Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol. Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows: (Table: see text). The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine alpha-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine alpha-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.     

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.     

Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.     

Conformation of tachyplesin I from Tachypleus tridentatus when interacting with lipid matrices.

The mode of action of tachyplesin I, an antimicrobial cationic heptadecapeptide amide isolated from the hemocyte debris of a horseshoe crab, Tachypleus tridentatus, toward lipid matrices was studied with synthetic tachyplesin I, its analogs with Phe in place of Trp or Tyr, a linear analog with no disulfide bonds, and two linear short fragments. Circular dichroism spectra showed that tachyplesin I took an antiparallel beta-structure in buffer solution and a certain less ordered structure in acidic liposomes composed of egg phosphatidylcholine and egg phosphatidylglycerol (3:1). Spectrophotometric titration of the peptides with laurylphosphorylcholine revealed that both Trp and Tyr residues orient toward the inside of lipid matrices, suggesting that they are on the same side of the peptide backbone. The carboxyfluorescein leakage experiment and fluorescence data indicated that tachyplesin I interacted strongly with neutral and acidic lipid bilayers and an aromaticity-rich hydrophobic part of the peptide was embedded in lipid membranes. All the peptides except for the short fragments were almost equally active in lipopolysaccharide binding. The energy-transfer experiment showed that a conformational change occurred such that the Tyr and Trp residues are positioned more closely to each other in acidic liposomes than in buffer solution. The present study strongly suggested that amphipathic lipid bilayers induced a conformational change of tachyplesin I from an energetically stable beta-structure to a less ordered, probably more amphipathic structure.     

Hemocyanin from Tachypleus gigas. I. Oxygen-binding properties

Hemocyanin was prepared from an Asian horseshoe crab, Tachypleus gigas. The hemocyanin was found to be similar to Limulus hemocyanin in the size of native molecules (48-mer) and dissociation under nonphysiological conditions. It also showed the reverse Bohr effect. The O2 affinity of the dissociated monomer was higher than that of the native molecule. Equilibrium O2 binding to T. gigas hemocyanin was studied with special attention to the effect of inorganic ions. Neutral salts decreased the O2 affinity of the associated hemocyanin. In the presence of CaCl2 the strength of the effect was in the order of Na+ greater than Cs+ not equal to K+ for the series of chlorides, and Br- not equal to Cl- greater than SO4(2-) for the series of Na+ salts. A high concentration of CaCl2 (50-500 mM) considerably increased the Hill coefficient. The O2 binding data obtained under various ionic conditions were analyzed by model fitting. The two-state concerted model could be fitted to the data, if the ligand affinity of the states was allowed to vary. Statistical tests of the fitting showed that the hexameric structure can be regarded as the functional unit under physiological conditions.     

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.     

Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives

Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachypleus tridentatus.It has high antimicrobial activity and adopts a beta-hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar beta-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the beta-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the beta-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity: a beta-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association.     

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.     

An antimicrobial peptide tachyplesin acts as a secondary secretagogue and amplifies lipopolysaccharide-induced hemocyte exocytosis

In the horseshoe crab, bacterial lipopolysaccharide (LPS) induces exocytosis by granular hemocytes, resulting in the secretion of various defense molecules, such as lectins and antimicrobial peptides, via a G protein-mediating signaling pathway. This response is a key component of the horseshoe crab innate immune response against infectious microorganisms. Here, we report an endogenous amplification mechanism for LPS-induced hemocytes exocytosis. The concentration of LPS required for maximal secretion decreased in proportion to the density of hemocytes, suggesting the presence of a positive feedback mechanism for secretion via a mediator secreted from hemocytes. The exocytosed fluid of hemocytes was found able to induce hemocyte exocytosis in the absence of LPS. Furthermore, tachyplesin, a major antimicrobial peptide of hemocytes, was able to trigger exocytosis in an LPS-independent manner, which was inhibited by a phospholipase C inhibitor, U-73122, and a G protein inhibitor, pertussis toxin. Surface plasmon resonance analysis showed that tachyplesin directly interacts with bovine G protein. These findings suggest that the tachyplesin-induced hemocyte exocytosis also occurs via a G protein-mediating signaling pathway. We concluded that tachyplesin functions not only as an antimicrobial substance, but also as a secondary secretagogue of LPS-induced hemocyte exocytosis, leading to the amplification of the innate immune reaction at sites of injury.     

The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine-knot motif.

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.     

Antimicrobial tachyplesin peptide precursor. cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus)

The hemocytes of the horseshoe crab have been found to contain a new family of Arthropodous antibiotics, termed the "tachyplesin family." These peptides are composed of 17-18 amino acid residues with a carboxyl-terminal arginine alpha-amide. We report here the entire cDNA sequence coding for the tachyplesin precursors and their distribution in various tissues of the horseshoe crab. Sequence analysis of the cloned cDNAs revealed that the tachyplesin precursors consist of 77 amino acids with 23 residues in a presegment, and that there are two types of mRNAs corresponding to the isopeptides tachyplesins I and II. Both precursors contain a putative signal peptide, a processing peptide sequence and a carboxyl-terminal amidation signal "Gly-Lys-Arg" connected to the mature tachyplesin peptide. Moreover, an unusual acidic amino acid cluster, Asp-Glu-Asp-Glu-Asp-Asp-Asp-Glu-Glu-COOH, is present in the carboxyl-terminal portions of both precursors. These results suggest that the two types of tachyplesin precursors are first synthesized as preproproteins and are then incorporated into the intracellular organelle, accompanied by various processing events. Northern blot analysis on a total RNA from various tissues of the horseshoe crab revealed that the tachyplesin precursors are expressed mainly in hemocytes and cardiac and brain tissues. Tachyplesin was immunohistochemically localized in the smaller dense granules rather than the typical large granules present in abundance in the hemocytes.     

Bacteria-selective synergism between the antimicrobial peptides alpha-helical magainin 2 and cyclic beta-sheet tachyplesin I: toward cocktail therapy.

Magainin 2 and tachyplesin I (T-SS) are membrane-permeabilizing antimicrobial peptides discovered from frog skin and horseshoe crab hemolymph, respectively. They are classified into different secondary structural classes, i.e., alpha-helix and cyclic beta-sheet, respectively. We found that F5W-magainin 2 (MG2) and T-SS exhibited marked synergistic effects against Gram-negative and Gram-positive bacteria without enhancing hemolytic activity as a measure of toxicity. Dye release experiments using liposomes suggested that the selective synergism is mainly due to anionic phospholipid-specific synergism in membrane permeabilization. Furthermore, the cyclic structure of T-SS was found to be necessary for synergism because a linear analogue of T-SS did not show good synergism with MG2. These novel observations suggested the possibility of the development of cocktail therapeutic regimens using combinations of antimicrobial peptides.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk L-alpha-phosphatidylglycerol/egg yolk L-alpha-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Action mechanism of tachyplesin I and effects of PEGylation

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.     

Heterogeneity of the minimum functional unit of hemocyanins from the spider (Argiope bruennichii), the scorpion (Heterometrus sp.), and the horseshoe crab (Tachypleus tridentatus).

The heterogeneity of arthropod hemocyanins was studied by polyacrylamide gel electrophoresis and immunochemical techniques. The spider (Argiope bruennichii), the scorpion (Heterometrus sp.), and the horseshoe crab (Tachypleus tridentatus) were found to have 4, 5, and 5 minimum functional units of hemocyanin, respectively, the apparent molecular weights of which were 79,000, 81000, and 80,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas

The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.     

Hemocyanin from Tachypleus gigas. II. Cooperative interactions of the subunits

Six subunits (I to VI) were isolated from hemocyanin of an Asian horseshoe crab, Tachypleus gigas, by anion exchange chromatography of the dissociated hemocyanin. The subunit preparations were nearly homogeneous as judged by alkaline electrophoresis, but they still showed the presence of isoproteins in isoelectric focusing. The subunits were reassembled (in 10 mM CaCl2 at pH 7.5) and tested for restoration of the cooperativity in O2 binding. The reassembly of the subunits gave equilibrium mixtures of the monomer and hexamer with small amounts of larger molecules. Homogeneous and heterogeneous hexamers were prepared by reassembling a single kind or two kinds of subunits, followed by isolation of the hexamer fraction by gel filtration. Among the homohexamers, only the subunit V hexamer showed cooperativity in O2 binding with the Hill coefficient of 1.6. Among the heterohexamers the subunit I/V hybrid was most noteworthy, showing a Hill coefficient (1.7) higher than that of any other heterohexamer examined. It was concluded that there are specific interactions between the subunits I and V. It is suggested that their interactions are important for the cooperativity in the native hemocyanin.     

Hemocyanins and the immune response: defense against the dark arts

The innate immune response is a conserved trait shared by invertebratesand vertebrates. In crustaceans, circulating hemocytes playsignificant roles in the immune response, including the releaseof prophenoloxidases. Activated phenoloxidase (tyrosinase) participatesin encapsulation and melanization of foreign organisms as wellas sclerotization of the new exoskeleton after wound-repairor molting. Hemocyanin functions as a phenoloxidase under certainconditions and thus also participates in the immune responseand molting. The relative contributions of hemocyte phenoloxidaseand hemocyanin in the physiological ratio at which they occurin hemolymph have been investigated in the crab Cancer magister.Differences in activity, substrate affinity, and catalytic abilitybetween the two enzymes indicate that hemocytes are the predominantsource of phenoloxidase activity in crabs. In contrast, hemocyaninis the primary source of phenoloxidase activity in isopods andchelicerates whose hemocytes show no phenoloxidase activity.Quantitative PCR studies on the distribution of prophenoloxidasemRNA in the tissues of Carcinus maenas showed little effectrelative to salinity stress. Phylogenetic analysis of hemocyanin,phenoloxidase, and other members of this arthropod gene familyare consistent with the possibility that a common ancestralmolecule had both phenoloxidase and oxygen-binding capabilities.     

A link between blood coagulation and prophenol oxidase activation in arthropod host defense

Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade.     

鲎源抗菌肽的研究及其潜在应用价值

鲎血细胞来源的抗菌肽,在鲎天然免疫中起至关重要的作用,它对外源病原菌具有抗性作用,降低了外源病原菌对鲎活体的致病性,增强了鲎的天然免疫能力。鲎源抗菌肽有一些不同于其他来源的抗菌肽的优势,对鲎源抗菌肽的研究有很重要的实用意义。概述了鲎源抗菌肽的性质、分子结构、基因序列及其制备,并对它们的潜在应用价值进行了论述。