPS modulins suggest that clinical intervention strategies based solely on abrogating the effects of LPS are likely to be only partially effective.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.