cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthia aurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5′-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Ciona intestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (−) bacteria, while evidencing no hemolytic activity toward human erythrocytes.     

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.     

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.     

Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys16 to Gly16 mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys16 to Gly16 mutant (with MICs>100 microM). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys16 to Gly16 mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 microM, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments.     

Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity

In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12-26) (GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction with bacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12-25) and Mel(12-24), were synthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12-26). If both of the Gln residues of Mel(12-26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increased slightly. If the Gln residue of Mel(12-25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, although the substituted peptide possessed much higher hydrophobicity and higher alpha-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminal residues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletion may be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes because of the lower molecular weights of the deletion peptides.     

戊型肝炎病毒衣壳蛋白同源二聚体的相互作用结构域

为了探讨戊型肝炎病毒衣壳蛋白同源二聚体形成的关键区域和相互作用结构域,以及二聚体形成与主要天然中和表位的形成之间的关系,通过末端缺失、定点突变技术研究戊型肝炎病毒（HEV）ORF2的aa394-aa606片段NE2的聚合现象,发现其C端的aa597-aa602（AVAVLA）疏水区是该片段同源聚合的核心区域,提高该区域氨基酸的亲水性将妨碍聚合现象的发生;半胱氨酸化学交联实验表明NE2形成同源二聚体时,aa597在空间位置上相接近,处于可生成化学键的距离,提示所处区域为疏水聚合的作用结构域;通过Blast程序估算核心区域的天然突变率,发现其疏水性高度保守;N端缺失实验表明,至少65个氨基酸既不影响同源聚合也不直接参与主要的天然中和表位的形成,但可协助中和表位构象的形成,而这种协助作用可被ORF2的末端肽段所代替。Aa597-aa602（AVAVLA）疏水区为戊肝病毒衣壳组装的第一步骤的核心区域,并与重要的天然中和表位的形成直接相关,从而为戊肝病毒疫苗的研究提供更详细的信息。     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

Dimerization of resistin and resistin-like molecules is determined by a single cysteine

Resistin is a peptide hormone secreted by adipocytes. Cysteine residues comprise 11 of 94 (12%) amino acids in resistin. The arrangement of these cysteines is unique to resistin and its recently discovered family of tissue-specific secreted proteins, which have been independently termed resistin-like molecules (RELMs) and the FIZZ (found in inflammatory zone) family. Here we show that resistin is a disulfide-linked homodimer that can be converted to a monomer by reducing conditions. The intestine-specific RELM beta has similar characteristics. Remarkably, however, the adipose-enriched RELM alpha is a monomer under non-reducing conditions. We note that RELM alpha lacks a cysteine residue, closest to the cleaved N terminus, that is present in resistin and RELM beta in multiple species. Conversion of this cysteine to alanine abolishes dimerization of resistin. Thus, a single disulfide bond is necessary to connect two resistin subunits in a homodimer. The additional 10 cysteines most likely participate in intramolecular disulfide bonds that define the conserved structure of the family members. The monomeric nature of RELM alpha suggests structural and potentially functional divergence between resistin and this close family member.     

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.     

Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

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.     

Amino acid sequence of locust neuroparsins

Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues.     

N-linked glycans of the human insulin receptor and their distribution over the crystal structure

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Coexpression of rumen microbial β-glucanase and xylanase genes in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial peptides are promising candidates for therapeutic and industrial application owing to their broad spectrum. In this work, a cost-effective method for expression of a potent antimicrobial peptide, bovine lactoferricin derivative LfcinB15-W4,10, has been developed. The oligonucleotide encoding the peptide was linked to generate different oligomeric oligonucleotide segments containing from one to nine but eight tandem copies which was inserted individually to the E. coli expression vector pET32a. The thioredoxin fusion peptides were successfully expressed and detected with different molecular weight on SDS gel, respectively. Among the monomer and other multimeric peptides, the tetramer was expressed at the highest level. After purification, more than 10 mg of tetramer with 99% purity was obtained from 1 l culture and exhibited similar antimicrobial activity as synthetic LfcinB15-W4,10 monomer. The expression system in this study provides a potential production method for lactoferricin derivatives and other antimicrobial peptides in research and industrial applications.     

Dimer structure of a neuropeptide precursor established: consequences for processing

A prohormone (P1) of locust adipokinetic hormone I (AKH I) is shown here to be a homodimer of a 41 residue subunit called the A-chain. The A-chain, from the N terminal, consists of AKH I (10 amino acids starting with pyroglutamate) followed by a Gly-Lys-Arg processing site and then a 28 residues called the alpha chain containing a single cysteine and a potential Arg-Lys processing site. When processed each molecule of the homodimer precursor yields two copies of AKH I and one alpha chain homodimer. We call the alpha-alpha homodimer product of P1 processing AKH precursor related peptide 1 or APRP 1. The Arg-Lys dibasic pair found within the alpha chain is not cleaved in vivo. Our results show that neuropeptide precursors can be dimers and that dimer products can be synthesized by processing of a preformed dimer precursor rather than by dimerization of independent subunits.