Interaction of basic extension peptide fragments of adrenodoxin precursor with phospholipid vesicles

Two extension peptide fragments PA_1-4 and PA_17-32, which correspond to the residues 1–14 and 17–32, respectively, of adrenodoxin precursor, were synthesized by the solution method to find a sequence necessary for the import of the precursor into mitochondria. Biological assay showed that PA_1-14 inhibited the import of two mitochondrial enzyme precursors, but PA_17-32 showed no inhibition, indicating that the N-terminal sequence has important information for import. CD spectra of the peptides demonstrated that PA_1-14 formed α-helical structure in Tris-HCl buffer (pH 7.4) containing acidic phospholipid liposomes. Furthermore, PA_1-14 induced the moderate leakage of carboxyfluorescein from phospholipid vesicles. The relationship between the structure and function of the peptides is discussed.     

Influence of lipophilic groups in cationic α-helical peptides on their abilities to bind with DNA and deliver genes into cells

Abstract: For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic α-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH₂) obtained from wasp venom and an α-helix model peptide (LARL-LARL-LARL-NH₂). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.     

Interaction of α-helical peptides with phospholipid membrane: effects of chain length and hydrophobicity of peptides

To investigate the interaction of amphiphilic α-helical peptides with phospholipid membranes, we synthesized Ac-(Leu-Ala-Arg-Leu)₃-NHcH₃ (4₃) and three derivatives, in which the chain length and the size of the hydrophobic region of the peptides were different from each other. These peptides formed an α-helical structure in the presence of vesicles. In the membrane-perturbation measurement, only 4₃ showed a strong membrane-perturbation activity below phase-transition temperature (25°C), but above phase-transition temperature (50°C), most peptides showed similar strong activities. On the other hand, in membrane-fusion measurement the long peptides, e.g., Ac-(Leu-Ala-Arg-Leu)₃-(Leu-Arg-Ala-Leu)₃-NHCH₃, had strong activities at low peptide concentrations at 25°C. The present study indicated a parallel relationship did not always exist between membrane fusion and perturbation caused by peptides.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom

Abstract: Eumenine mastoparan‐AF (EMP‐AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP‐AF peptide presented low cytolytic activities in human erythrocytes and in RBL‐2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad‐spectrum inhibitory activity against Gram‐positive and Gram‐negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C‐terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N‐terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.     

Interaction of calmodulin with synthetic deletion peptides of melittin

The 26-residue peptide melittin present in bee venom has been shown to bind calmodulin tightly. In this study we synthesized the following series of deletion peptides of melittin by the solid-phase method: Mel12, Mel13, Mel 14, Mel 15, Mel15F. The results of this study show that the deletion peptides Mel 14 and Mel 15 have almost the same binding activity as the intact native peptide. Each deletion peptide forms a 1:1 complex with calmodulin according to electrophoresis analysis. When the tryptophanyl residue of Mel15 was replaced by the phenylalaninyl residue, the dissociation constant of the peptide—calmodulin complex increased. This shows the importance of the tryptophanyl residue for binding to calmodulin.     

Interaction of acylated and substituted antimicrobial peptide analogs with phospholipid-polydiacetylene vesicles. Correlation with their biological properties

A series of peptide analogs based on region 6-22 of Plantaricin 149 sequence were synthesized. The interaction between these analogs and phospholipid-polydiacetylene vesicles was investigated to evaluate the ability of the bioassay to detect differences in the interaction of the peptides with dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles, associated with amino acid substitution and N-terminal conjugation of the sequences with short fatty acids (8 and 12 carbon atoms). Fatty acid conjugation of peptides with low antimicrobial activity resulted in lipopeptides with improved activity against strains of Staphylococcus aureus and Listeria monocytogenes. The length of the fatty acid determined the bacterial specificity, and the conjugation with n-octanoic acid yielded the most active analog (C8-CT) against Staphylococcus aureus strain (MIC: 1.0 μm) while the conjugation with n-dodecanoic acid (C12-CT) was optimal for Listeria monocytogenes strain (MIC: 2.0 μm). In contrast, the substitution of Phe by Trp had an unfavorable effect on the antimicrobial activity. Hemolysis tests and membrane interaction studies with dipalmitoylphosphatidylcholine-polydiacetylene vesicles showed that lipopeptides interact to a greater extent with both biological and biomimetic membranes. Also, a good correlation was found between antimicrobial activity against Staphylococcus aureus strain and % colorimetric response values with dipalmitoylphosphatidylglycerol-polydiacetylene vesicles.     

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure).

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences: Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2 All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation.     

Design of 16-residue peptides possessing antimicrobial and hemolytic activities or only antimicrobial activity from an inactive peptide

We have explored the possibility of generating peptides having antimicrobial and hemolytic activities or only antimicrobial activity, from a 16-residue peptide, GFFALIPKIISSPLFK, corresponding to the N-terminal region of the toxin pardaxin. This peptide does not exibit these activities, although it can permeabilize model membranes. Peptides were synthesized wherein either A4 or P7 were substituted by K and S11 replaced by K , Peptides in which P7 and S11 were replaced with K, ( AK ) and A4 and S11 replaced with K and A instead of P at position 7, ( KA ) showed potent antimicrobial and hemolytic activities. However, the peptide where S11 and A4 were replaced with K, ( KP ) showed pronounced antimicrobial activity with very weak hemolytic activity. Circular dichroism studies indicated that peptides AK and KA had a strong propensity to occur in a helical conformation, whereas KP did not. Peptides AK and KA were very effective in permeabilizing model membranes, whereas KP was relatively ineffective. Our studies thus suggest the requirements for a peptide to have only antimicrobial activity and also that selectivity in activity can be rationalized on the basis of biophysical principles. Thus, by judicious positioning of amino acids, especially positively charged ones, it should be possible to generate biologically active peptides without taking recourse to a combinatorial approach. © Munksgaard 1995.     

Synthesis and conformation of constrained peptides with hypoglycaemic activity derived from human growth hormone

The α-aminosuccinimide (Asu¹¹) octapeptide analogue of human growth hormone hGH[6-13] (Leu⁶-Ser-Arg-Leu-Phe-Asu-Asn-Ala¹³) has been reported [Robson et al. (1990) Biol. Chem. Hoppe Seyler 371 , 423-431] to have hypoglycaemic activity whilst the corresponding peptide with Asp at position 11 is inactive. In order to determine whether this change in activity is caused by conformational and/or stereo-electronic effects, the incorporation of two different isomeric γ-lactam structures at position 11 has been investigated. One lactam structure (i) is of the type developed by Freidinger and coworkers [Freidinger et al. (1982) J. Org. Chem. 47 , 102-107], whilst the isomeric γ-lactam structure (ii) represents a new type of constrained synthon for use in peptide synthesis. The chiral type-ii γ-lactam was synthesized via a suitably protected desoxo-dipeptide prepared in several ways from L-aspartic acid. The solution conformations of the [Asu¹¹]- and the [γ-lactam¹¹]-containing hGH[6-13] peptide analogues were investigated with the aid of two-dimensional NMR (COSY and NOESY) spectroscopy. Conformational similarities were found for these hGH[6-13] peptide analogues. For example, for all peptide analogues studied, weak NOEs were evident between the Phelo ring protons and protons of the amino acid residues at the C-terminus. Overall, however, the NOESY NMR spectra of the [Asu¹¹]- and the [γ-lactam¹¹]-containing peptides related to hGH[6-13] suggest the presence of an extended structure in solution with a possible weak type II′β-turn at position 11. The extent of conformational constraint introduced into these hGH[6-13] peptide analogues by substitution of the Asu¹¹ residue with either isomeric γ-lactam structure was reflected as differences in their hypoglycaemic activity. In particular, the hGH[6-13] peptide analogue derived from the new chiral type-ii γ-lactam exhibits both lower activity in intravenous insulin tolerance tests in vivo and weaker NOEs than the isomeric hGH[6-13] peptide analogue derived from the type (i) γ-lactam structure. The relative change in blood glucose levels from 20 to 90 min for the racemic (R,S)-form of the type-ii γ-lactam compared to the control values was approximately half that of the (S)-stereoisomer. © Munksgaard 1994.     

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity

Abstract: A 25‐mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure–activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an α‐helical structure in the presence of DOPC/DOPG (3 : 1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye‐leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye‐leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that α‐helicity, rather than hydrophobicity, affects the properties and activity of the peptide.     

Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA-Zn(II) column was observed for P1 protamines with one histidine in the N-terminal sequence (ram and boar protamines). The strong binding was found to be related to the presence of tyrosine, serine and threonine closely spaced to the histidyl side chain. In the case of human protamine P2, the strong retention on the IDA-Zn(II) column seems to result from the additive contribution of all the histidine residues of the molecule. Thus, strong retention of protamines in IMAC seems to depend on an additive contribution of amino-acid side chains: histidine, tyrosine, serine, threonine and perhaps arginine. The high affinity of protamines, more especially P2 protamines, for zinc suggests that this metal ion could play a role for their correct folding and binding to DNA.     

杀菌性/通透性增加蛋白(BPI)模拟肽对铜绿假单胞菌形态学的影响

目的　探讨杀菌性 /通透性增加蛋白 (BPI)模拟肽 10 3 42的体外杀菌作用及其对铜绿假单胞菌形态学的影响。方法　培养铜绿假单胞菌PA10 3、PSPNC49619、大肠埃希氏菌J5、V5 17和金葡球菌MSSA2 5 92 3于对数生长期 ,调整细菌浓度为 10 5CFU/ml ,加入BPI模拟肽 10 3 42 (10 0 μg/ml) ,测定其对细菌的杀菌曲线。透射电镜和扫描电镜下观察BPI模拟肽 10 3 42 (10 0 μg/ml)与铜绿假单孢菌PA10 3 3 7℃共培养 5、10、15、3 0min后细菌形态学的变化。结果　BPI模拟肽 10 3 42具有明显的杀菌作用。与细菌作用 5min后 ,细菌外膜出现轮廓模糊 ,10min后细菌内膜出现不完整 ,至 15min细菌胞膜不清 ,胞质外流 ,3 0min能见到细菌完全裂解。结论　BPI模拟肽 10 3 42具有明显的杀菌活性 ,该活性与破坏铜绿假单胞菌的膜结构密切相关。     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30α, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30α, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a β-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an α-helical pattern though the a-helical content calculated was low (25–30%). α-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248–252 that residues 90–111 of PH-30α may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30α in membranes.     

Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein

Starting from the α-(2,4-diniethoxybcnzyl) ester of N-(9-fluorenylniethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N^c-l-(4,4-dimethyl-2,6-dioxocyclohexylidenc)ethyl lysY1 [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetarnide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylinercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue. and cleaved from the solid support with trifluoroacetic acid/anisole/ water, 95/2.5/2.5. Cyclic peptides comprising 7–14 amino acid residues were obtained employing this procedure. As a model conjugation. cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide1 peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.     

Purification,characterization, in vitro anti-hepatic fibrosis activity of bioactive peptides derived from Carapax Trionycis hydrolysates

Anti-hepatic fibrosis peptide from Carapax Trionycis was purified, characterized, and inhibitory effect was assessed. Carapax Trionycis extract peptide hydrolysates (CTEPHs) were separated by ultrafiltration, Sephadex G-15 gel chromatography and RP-HPLC. One novel anti-hepatic fibrosis peptide (CTEPH-1: Asn-Pro-Asn-Pro-Thr) was obtained and identified. MTS assay and enzyme-linked immunosorbent assay were applied to evaluate the anti-fibrotic effect of CTEPH-1 on activated hepatic stellate cells (HSCs) in vitro. CTEPH-1 efficiently inhibited activation and proliferation of cultured HSC-T₆ cells via lowering the contents of collagen and TIMP-1 except for matrix metalloproteinase-1 (MMP-1). The purified peptide might be beneficial as functional food or potential drug for treatment of liver fibrogenesis.     

Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog,Hydrophylax bahuvistara (Anura: Ranidae)

Two novel peptides (brevinin1 HYba1 and brevinin1 HYba2) were identified from the skin secretion of the frog Hydrophylax bahuvistara, endemic to Western Ghats, India, and their amino acid sequences were confirmed using cDNA cloning and LC/MS/MS. Antibacterial, hemolytic, and cytotoxic activities of brevinin1 peptides and their synthetic analogs (amidated C‐terminus) were investigated and compared. All the peptides except the acidic forms showed antibacterial activity against all tested Gram‐positive and Gram‐negative bacteria. They exhibited low hemolysis on human erythrocytes and showed potent cytotoxic activity against Hep 3B cancer cell line. Upon amidation, the peptides showed increased activity against the tested microbes without altering their hemolytic and cytotoxic properties. The study also emphasizes the need for screening endemic amphibian fauna of Western Ghats, as a potential source of host defense peptides with possible therapeutic applications in the future.     

Purification and identification of anti-inflammatory peptides derived from simulated gastrointestinal digests of velvet antler protein (Cervus elaphus Linnaeus)

The objective of this study was to identify anti-inflammatory peptides from simulated gastrointestinal digest (pepsin–pancreatin hydrolysate) of velvet antler protein. The hydrolysate was purified using ultrafiltration and consecutive chromatographic methods. The anti-inflammatory activity of the purified fraction was evaluated by the inhibition of NO production in lipopolysaccharide-induced RAW 264.7 macrophages. Four anti-inflammatory peptides, VH (Val–His), LAN (Leu–Ala–Asn), AL (Ala–Leu), and IA (Ile–Ala), were identified by liquid chromatography/tandem mass spectrometry. Each of these peptides demonstrated a U-shaped dose–effect relationship. VH, LAN, AL and IA showed the strongest anti-inflammatory activities at 200 μg/mL, that is, 15.5%, 13.0%, 16.0% and 11.2% inhibition of lipopolysaccharide-induced NO production, respectively. Additionally, the enhanced NO inhibitory activity was observed for the peptides mixture, indicating the possible synergistic effects. These results suggested that the peptides derived from velvet antler protein could potentially be used as a promising ingredient in functional foods or nutraceuticals against inflammatory diseases.     

Design and characterization of short hybrid antimicrobial peptides from pEM‐2, mastoparan‐VT1, and mastoparan‐B

Antimicrobial peptides are considered to be excellent templates for designing novel antibiotics because of their broad‐spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance. In this study, for the first time, a series of short hybrid antimicrobial peptides combined by different fragments of venom‐derived alpha‐helical antimicrobial peptides pEM‐2, mastoparan‐VT1, and mastoparan‐B were designed with the intent to improve the therapeutic index of the parental peptides. Short hybrid antimicrobial peptides PV, derived from pEM‐2 and mastoparan‐VT1, was found to possess the highest antibacterial, hemolytic, and cytotoxic activity. Short hybrid antimicrobial peptides PV3, derived from pEM‐2 and three fragments of mastoparan‐VT1, showed more than threefold improvement in therapeutic index compared with parental peptides pEM‐2 and mastoparan‐VT1. PV had the highest antimicrobial activity in stability studies. Except BVP, designed based on all three parental peptides, the other short hybrid antimicrobial peptides at their minimal inhibitory concentration and 2× minimal inhibitory concentration required less than 120 and 60 min to reduce >3log10 the initial inoculum, respectively. All peptides had membrane‐disrupting activity in a time‐dependent manner. Collectively, this study highlights the potential for rational design of improved short hybrid antimicrobial peptides such as PV3 that was an ideal candidate for further assessment with the ultimate purpose of development of effective antimicrobial agents.     

Lipophilic derivatization of synthetic peptides belonging to NS3 and E2 proteins of GB virus‐C (hepatitis G virus) and its effect on the interaction with model lipid membranes

Abstract: The synthesis by solid‐phase methodologies of peptides belonging to structural and non‐structural proteins of GB virus C as well as its N‐α‐acylation with myristate and palmitate fatty acids is described. To explore the peptide–lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non‐structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.