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Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

ABSTRACT: Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

SUBMITTER: Cole AM

PROVIDER: S-EPMC90011 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

Cole A M AM Darouiche R O RO Legarda D D Connell N N Diamond G G

Antimicrobial agents and chemotherapy 20000801 8

Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcr ...[more]

PMID: 10898673

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Project description:BackgroundEpstein-Barr virus (EBV) is a universal herpes virus which can cause a life-long and largely asymptomatic infection in the human population. However, the exact pathogenesis of the EBV infection is not well known.ObjectiveA comprehensive bioinformatics prediction was carried out for investigating the molecular properties of the BGLF2 and to afford a foundation for future research of the role and instrument of BGLF2 in the course of EBV infection.Materials and methodsA 1011-base-pair sequence of BGLF2 gene from the Epstein-Barr virus (EBV) Akata strain genome was amplified using polymerase chain reaction and was further characterized by cloning, sequencing, and subcellular localization in the COS-7 cells.ResultsThe bioinformatics analysis demonstrated that EBV BGLF2 gene encodes a putative BGLF2 polypeptide which contains a conservative Herpes_UL16 domain. It was established that the polypeptide shows a close relationship with the Herpes UL16 tegument protein family and is extremely conserved among its homologues proteins encoded by UL16 genes. Multiple sequence alignments of the nucleic acid and amino acid sequence showed that the gene product of EBV BGLF2 contains a comparatively higher homology with the BGLF2-like proteins of the subfamily Gammaherpesvirinae than that of other subfamilies of the herpes virus. Moreover, the phylogenetic analyses suggested that EBV BGLF2 has a close genetic relationship with the member of Gammaherpesvirinae; in particular with the members of Cercopithecine herpesvirus 15 and Callitrichine herpesvirus 3. An antigen epitope analysis indicated that BGLF2 contains several potential B-cell epitopes. In addition, the secondary structure, as well as the three dimensional structure prediction suggests that BGLF2 consists of the both α-helix and β-strand. Besides, the subcellular localization prediction revealed that BGLF2 localizes in both nucleus and cytoplasm.ConclusionsIllustrating the relevance of the molecular properties and genetic evolution of EBV, BGLF2 will offer the perspectives for further study on the role and mechanism of the BGLF2 in course of EBV infection. These works will also conduct our understanding of the EBV at the molecular level as well as enriching the herpesvirus database.

Project description:Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins.

Dataset Information

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

Publications

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

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