Dataset Information

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

ABSTRACT: Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed ?-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the ?-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the ?-1 and ?-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

SUBMITTER: Jung W

PROVIDER: S-EPMC4838330 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

Jung Woongsic W Campbell Robert L RL Gwak Yunho Y Kim Jong Im JI Davies Peter L PL Jin EonSeon E

PloS one 20160420 4

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and war ...[more]

PMID: 27097164

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Project description:Ice-binding proteins (IBPs) have been identified in numerous polar algae and bacteria, but so far not in any cyanobacteria, despite the abundance of cyanobacteria in polar regions. We previously reported strong IBP activity associated with an Antarctic Nostoc species. In this study, to identify the proteins responsible, as well as elucidate their origin, we sequenced the DNA of an environmental sample of this species, designated Nostoc sp. HG1, and its bacterial community and attempted to identify IBPs by looking for known IBPs in the metagenome and by looking for novel IBPs by tandem mass spectrometry (MS/MS) proteomics analyses of ice affinity-purified proteins. The metagenome contained over 116 DUF3494-type IBP genes, the most common type of IBP identified so far. One of the IBPs could be confidently assigned to Nostoc, while the others could be attributed to diverse bacteria, which, surprisingly, accounted for the great majority of the metagenome. Recombinant Nostoc IBPs (nIBPs) had strong ice-structuring activities, and their circular dichroism spectra were consistent with the secondary structure of a DUF3494-type IBP. nIBP is unusual in that it is the only IBP identified so far to have a PEP (amino acid motif) C-terminal signal, a signal that has been associated with anchoring to the outer cell membrane. These results suggest that the observed IBP activity of Nostoc sp. HG1 was due to a combination of endogenous and exogenous IBPs. Amino acid and nucleotide sequence analyses of nIBP raise the possibility that it was acquired from a planctomycete.IMPORTANCE The horizontal transfer of genes encoding ice-binding proteins (IBPs), proteins that confer freeze-thaw tolerance, has allowed many microorganisms to expand their ranges into polar regions. One group of microorganisms for which nothing is known about its IBPs is cyanobacteria. In this study, we identified a cyanobacterial IBP and showed that it was likely acquired from another bacterium, probably a planctomycete. We also showed that a consortium of IBP-producing bacteria living with the Nostoc contribute to its IBP activity.

Project description:Peptide toxins isolated from animal venom secretions have proven to be useful pharmacological tools for probing the structure and function of a number of molecular receptors. Their molecular structures are stabilized by posttranslational formation of multiple disulfide bonds formed between sidechain thiols of cysteine residues, resulting in high thermal and chemical stability. Many of these peptides have been found to be potent modulators of ion channels, making them particularly influential in this field. Recently, several peptide toxins have been described that have an unusual tandem repeat organization, while also eliciting a unique pharmacological response toward ion channels. Most of these are two-domain peptide toxins from spider venoms, such as the double-knot toxin (DkTx), isolated from the Earth Tiger tarantula (Haplopelma schmidti). The unusual pharmacology of DkTx is its high avidity for its receptor (TRPV1), a property that has been attributed to a bivalent mode-of-action. DkTx has subsequently proven a powerful tool for elucidating the structural basis for the function of the TRPV1 channel. Interestingly, all tandem repeat peptides functionally characterized to date share this high avidity to their respective binding targets, suggesting they comprise an unrecognized structural class of peptides with unique structural features that result in a characteristic set of pharmacological properties. In this article, we explore the prevalence of this emerging class of peptides, which we have named Secreted, Cysteine-rich REpeat Peptides, or "SCREPs." To achieve this, we have employed data mining techniques to extract SCREP-like sequences from the UniProtKB database, yielding approximately sixty thousand candidates. These results indicate that SCREPs exist within a diverse range of species with greatly varying sizes and predicted fold types, and likely include peptides with novel structures and unique modes of action. We present our approach to mining this database for discovery of novel ion-channel modulators and discuss a number of "hits" as promising leads for further investigation. Our database of SCREPs thus constitutes a novel resource for biodiscovery and highlights the value of a data-driven approach to the identification of new bioactive pharmacological tools and therapeutic lead molecules.

Dataset Information

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

Publications

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

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