Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576bp and coded for 191 amino acid residue protein precursors. Each of the deduced elapid cathelicidin has a 22 amino acid residue signal peptide, a conserved cathelin domain of 135 amino acid residues and a mature antimicrobial peptide of 34 amino acid residues. Unlike the highly divergent cathelicidins in mammals, the nucleotide and deduced protein sequences of the three cloned elapid cathelicidins were remarkably conserved. All the elapid mature cathelicidins were predicted to be cleaved at Valine157 by elastase. OH-CATH, the deduced mature cathelicidin from king cobra, was chemically synthesized and it showed strong antibacterial activity against various bacteria with minimal inhibitory concentration of 1-20microg/ml in the presence of 1% NaCl. Meanwhile, the synthetic peptide showed no haemolytic activity toward human red blood cells even at a high dose of 200microg/ml. Phylogenetic analysis of cathelicidins from vertebrate suggested that elapid and viperid cathelicidins were grouped together in the tree. Snake cathelicidins were evolutionary closely related to the neutrophilic granule proteins (NGPs) from mouse, rat and rabbit. Snake cathelicidins also showed a close relationship with avian fowlicidins (1-3) and chicken myeloid antimicrobial peptide 27. Elapid cathelicidins might be used as models for the development of novel therapeutic drugs.     

Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. But so far it has not yet been found in amphibians although a large number of other groups of AMPs have been identified. In the current work, the first amphibian cathelicidin (cathelicidin-AL) has been characterized from the frog skin of Amolops loloensis. Cathelicidin-AL (RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA) is a cationic peptide containing 48 amino acid residues (aa) with 12 basic aa and no acidic aa. The chemical synthesized peptide efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. The cDNA encoding cathelicidin-AL precursor was cloned from the skin cDNA library of A. loloensis. As other cathelicidins, the precursor of cathelicidin-AL also contains highly conserved anionic cathelin domain of cysteine proteinase inhibitor followed by the AMP fragment at C-terminus. Phylogenetic analysis revealed that as connecting link, the amphibian cathelicidin predates reptilia but postdates fish cathelicidin. The peptide purification combined with gene cloning results confirms the presence of cathelicidin in amphibians and filled the evolutionary gap of cathelicidin in vertebrate, considering amphibians' special niche as the animals bridging the evolutionary land-water gap.     

香鱼hepcidin基因的克隆、序列分析及组织表达特征

Hepcidin是一类富含半胱氨酸的抗菌肽,在鱼类非特异性免疫中起重要作用,具有调节铁代谢的功能。该研究克隆了香鱼hepcidin基因cDNA序列,全长763个核苷酸,包含一个完整的开放阅读框,推测编码一个由85个氨基酸组成的相对分子质量为9.7k的多肽,N端24个氨基酸是信号肽序列。香鱼hepcidin的成熟肽序列由25个氨基酸组成,含有8个半胱氨酸,可形成4个分子内二硫键结构。系统进化树分析表明,hepcidin的物种进化关系与目前接受的物种分类关系基本一致,香鱼hepcidin位于鱼类hepcidin簇中,与大西洋鲑hepcidin的亲缘关系最近,达60%。香鱼hepcidin在肝脏中表达量最大,在脾、肾、心脏和肌肉中也有表达。实时荧光定量PCR(qRT-PCR)结果表明,鳗利斯顿氏菌(Listonella anguillarum)感染以后香鱼肝组织中hepcidin基因mRNA表达量显著增加,在12h增加了8.26倍。hepcidin基因可能在香鱼抗外界病原物感染过程中起重要作用。     

香鱼凝血因子X基因表达与鳗利斯顿氏菌感染的相关性

凝血途径的关键因子——凝血因子X(coagulation factor X,FX),是一种与免疫调控密切相关的维生素K依赖型丝氨酸蛋白酶.该研究克隆了香鱼(Plecoglossus altivelis) FX基因全长cDNA序列,它由1817个核苷酸组成,包含一个大的开放阅读框,编码一个由453个氨基酸组成的相对分子质量为5.07×104的蛋白.香鱼FX与已知的哺乳动物FX的结构相似,N端24个残基为信号肽序列.序列比较表明,香鱼FX与斑马鱼FX的氨基酸同一性最高,为53％.在健康香鱼中,FX基因mRNA主要在肝组织中表达,脑和鳃中也有少量表达.实时荧光定量PCR分析揭示,鳗利斯顿氏菌感染后香鱼肝组织中FX基因mRNA表达显著上调,16h时达到5.43倍.通过原核表达系统表达了香鱼FX丝氨酸蛋白酶结构域,并制备了抗血清.Western blotting分析显示,鳗利斯顿氏菌感染后香鱼血清中FX含量显著增加,并随着时间延长上调倍数不断增大,在36h时达到3.68倍.据此,FX基因mRNA及蛋白的表达与鳗利斯顿氏菌感染香鱼的过程紧密相关,揭示它可能在鱼类抗细菌感染的免疫反应中起重要作用.     

A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor.     

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.     

香鱼补体成分C9基因的克隆、序列分析及表达

补体成分C9是构成膜攻击复合体引起靶细胞溶解破坏的重要组成成分。该文测定了香鱼C9(aC9)基因的cDNA全序列,序列全长2125个核苷酸,编码一个由592个氨基酸组成、相对分子质量为6.56×104的前体蛋白,N端22个氨基酸为信号肽序列。序列分析表明,aC9与虹鳟C9的氨基酸同源性最高,达56.8%,与其它鱼类C9的同源性介于40.9%~53.8%之间。aC9在健康香鱼肝、脾、肠、鳃和肌肉有表达,其中在肝内的表达量最高。实时荧光定量PCR的结果显示,鳗利斯顿氏菌侵染4h后,肝中aC9mRNA表达量显著上调,并随着时间的推移在16h时达到峰值。Westernblotting分析的结果显示,鳗利斯顿氏菌侵染后香鱼血清中的aC9蛋白随着时间的推移呈显著上调。以上结果表明,香鱼肝组织C9基因表达变化与鳗利斯顿氏菌的侵染密切相关,揭示了C9在鱼类抗细菌免疫反应中具有重要的作用。     

景东湍蛙抗菌肽jindongenin-1d的分析和活性测定

新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。     

Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Five novel antimicrobial peptides from the Kuhl’s wart frog skin secretions, Limnonectes kuhlii

Five novel antimicrobial peptides (temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin-LK1, and gaegurin-LK2) are purified and characterized from Kuhl’s wart frog skin secretions, Limnonectes kuhlii. They share obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors are composed of a predicted signal peptide, an acidic spacer peptide, and a mature antimicrobial peptide. Most of them showed strong antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. The current work identified and characterized three families of antimicrobial peptides from L. kuhlii skins and confirmed that the genus of Limnonectes amphibians share similar antimicrobial peptide families with the genus of Rana amphibians. In addition, a unique antimicrobial peptide (temporin-LK1) with 17 residues including four phenylalanines, which is significantly different from other temporins (16 residues, one or two phenylalanines), was identified in this work. Such unique structure might provide novel template or leading structure to design antimicrobial agents.     

家蝇防御素基因的cDNA克隆及序列分析

Defensin is a kind of cationic.inducible antimicrobial peptide found in a large range of living organisms that contributes to host defense by disrupting the cytoplasmic membrane of microorganisms.with their broad antimicrobial spectrum and strong pharmaceutical effects.antimicrobial peptides,including defensins,represent a source of novel antibiotic agents.A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDnA library of houseflies(Musca domestica) that had been challenged by E.coli and staphylococcus taincd an NH2-terminal signal sequence(1-22)followed by a propeptide and the mature peptide(53-92),The sequence identity with other insect defensin is between 51% and 73%.The mature peptide,with a predicted molecular weight of 4.0kDa,and pI of 8.69,has 1 negative charged amino acid and 4 positice ones,the putative housefly defensin is characterized by 6 invariant cysteine residues forming 3 disulfide bonds,Cys1-Cys4,Cys2-Cys5 and Cys3-Cys6,These results suggest that the novel full-length cDNA of the defensin gene.Denominated Mdde,has been successfully cloned from houseflies.     

Molecular characterization of cDNA encoding B. taurus cathelicidin-7 like antibiotic peptide from bone marrow cells of Bubalus bubalis.

Cathelicidins represent a diverse family of endogenous cationic antibiotic peptide present in all mammalian species. In the present study, a novel cathelicidin cDNA was identified and characterized from bone marrow cells of buffalo (Bubalus bubalis) using RT-PCR based approach. The cDNA encodes a propeptide of 1.18 kDa with net positive charge at neutral pH. The precursor peptide possesses a signal peptide of 29 amino acids and a biologically active peptide of 34 residues. Comparison of sequences indicates only 66.1 and 64.1% identity at nucleotides and amino acids level respectively, with the already reported cathelicidin congener from the same species. However, high degree of similarity (92.8% nucleotides and 81.9% amino acids) was noticed with cathelicidin 7 sequence of Bos taurus suggesting interspecies conservation of cathelicidin peptides rather than intra-species within bovidae family. Phylogenetic trees analyses also support these data. Our findings, further justify the cloned cDNA as a unique cathelicidin member of B. bubalis, and may reasonably considered to be another example of structural diversity exhibited by cathelicidin-derived peptides as reported from other mammals.     

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide,Hepcidin (<Emphasis Type="Italic">Le</Emphasis>-Hepc), from <Emphasis Type="Italic">Leiognathus equulus</Emphasis> and Analysis of Its Functional Properties In Silico

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

Three novel antimicrobial peptides from the skin of Rana shuchinae

Intensive studies have demonstrated that there are many antimicrobial peptides in amphibian skins. Three novel antimicrobial peptides were identified from the skin of the frog, Rana shuchinae. They are named shuchins 3–5. Their sequences were determined as KAYSMPRCKGGFRAVMCWL-NH₂, KAYSTPRCKGLFRALMCWL-NH₂, and KAYSMPRCKYLFRAVLCWL-NH₂ by Edman degradation and mass spectrometry analysis, respectively. They are composed of 19 amino acids (aa) with unique sequences. BLAST search indicated that they showed no similarity to any known peptides or proteins. They are a novel family of antimicrobial peptide. These peptides showed antimicrobial activities against all of tested microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi. The cDNAs encoding precursors of these peptides were cloned from the skin cDNA library of R. shuchinae. The precursors are composed of 64 amino acid residues including predicted signal peptides, acidic spacer peptides, and mature antimicrobial peptides. The current work identified a novel antimicrobial peptide family.     

A novel bradykinin-like peptide from skin secretions of rufous-spotted torrent frog, Amolops loloensis

A bradykinin-like peptide has been isolated from skin secretions of rufous-spotted torrent frog, Amolops loloensis. This bradykinin-like peptide was named amolopkinin. Its primary structure, RAPVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Amolopkinin is composed of 12 amino acid residues and is related to bradykinin composed of nine amino acid residues, identified from the skin secretions of Odorrana schmackeri. Amolopkinin was found to elicit concentration-dependent contractile effects on isolated guinea pig ileum. cDNA clones encoding the precursor of amolopkinin were isolated by screening a skin cDNA library of A. loloensis and then sequenced. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an18-nucleotide fragment (TCAAGAATGATCAGACGC in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in absence of a di-basic site for trypsin-like proteinases and an unusual - APV - insertion in the N-terminal part of amolopkinin. This is the first report of a bradykinin-like peptide comprised of bradykinin with an insertion in its N-terminal part. Our results demonstrate the hypervariability of amphibian bradykinin-like peptides, as well as the diversity of antimicrobial peptides in amphibians.