UNIPROT:P43026 - FACTA Search

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Query: UNIPROT:P43026 (lipopolysaccharide)
62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes. 128 92

Tumor necrosis factor (TNF) induced by bacterial lipopolysaccharide (LPS) was shown to have an important role in precipitation of septic shock and disseminated intravascular clotting (DIC). At the endothelial level TNF down-regulates thrombomodulin (thus preventing protein C formation) and inhibits the production of tissue plasminogen activator (t-PA), thus impairing anticoagulant mechanisms. On the other hand, TNF up-regulates the production of procoagulant factors such as t-PA inhibitor (PAI), tissue factor and platelet activating factor (PAF). These effects create an imbalance between procoagulant and anticoagulant mechanisms, in favor of the former. TNF also activates polymorphonuclears (PMNs), and increases their chemotaxis and adherence to endothelial surfaces by up-regulation of specific endothelial (ELAM-1) and PMN (CDw18) adherence proteins. The damage inflicted by activated PMN to the endothelial cell promotes tissue factor exposure and PAI release, with initiation of the characteristic explosive coagulation process of DIC, facilitated by the dissociation between pro- and anticoagulant mechanisms induced by TNF. These newly discovered mechanisms precipitating septic shock and DIC enable consideration of new treatments for this condition as anti-TNF antibodies or TNF inhibitors, anti-ELAM-1 antibodies anti-tissue factor antibodies, administration of activated factor C, etc. These therapeutic approaches may revolutionize the treatment of septic shock and DIC in the next decade.
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PMID:Role of tumor necrosis factor in the pathogenesis of intravascular coagulopathy of sepsis: potential new therapeutic implications. 199 4

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the limulus hemolymph. We have determined the entire amino acid sequence of factor C using recombinant DNA technique. The zymogen consisted of 994 amino acid residues with a calculated molecular mass of 109,648 Da. Most interestingly, factor C has five repeating units ("Sushi" domain or short consensus repeat) of about 60 amino acid residues each, which have been found in many proteins participating in the mammalian complement system. In addition to a typical serine protease domain in the carboxyl-terminal portion, characteristic segments with an epidermal growth factor-like, a lectin-like, a cysteine-rich, and a proline-rich domain were also found, revealing a unique mosaic protein structure. The serine protease domain was most analogous to human thrombin. Factor C was identified to localize in large granules in the cell, indicating that it is released from the cell by lipopolysaccharide stimulation. Furthermore, we identified a transcript possibly derived by alternative splicing of factor C mRNA, which encodes a protein sharing the amino-terminal portion of factor C. We suggest that factor C, a newly discovered type of serine protease zymogen, is a "coagulation-complement factor" which may play important roles in both hemostasis and host defense mechanisms.
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PMID:Limulus factor C. An endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains. 200 2

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.
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PMID: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. 201 64

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.
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PMID:Interaction between lipopolysaccharide and intracellular serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes. 316 90

An intracellular clotting factor, factor C, found in the horseshoe crab hemocytes is a lipopolysaccharide-sensitive serine-protease zymogen, which participates in the initiation of the hemolymph clotting system [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. The subsequent study of this zymogen, using various synthetic lipid A analogues, revealed that the zymogen factor C is rapidly activated by acylated (beta 1-6)-D-glucosamine disaccharide bisphosphate (synthetic Escherichia coli-type lipid A), and the corresponding 4'-monophosphate analogues. However, the corresponding non-phosphorylated lipid A did not activate factor C, indicating that a phosphate ester group linked with the (beta 1-6)-D-glucosamine disaccharide backbone is required for the zymogen activation. During these studies we also found that the zymogen factor C is significantly activated by acidic phospholipids, such as phosphatidylinositol, phosphatidylglycerol and cardiolipin, but not at all by neutral phospholipids. The rate of this activation, however, was affected markedly by ionic strength in the reaction mixture, although such an effect was not observed in the lipid-A-mediated activation of factor C. A variety of negatively charged surfaces, such as sulfatide, dextran sulfate and ellagic acid, which are known as typical initiators for activation of the mammalian intrinsic clotting system, did not show any effect on the zymogen factor C activation. These results suggest that lipid A is the most effective trigger to initiate the activation of the horseshoe crab hemolymph clotting system.
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PMID:Intracellular serine-protease zymogen, factor C, from horseshoe crab hemocytes. Its activation by synthetic lipid A analogues and acidic phospholipids. 316 24

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.
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PMID:Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease. 330 57

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.
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PMID:Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Isolation and characterization. 351 66

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.
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PMID:Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes. 351 94

Exposure of limulus hemocytes to bacterial endotoxins (lipopolysaccharide, LPS) results in the activation of the intracellular clotting system, consisting of several protein components. During the separation of these components, a potent anticoagulant, named anti-LPS factor, which inhibits the endotoxin-mediated activation of the coagulation cascade, was found in hemocytes from both Tachypleus tridentatus and Limulus polyphemus (Tanaka, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 717-723). The principle isolated from the Tachypleus hemocyte lysate by column chromatographies on dextran sulfate-Sepharose CL-6B and Sephadex G-50 under sterile conditions was a simple basic protein with an apparent molecular weight of 15,000. It consisted of a single chain polypeptide containing a total of 128 amino acids. The COOH-terminal end was presumed to be histidine, but no NH2-terminal end reactive to phenylisothiocyanate was detected. The isolated anti-LPS factor specifically inhibited the endotoxin-mediated activation of factor C, which has recently been identified as an LPS-sensitive serine protease zymogen in the hemocytes. This inhibition appeared to be due to the binding of anti-LPS factor with LPS. Moreover, anti-LPS factor had an antibacterial effect on the growth of Gram-negative bacteria (Salmonella minnesota R595 and 1114W) but not on that of Gram-positive bacteria (Staphylococcus aureus 209P). These biological activities of the isolated anti-LPS factor suggest an important role in cellular defence of limulus against microbial invasion.
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PMID:Isolation and biological activities of limulus anticoagulant (anti-LPS factor) which interacts with lipopolysaccharide (LPS). 403 Jul 41

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