Molecular cloning of novel antimicrobial peptide genes from the skin of the Chinese brown frog, Rana chensinensis

One species of the Chinese brown frog, Rana chensinensis, is widely distributed in north-central China. In this study, a cDNA library was constructed to clone the antimicrobial peptides' genes from the skin of R. chensinensis. Twenty-three prepropeptide cDNA sequences encoding twelve novel mature antimicrobial peptides were isolated and characterized. Six peptides belonged to three known families previously identified from other Ranid frogs: temporin (4 peptides), brevinin-2 (1 peptide), and palustrin-2 (1 peptide). The other six peptides showed little similarity to known antimicrobial peptides. According to the amino acid sequences, with or without α-helix structure, and either hydrophilic or hydrophobic, these were organized into four new families: chensinin-1 (3 peptides), chensinin-2 (1 peptide), chensinin-3 (1 peptide), and chensinin-4 (1 peptide). Five peptides from different families were chemically synthesized, and their antimicrobial, cytolytic, and hemolytic activities were evaluated. Of these, brevinin-2CE showed strongest antimicrobial activities against both the Gram-positive and Gram-negative bacteria with a slight hemolysis. Temporin-1CEe and palustrin-2CE also displayed a slight hemolysis, but they had different activities to prokaryotic cells. Temporin-1CEe showed higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, whereas it was contrary to palustrin-2CE. Chensinin-1 CEb and chensinin-3CE only had moderate antimicrobial activity against microorganisms. In addition, the brevinin-2 peptides from different brown frogs were analyzed to reveal the taxonomy and phylogenetic relationships of R. chensinensis.     

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis)

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH₂, and exhibits 50–56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.     

Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica

Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.     

Interactions between chensinin‐1, a natural antimicrobial peptide derived from Rana chensinensis,and lipopolysaccharide

Lipopolysaccharide (LPS) plays a critical role in the pathogenesis of sepsis caused by gram‐negative bacterial infections. Therefore, LPS‐neutralizing molecules would have important clinical applications. Chensinin‐1, a novel antimicrobial peptide with atypical structural features, was found in the skin secretions of the Chinese brown frog Rana chensinensis. To understand the role of LPS in the bacterial susceptibility to chensinin‐1 and to investigate its anti‐endotoxin effects, the interactions of chensinin‐1 with LPS were investigated in this study using circular dichroism, in situ IR, isothermal titration calorimetry, and zeta potential. This study is the first to use in situ IR spectroscopy to evaluate the secondary structural changes of this peptide. The capacity of chensinin‐1 to block the LPS‐dependent cytokine secretion of macrophages was also investigated. Our results show that chensinin‐1 can form α‐helical structures in LPS suspensions. LPS can affect the antimicrobial activity of chensinin‐1, and chensinin‐1 was able to mitigate the effects of LPS. These data may facilitate the development of antimicrobial peptides with potent antimicrobial and anti‐endotoxin activities. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 719–726, 2015.     

中国林蛙皮肤抗菌肽基因的cDNA克隆及抗菌、抗癌和溶血活性的测定

采用RT-PCR和3￠RACE方法, 从中国林蛙皮肤总RNA中克隆出了6条编码不同抗菌肽前体的cDNA序列, 分别命名为: preprotemporin-1CEa、preprotemporin-1CEb、preprotemporin-1CEc、preprobrevinin-1CEa、preprobrevinin-1CEb和preprochensinin-1。cDNA碱基序列全长为289~315 bp, 编码59~65个氨基酸。6个抗菌肽前体由3部分结构域组成: 22个氨基酸残基组成的信号肽、多个酸性氨基酸残基组成的中间序列、高度变异的成熟肽。preprotemporin-1CEa、preprotemporin-1CEb和preprotemporin-1CEc属于temporin-1家族抗菌肽前体, 具有肽链短, C-端酰胺化的特点; preprobrevinin-1CEb和preprobrevinin-1CEa属于brevinin-1家族抗菌肽前体, 在肽链的C-端含有RANA盒结构, 可在2个半胱氨酸残基间形成二硫键, 组成7肽环; preprochensinin-1在已知多种数据库中没有发现序列同源的多肽, 被鉴定为新抗菌肽。人工合成temporin-1CEa、temporin-1CEb、brevinin-1CEa和chensinin-1四种中国林蛙皮肤抗菌肽, 活性检测结果表明: 它们对金黄葡萄球菌等3种革兰氏阳性细菌的生长具有明显抑制作用, 同时抑制乳腺癌MCF-7细胞和宫颈癌HeLa细胞生长。     

A melittin-related peptide from the skin of the Japanese frog,Rana tagoi,with antimicrobial and cytolytic properties

Two peptides with antimicrobial and cytolytic properties were purified from an extract of the skin of Tago's brown frog Rana tagoi. The primary structure of one peptide (FLPILGKLLS(10)GIL.NH(2)) identifies it as a member of the temporin family, whereas the second peptide (AIGSILGALA(10)KGLPTLISWI(20)KNR.NH(2)) displays 78% sequence identity to melittin from the venom of the honeybee Apis florea. Compared with melittin, the melittin-related peptide (MRP) was equipotent in inhibiting the growth of the Gram-positive bacterium Staphylococcus aureus, 5-fold less potent against the Gram-negative bacterium Escherichia coli and against the fungal pathogen, Candida albicans. MRP was 13-fold less hemolytic than melittin against human erythrocytes and 4- and 5-fold less cytolytic against mouse EL4 T-lymphoma-derived cells and L929 fibroblasts, respectively. However, at non-cytotoxic concentrations (相似文献   

Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria.

A peptide, termed ranatuerin 1T, with growth-inhibiting activity toward Staphylococcus aureus, was isolated from an extract of the skin of the European brown frog, Rana temporaria. The primary structure of the peptide was established as: GLLSGLKKVG10 KHVAKNVAVS20LMDSLKCKIS30GDC. In common with other anti-microbial peptides from Ranid frogs, (e.g., ranalexin, ranatuerins, gaegurins, brevinins, esculetins, rugosins), ranatuerin IT contains an intramolecular disulfide bridge forming a heptapeptide ring but there is little structural similarity outside this cyclic region. The minimum inhibitory concentration (MIC) of ranatuerin 1T was 120 microM against the Gram-positive bacterium S. aureus and 40 microM against the Gram-negative bacterium Escherichia coli, but the peptide was not active against the yeast Candida albicans.     

Molecular cloning, expression, purification, and functional characterization of palustrin-2CE, an antimicrobial peptide of Rana chensinensis

Antimicrobial peptides are effector molecules of the innate immunity of amphibians. Here, one antimicrobial peptide cDNA precursor, prepropalustrin-2CE3, from the tadpole of the Chinese brown frog Rana chensinensis was cloned. The coding sequence corresponding to the mature palustrin-2CE peptide was subcloned into pGEX-6p-1. The soluble GST-palustrin-2CE fusion protein was successfully expressed in the BL21(DE3)pLysS strain at 16 °C, and the proportion of the fusion protein reached 35%-39% of the total cellular protein. After removal of the GST-fusion tag, the purity of the palustrin-2CE obtained by Sephadex G50 chromatography was about 97%. Moreover, the purified palustrin-2CE displayed obviously inhibitory activities against the sensitive bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and multi-drug resistant S. aureus and E. coli. These findings suggest that the tadpole of the Chinese brown frog is a unique source of antimicrobial peptides and indicates the therapeutic potential of the palustrin-2CE peptide.     

Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.     

Solution structure of antimicrobial peptide esculentin-1c from skin secretion of Rana esculenta

Granular glands in the skins of frogs synthesize and secrete a remarkably diverse range of peptides capable of antimicrobial activity. These anuran skin antimicrobial peptides are commonly hydrophobic, cationic and form an amphipathic α-helix in a membrane mimetic solution. Recently, they have been considered as useful target molecules for developing new antibiotics drugs. Esculentin-1c is a 46-amino acid residue peptide isolated from skin secretions of the European frog, Rana esculenta. It displays the most potent antimicrobial activity among bioactive molecules. Esculentin-1c has the longest amino acids among all antimicrobial peptides. The present study solved the solution structure of esculentin-1c in TFE/water by NMR, for the first time. We conclude that this peptide is comprised of three α-helices with each helix showing amphipathic characteristics, which seems to be a key part for permeating into bacterial membranes, thus presenting antimicrobial activity.     

Synthetic mimic of antimicrobial peptide with nonmembrane-disrupting antibacterial properties

Polyguanidinium oxanorbornene ( PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents.     

Functional characterisation of the 1-18 fragment of esculentin-1b, an antimicrobial peptide from Rana esculenta

Esculentin-1 is a 46-amino acid residue peptide isolated from skin secretions of Rana esculenta, displaying the most potent antimicrobial activity among the bioactive molecules found in the secretion, with negligible effects on eukaryotic cell membranes. From skin secretions, the 19-46 fragment of esculentin-1, devoid of antibacterial activity, was also isolated. We studied in detail the activity of the N-terminal fragment (1-18) of esculentin-1 using a synthetic amidated analogue. The results show that this fragment is highly active against most bacterial and fungal species, although at a lower extent than the full-length peptide, being four-fold more potent against Phytophthora nicotianae. It has a reduced activity against human erythrocytes with respect to the full-length peptide. The killing curves in liquid medium are similar for the two molecules and the shorter peptide is able to increase the bacterial outer and inner membrane permeability. Overall these data indicate that the antimicrobial properties of esculentin-1 are exerted by its N-terminal 1-18 region and that the positively charged residue distribution as well as peptide length represent important determinants for cell selectivity.     

抗菌肽的结构特征、生物活性及应用

抗菌肽分子呈双亲性的仅一螺旋结构可使细菌、真菌、寄生虫的质膜形成离子通道,从而导致这些病原体死亡。抗菌肽分子也可通过影响它们的能量转运和代谢,损害它们的呼吸链的功能,抑制它们的蛋白质和DNA的合成,以及干扰病毒的侵染过程和抑制病毒的繁殖等机制,对病原体产生抑制或杀灭作用。此外,抗菌肽还可杀伤肿瘤细胞并提高机体的免疫功能。因此,抗菌肽有望开发为防治人和动物疾病的药物,应用前景广阔。     

An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina

A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1 Da, against the Gram-positive bacterium Staphylococcus aureus was 7 microM and against the Gram-negative bacterium Escherichia coli was 30 microM.     

Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic Brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).     

Purification and properties of laticeptin, an antimicrobial peptide from skin secretions of the South American frog Leptodactylus laticeps

Norepinephrine-stimulated skin secretions from the Sante Fe frog Leptodactylus laticeps contained high concentrations of a peptide, termed laticeptin, with the primary structure Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Leu-Ala-Gly-His-Leu-Ala-Thr-Lys-Val-Met-Asn-Lys-Leu.NH(2). Laticeptin inhibited the growth of selected Gram-negative bacteria but the lack of activity against Gram-positive bacteria and the very low hemolytic activity is probably a consequence of the weak amphipathicity of the peptide in its alpha-helical conformation.     

Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins

A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.     

Two novel antimicrobial peptides from skin secretions of the frog,Rana nigrovittata

Two novel antimicrobial peptides with similarity to brevinin‐2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin‐2‐RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin‐2‐RN2), respectively, by Edman degradation. Different from brevinin‐2, which is composed of 33 amino acid residues (aa), both brevinin‐2‐RN1 and ‐RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465‐9) encoding precursors of brevinin‐2‐RN1 and ‐RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin‐2‐RN. Both brevinin‐2‐RN1 and ‐RN2 showed strong antimicrobial activities against gram‐positive and gram‐negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.