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.     

鲎源抗菌肽对大肠杆菌的抑杀机理

【目的】研究鲎源抗菌肽鲎素对大肠杆菌抑杀的作用机理,为防治大肠杆菌引起的肠道疾病提供新的潜在抗菌药物。【方法】利用牛津杯法和微量MH肉汤稀释法测定其抗菌活性,激光共聚焦扫描显微镜观察鲎素对大肠杆菌的结合、分布及杀伤过程,透射电镜观察鲎素对大肠杆菌超微结构的影响,并采用琼脂糖凝胶阻滞电泳研究其对大肠杆菌基因组DNA和RNA的影响。【结果】鲎素对大肠杆菌的最小抑菌浓度与最低杀菌浓度分别为5 mg/L和20 mg/L;激光共聚焦扫描显微镜和透射电镜观察发现鲎素能快速作用于细胞表面,并发生聚集现象,随着作用时间的延长能导致细胞膜结构的破坏和细胞内含物的释放;通过琼脂糖电泳结果显示鲎素也能够作用于大肠杆菌基因组DNA,并呈浓度依赖关系,10 mg/L鲎素对基因组DNA无明显影响,80 mg/L鲎素能导致DNA断裂;凝胶阻滞电泳显示鲎素也能与基因组DNA和RNA发生结合。【结论】研究结果为深入探讨鲎素抑杀大肠杆菌的分子机制提供重要的理论依据。     

Influences of disulfide connectivity on structure and antimicrobial activity of tachyplesin I

Tachyplesin I is a potent antimicrobial peptide with broad spectrum of antimicrobial activity. It has 2 disulfide bonds and can form 3 disulfide bond isomers. In this study, the structure and antimicrobial activity of 3 tachyplesin I isomers (tachyplesin I, 3C12C, 3C7C) were investigated using molecular dynamic simulations, circular dichroism structural study, as well as antimicrobial activity and hemolysis assay. Our results suggest that in comparison to the native peptide, the 2 isomers (3C12C, 3C7C) have substantial structural and activity variations. The native peptide is in the ribbon conformation, while 3C12C and 3C7C possess remarkably different secondary structures, which are referred as “globular” and “beads” isomers, respectively. The substantially decreased hemolysis effects for these 2 isomers is accompanied by significantly decreased anti‐gram‐positive bacterial activity.     

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.     

Interaction between tachyplesin I,an antimicrobial peptide derived from horseshoe crab,and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program.CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.     

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.     

PEGylation of therapeutic proteins

Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.     

Action mechanism of insulin-mimetic vanadyl-allixin complex

In the 21st century, there has been a dramatic worldwide increase in the prevalence of metabolic syndromes, including diabetes mellitus (DM). Several synthetic pharmaceutical agents have been developed and used for the treatment of type-2 DM; however, these compounds have several problems such as side effects, hypoglycemia, and weight gain. Therefore, new drugs are required for DM therapy. We have proposed that some vanadyl complexes function as potent insulin-mimetic and antidiabetic agents in type-1 and type-2 DM animal models. In this article, we review the possible action mechanism of insulin-mimetic and antidiabetic vanadyl complexes, focusing on a recently proposed complex, bis(allixinato)oxovanadium(IV), with respect to the insulin-signaling pathway in cultured adipocytes.     

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.     

Studies on the reaction mechanism of DT diaphorase. Action of dead-end inhibitors and effects of phospholipids.

The relationship between the inhibition of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC.1.6.99.2] by 4-hydroxycoumarin and the 1,3-indandione derivates was investigated. Evidence is presented that these two classes of anticoagulants, although both acting as competitive inhibitors with respect to NAD(P)H, bind to different sites of the enzyme in a synergistic fashion. These findings are interpreted as indicative of a cooperativity between different substrate-binding sites of the enzyme.Neutral phospholipids exert effects on partially purified rat-liver DT diaphorase similar to those earlier obtained with nonionic detergents. The effects concern several kinetic parameters of the enzyme, including V, K_m for electron donor and acceptor, and K_i for various inhibitors. The changes in the kinetic parameters vary in extent and direction according to the individual phospholipids.     

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.     

溶葡球菌酶的定点突变与PEG巯基定点修饰

为了建立聚乙二醇 (PEG) 巯基定点修饰溶葡球菌酶的方法,并检验假定连接区的突变与修饰对酶活的影响,对溶葡球菌酶的假定连接区进行了巯基聚乙二醇定点修饰研究。通过分析溶葡球菌酶的结构特征,选择两个结构域之间的氨基酸 (133-154aa) 进行定点突变引入半胱氨酸残基。使用单甲氧基聚乙二醇马来酰亚胺 (mPEG-MAL) 进行定点修饰,对修饰后的酶进行纯化并测定酶活性。结果表明定点突变的半胱氨酸残基PEG修饰效率高、产物单一,运用简便的Ni2+-NTA柱亲和层析法实现了一步分离,获得了高纯度的目标蛋白,但在连接区进行定点突变及PEG定点修饰后的酶活有不同程度的降低,表明假定连接区部分位点的PEG修饰会对溶葡球菌酶的催化活性产生一定影响。