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.     

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca²⁺ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and $\text{p-}\text{nitrophenyl}\text{-p′-}\text{guanidobenzoate}$ blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.     

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.     

Conditions for the association of the two clotting proteins of the cockroachRhyparobia (Leucophaea) maderae (Blattaria)

Summary The clotting system ofRhyparobia (Leucophaea) maderae comprises two clotting proteins, plasma coagulogen and hemocyte coagulogen, which during clotting become crosslinked. Cross-linking is thought to be preceded by an association of the two coagulogens. This paper reports an attempt to elucidate the mechanism of association, using an aged hemocyte coagulogen (=hemocyte gel).In a first series of experiments association was studied with a normal, unmodified gel under various conditions (ionic strength, pH, inhibitors). Association is optimal at low ionic strength and a slightly acidic to neutral pH. When the associated proteins are subjected to increased ionic strength or higher pH they dissociate again. Association is not influenced by crosslinking inhibitors such as EDTA, iodoacetamide, hydroxylamine, and hydrazine up to concentrations of 0.01M.In a second series of experiments association was tested with hemocyte gels which had been treated with a variety of chemicals in order to modify the amino acid side chains. Association is inhibited only when carboxyl groups of the gel are modified.The results of both series of experiments suggest that during association the two proteins are held together mainly by electrostatic attractions between negatively charged carboxyl groups of the hemocyte gel and positively charged amino and/or guanidino groups of the plasma coagulogen.     

甲壳动物血液凝固的分子机制

甲壳动物的防御机制完全依赖于非特异性免疫系统,血淋巴的凝固在宿主防御和阻止血淋巴渗漏中起重要的作用.甲壳动物的凝固酶的激活有两种独立的方式.在鳌虾中,依赖于Ca2+的转谷氨酰胺酶催化可溶性的凝固蛋白原转变成共价交联的凝固蛋白多聚物,不需要蛋白裂解反应.相反,在鲎中,位于淋巴细胞中凝固反应的所有因子都在受到脂多糖激活分泌到胞外,一系列的蛋白裂解反应使得凝固蛋白原转变成凝固蛋白,凝固蛋白间通过非共价的交联结合,而有转谷氨酰胺酶催化的凝固蛋白和淋巴细胞表面proxin间的结合则更加稳定了凝固蛋白反应.     

Crystal structure of a 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.     

Isolation and purification of a cell adhesion factor from crayfish blood cells

Isolated granular haemocytes (blood cells) from the crayfish Pacifastacus leniusculus attached and spread in vitro on coverslips coated with a lysate of crayfish haemocytes. No cell adhesion activity was detected in crayfish plasma. The cell adhesion activity was only present in haemocyte lysates in which the prophenoloxidase (proPO) activating system (Soderhall and Smith, 1986a, b) had been activated; either by lipopolysaccharide (LPS), the beta-1,3-glucan laminarin, or by preparing the lysate in 5 mM Ca2+. Both lysates of granular or of semigranular haemocytes could mediate adhesion. After A23187-induced exocytosis of the granular cells, cell adhesion activity could be generated in the secreted material if it was incubated with laminarin. The factor responsible for cell adhesion was isolated from an active haemocyte lysate and purified by ammonium sulfate precipitation, cation exchange chromatography and Con A-Sepharose; it had a molecular mass of approximately 76 kD on an SDS-polyacrylamide gel. An antibody to this 76-kD band inhibited cell adhesion. Ca2+ was necessary in the medium for the cells to adhere to the adhesion factor. With cyanide or azide, the cells attached but failed to spread. It is suggested that in vivo the cell adhesion factor is stored in the secretory granules of the semigranular and the granular cells in a putative inactive pro-form, which can be released during exocytosis and, in the presence of beta- 1,3-glucans or LPS, be activated outside the cells to mediate cell attachment and spreading, processes of essential importance in arthropod host defense.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Abstract Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-β-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica . The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-β-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-β-glucan synthase activity.     

Haemocytic encapsulation and the prophenoloxidase-activation pathway in the locust Schistocerca gregaria forsk

Negatively-charged Sepharose beads are not encapsulated in vivo by haemocytes of the locust Schistocerca gregaria. It has been suggested by other workers that components of the prophenoloxidase activation pathway of haemolymph might adhere to foreign surfaces and stimulate haemocyte adhesion, so one possible reason for the lack of encapsulation of beads might be due to failure of these components to adhere to the bead. Beads were thus incubated in locust haemocyte lysate supernatant, in which the prophenoloxidase pathway had been activated by Ca²⁺ or Zymosan supernatant, and were then injected into the haemocoeles of locusts. Although at least 5 proteins, including phenoloxidase, could be shown to be attached to the beads, these coated beads were not encapsulated suggesting either that the putative opsonin did not attach or that none of the components is opsonic in this system.In addition, it has been shown that the prophenoloxidase pathway in locust haemocyte lysate supernatant can be partially activated in the presence of Ca²⁺, strongly activated by β1,3-glucans and that production of phenoloxidase is not enhanced by the presence of bacterial LPS and is inhibited by a serine protease inhibitor. The changes in protein composition of unactivated and activated haemocyte lysate supernatant are discussed.     

Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.     

Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses.     

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.     

Protein kinase A activity and protein phosphorylation in the haemocytes of immune-challenged Galleria mellonella larvae

Protein kinase A (PKA) activity was detected in the haemocytes of greater wax moth, Galleria mellonella larvae using a specific peptide substrate--kemptide. The enzyme was activated in vitro by 1 microM concentration of cAMP, 8-Br-cAMP, 8-Chl-cAMP and BzcMP, whereas in the case of cGMP 10 microM concentration was necessary. Immune challenge of G. mellonella larvae with bacteria led to changes in haemocyte PKA activity. Gram-positive M. luteus was a better inducer of PKA activity than Gram-negative E. coli. The kinetics of activity changes was dependent on the bacteria used and considerably differed from that observed in water-treated insects. Inhibition of PKA activity by cell-permeable, specific inhibitor, Rp-8-Br-cAMPS, induced changes in haemocyte morphology resembling those caused by live bacteria. Four potential PKA substrates of 155 kDa, 44 kDa, 40 kDa and 22 kDa were recognized in the haemocytes of naive larvae by phospho-motif antibodies for PKA phosphorylation consensus site. The modification level of 40 kDa protein changed after water treatment and immune challenge of G. mellonella larvae, whereas that of 155 kDa protein changed only after E. coli and LPS injections. Additionally, in the haemocytes of bacteria- and LPS-challenged insects a transient phosphorylation of 36 kDa protein was detected.     

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P相似文献   

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in Cowpea

A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No cross-reaction occurred with Rhizopus stolonifer , Pythium ultimum , Mucor hiemalis , Fusarium oxysporum , Septoria nodorum , Rhizoctonia solani , Sclerotinia sclerotiorum , Phytophthora infestans and Aspergillus niger . In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-β-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-β-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds.     

Bacterial lipopolysaccharide modulates protein kinase C signalling in Lymnaea stagnalis haemocytes

Our knowledge of cell signalling pathways in the molluscan immune system and their response to immunological challenge is currently poor. The present study focused on the Protein Kinase C (PKC) pathway in the immune cells (haemocytes) of Lymnaea stagnalis and its response following exposure to bacterial lipopolysaccharide (LPS). Western blotting of haemocyte proteins with either anti-PKC (pan) or anti-phospho-PKC (Ser 660) antibodies revealed the presence of two PKC-like immuno-reactive proteins of approximately 76 and 85 kDa. Challenge of haemocytes with LPS transiently increased the phosphorylation of the 85 kDa isoform, with a 2.2-fold increase in phosphorylation levels at 5 min and a return to basal levels after 20 min. This LPS-mediated response was blocked following treatment of haemocytes with GF109203X. PKC activities measured in anti-phospho-PKC immunocomplexes following haemocyte treatment with LPS and GF109203X correlated well with the observed PKC phosphorylation levels. These data show for the first time that the activity of the PKC pathway in molluscan immune cells is modulated by LPS, as it is in mammals, and suggest that cell signalling in the innate immune response may have been conserved through evolution.