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.     

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.     

Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin I by solid-state NMR

The conformation and membrane topology of the disulfide-stabilized antimicrobial peptide tachyplesin I (TP) in lipid bilayers are determined by solid-state NMR spectroscopy. The backbone (phi and psi) torsion angles of Val(6) are found to be -133 degrees and 142 degrees , respectively, and the Val(6) CO-Phe(8) H(N) distance is 4.6 A. These constrain the middle of the N-terminal strand to a relatively ideal antiparallel beta-sheet conformation. In contrast, the phi angle of Gly(10) is +/-85 degrees , consistent with a beta-turn conformation. Thus, TP adopts a beta-hairpin conformation with straight strands, similar to its structure in aqueous solution but different from a recently reported structure in DPC micelles where bending of the two beta-strands was observed. The Val(6) and Gly(10) CO groups are both 6.8 A from the lipid (31)P, while the Val(6) side chain is in (1)H spin diffusion contact with the lipid acyl chains. These results suggest that TP is immersed in the glycerol backbone region of the membrane and is oriented roughly parallel to the plane of the membrane. This depth of insertion and orientation differs from those of the analogous beta-sheet antimicrobial peptide protegrin-1 and suggest the importance of structural amphiphilicity in determining the location and orientation of membrane peptides in lipid bilayers.     

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×10⁶ colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 g/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.Abbreviations AP acidic carboxyl terminal polypeptide - Eca Erwinia carotovora ssp. atroseptica - Ecc E. carotovora ssp. carotovora - Ech E. chrysanthemi - IF intercellular fluid - SP signal peptide - TPNI (tpnI) tachyplesin I     

Specific interactions of the antimicrobial peptide cyclic beta-sheet tachyplesin I with lipopolysaccharides

The cyclic beta-sheet antimicrobial peptide tachyplesin I (T-SS) was found to show 280-fold higher affinity for lipopolysaccharides (LPS) compared with acidic phospholipids, whereas the linear alpha-helical peptide F5W-magainin 2 (MG2) could not discriminate between LPS and acidic phospholipids. The recognition site was the lipid A moiety and the cyclic structure was crucial to this specific binding. The cyclic structure also endowed the peptide with very rapid outer membrane (OM) permeabilization.     

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.     

Interactions of an antimicrobial peptide, tachyplesin I, with lipid membranes.

Tachyplesin I, isolated from the acid extracts of hemocytes of Tachypleus tridentatus, is a cyclic broad-spectrum antimicrobial peptide forming a rigid, antiparallel beta-sheet because of two intramolecular S-S linkages. The strong binding of the peptide to lipopolysaccharides cannot explain the susceptibilities of gram positive bacteria and fungi to the peptide. We found that tachyplesin I caused a rapid K+ efflux from Escherichia coli cells, concomitant with a reduced cell viability. This result suggests that the peptide-induced permeability enhancement of the bacterial membranes may be a plausible action mechanism. Thus, we studied the interactions of tachyplesin I with various large unilamellar vesicles (LUVs) to reveal the molecular machinery of the antimicrobial activity. Tachyplesin I induced the leakage of calcein, a trapped fluorescent marker, from LUVs of acidic phospholipids, especially phosphatidylglycerol (PG), but not from phosphatidylcholine LUVs. A detailed analysis found that the affinity of the peptide to the PG membranes is very strong and that the binding of one peptide molecule to approx. 200 lipid molecules leads to a significant leakage. The location of tachyplesin I in membranes was estimated by use of the Trp-2 fluorescence of the peptide. The presence of PG LUVs caused a blue shift of the maximum wavelength, an increase in the quantum yield, and a complete protection from fluorescence quenching by an aqueous quencher, acrylamide. Moreover, the degree of fluorescence quenching of the Trp residue by n-doxylstearates was in the order n = 5 greater than 7 greater than 12 approximately equal to 16. These results show that the Trp residue of tachyplesin I seems to locate in a hydrophobic environment near the surface of the PG bilayers.     

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.     

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.     

Extension Arm Facilitated PEGylation of Hemoglobin: Correlation of the Properties with the Extent of PEGylation

Human hemoglobin (Hb) conjugated with six copies of PEG-5K is nonhypertensive. The hexaPEGylated Hb exhibits molecular size homogeneity in spite of the chemical heterogeneity with respect to the sites of conjugation (Manjula et al., 2005). In the present study, Hb conjugated with an average of 4, 6, 8 and 10 copies of PEG-5K chains have been generated using the extension arm facilitated PEGylation protocol. Except for the tetraPEGylated Hb, all the other products exhibit molecular size homogeneity. The molecular, colligative and functional properties of PEG-Hb conjugates have been correlated with the extent of PEGylation. The results imply that six copies of PEG-5K chains are accommodated on Hb without significant crowding on the molecular surface. As more copies of PEG-5K chains are conjugated to form octa and deca PEGylated Hb, the PEG-chains conjugated appear to undergo transition from a mushroom (compact) to a brush-like conformation (extended conformation) with a concomitant decrease in the propensity of the molecule to transition from oxy to deoxy conformation in the presence of allosteric effectors. The viscosity and the colloidal osmotic pressure of Hb increase with the number of the PEG-chains conjugated in an exponential fashion. The composition of the PEGylated Hb generated appears to be controlled by (i) high reactivity of thiol groups of the extension arms on Hb with maleimide-PEG, (ii) increase in the viscosity of the reaction mixture as the level of PEGylation increases and (iii) increased resistance induced by the PEG-shell of PEGylated Hb to accommodate more PEG-chains as the level of PEGylation increases. Potential implications of extent of PEGylation on the oxygen delivery by PEG-Hb conjugate in vivo have been discussed.     

In vitro activities of tachyplesin III against Pseudomonas aeruginosa

The in vitro activities of tachyplesin III were investigated against 20 multidrug-resistant Pseudomonas aeruginosa clinical isolates. Methods included minimal inhibitory concentrations, minimal bactericidal concentrations, time-kill studies, checkerboard titration method, endotoxin-binding activity and cytotoxicity assay. Overall the organisms were susceptible to the peptide at concentrations of 0.50-4 mg/l. Tachyplesin III completely inhibits the endotoxin procoagulant activity at 22.36 mg/l concentration. Fractional inhibitory concentration indexes demonstrated synergy between the peptide and betalactams or colistin. In conclusion, the intrinsic antibacterial and antiendotoxin activities and the synergistic interactions demonstrated with clinically used antibiotics make tachyplesin III valuable as potential candidate for new therapeutic strategies aimed to treat P. aeruginosa infection.     

Eukaryotic expression and antimicrobial spectrum determination of the peptide tachyplesin II

The ADAMs (a disintegrin and metalloproteases) are an important class of enzymes in the regulation of human disease. The pro domains of ADAMs are responsible for the latency and secretion of mature enzymes. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme after secretion. To understand the functions of human ADAM pro domains and to determine three-dimensional structures, we have screened promising targets for expression and purification properties when using Escherichia coli as the host. The pro domain of ADAM22 (ADAM22-P) expressed in E. coli was folded, as determined by CD and NMR spectroscopy. An ADAM22-P fragment encoding residues 26–199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of β-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P_N) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P_C).     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.