Identification and Characterization of an Isoform Antifreeze Protein from the Antarctic Marine Diatom, Chaetoceros neogracile and Suggestion of the Core Region.

Antifreeze proteins (AFPs) protecting the cells against freezing are produced in response to extremely low temperatures in diverse psychrophilic organisms, and they are encoded by multiple gene families. The AFP of Antarctic marine diatom Chaetoceros neogracile is reported in our previous research, but like other microalgae, was considered to probably have additional genes coding AFPs. In this paper, we reported the cloning and characterization of additional AFP gene from C. neogracile (Cn-isoAFP). Cn-isoAFP protein is 74.6% identical to the previously reported Cn-AFP. The promoter sequence of Cn-isoAFP contains environmental stress responsive elements for cold, thermal, and high light conditions. Cn-isoAFP transcription levels increased dramatically when cells were exposed to freezing (-20 °C), thermal (10 °C), or high light (600 µmol photon m-2 s-1) stresses. The thermal hysteresis (TH) activity of recombinant Cn-isoAFP was 0.8 °C at a protein concentration of 5 mg/mL. Results from homology modeling and TH activity analysis of site-directed mutant proteins elucidated AFP mechanism to be a result of flatness of B-face maintained via hydrophobic interactions.

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

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 ß-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the ß-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 ß-1 and ß-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.

Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum.

Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation. To coat Al with Cn-AFP, we used an Al-binding peptide (ABP) as a conjugator and fused it with Cn-AFP. The ABP bound well to the Al and did not considerably change the functional properties of AFP. Cn-AFP-coated Al (Cn-AFP-Al) showed a sufficiently low supercooling point. Additional trehalose coating of Cn-AFP-Al considerably delayed AFP denaturation on the Al without affecting its antifreeze activity. This metal surface-coating method using trehalose-fortified AFP can be applied to other metals important in the aircraft and cold storage fields where anti-icing materials are critical.

An intracellular antifreeze protein from an Antarctic microalga that responds to various environmental stresses.

The structure and function of the Antarctic marine diatom Chaetoceros neogracile antifreeze protein (Cn-AFP), as well as its expression levels and characteristics of the ice-binding site, were analyzed in the present study. In silico analysis revealed that the Cn-AFP promoter contains both light- and temperature-responsive elements. Northern and Western blot analyses demonstrated that both Cn-AFP transcript and protein expression were strongly and rapidly stimulated by freezing, as well as temperature and high light stress. Immunogold labeling revealed that Cn-AFP is preferentially localized to the intracellular space near the chloroplast membrane. Recombinant Cn-AFP had clear antifreeze activity. Protein-folding simulation was used to predict the putative ice-binding sites in Cn-AFP, and site-directed mutagenesis of the Cn-AFP b-face confirmed their identification.

Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis.

Haematococcus pluvialis cells predominantly remain in the macrozooid stage under favourable environmental conditions but are rapidly differentiated into haematocysts upon exposure to various environmental stresses. Haematocysts are characterized by massive accumulations of astaxanthin sequestered in cytosolic oil globules. Lipidomic analyses revealed that synthesis of the storage lipid triacylglycerol (TAG) was substantially stimulated under high irradiance. Simultaneously, remodelling of membrane glycerolipids occurred as a result of dramatic reductions in chloroplast membrane glycolipids but remained unchanged or declined slightly in extraplastidic membrane glycerolipids. De novo assembly of transcriptomes revealed the genomic and metabolic features of this unsequenced microalga. Comparative transcriptomic analysis showed that so-called resting cells (haematocysts) may be more active than fast-growing vegetative cells (macrozooids) regarding metabolic pathways and functions. Comparative transcriptomic analyses of astaxanthin biosynthesis suggested that the non-mevalonate pathway mediated the synthesis of isopentenyl diphosphate, as the majority of genes involved in subsequent astaxanthin biosynthesis were substantially up-regulated under high irradiance, with the genes encoding phytoene synthase, phytoene desaturase, and ß-carotene hydroxylase identified as the most prominent regulatory components. Accumulation of TAG under high irradiance was attributed to moderate up-regulation of de novo fatty acid biosynthesis at the gene level as well as to moderate elevation of the TAG assembly pathways. Additionally, inferred from transcriptomic differentiation, an increase in reactive oxygen species (ROS) scavenging activity, a decrease in ROS production, and the relaxation of over-reduction of the photosynthetic electron transport chain will work together to protect against photooxidative stress in H. pluvialis under high irradiance.

Isolation and characterization of antifreeze proteins from the antarctic marine microalga Pyramimonas gelidicola.

Antifreeze proteins (AFPs) play an important role in the psychrophilic adaptation of polar organisms. AFPs encoded by an Antarctic chlorophyte, identified as Pyramimonas gelidicola, were isolated and characterized. Two AFP isoforms were found from cDNAs and their deduced molecular weights were estimated to be 26.4 kDa (Pg-1-AFP) and 27.1 kDa (Pg-2-AFP). Both AFP cDNAs were cloned and expressed in Escherichia coli. The purified recombinant Pg-1-rAFP and Pg-2-rAFP both showed antifreeze activity based on the measurement of thermal hysteresis (TH) and morphological changes to single ice crystals. Pg-1-rAFP shaped ice crystals into a snowflake pattern with a TH value of 0.6 ± 0.02 °C at ~15 mg/ml. Single ice crystals in Pg-2-rAFP showed a dendritic morphology with a TH value of 0.25 ± 0.02 °C at the same protein concentration. Based on in silico protein structure predictions, the three-dimensional structures of P. gelidicola AFPs match those of their homologs found in fungi and bacteria. They fold as a right-handed ß-helix flanked by an α-helix. Unlike the hyperactive insect AFPs, the proposed ice-binding site on one of the flat ß-helical surfaces is neither regular nor well-conserved. This might be a characteristic of AFPs used for freeze tolerance as opposed to freeze avoidance. A role for P. gelidicola AFPs in freeze tolerance is also consistent with their relatively low TH values.

Frozen assembly of gold nanoparticles for rapid analysis of antifreeze protein activity.

We report the novel activity-based detection of antifreeze protein (AFP), also known as ice-binding protein (IBP), using freeze-labile gold nanoparticles (AuNPs) in order to overcome labor-intensive and low throughput issues of the current method based on thermal hysteresis (TH). Upon the addition of either CnAFP from the Antarctic diatom Chaetoceros neogracile or LeIBP from the Arctic yeast Leucosporidium sp. to mercaptosuccinic acid-capped AuNP, the self-assembly of AuNPs was highly inhibited after a freezing/thawing cycle, leading to no color change in the AuNP solution. As a result, the aggregation parameter (E(520)/E(650)) of AuNP presented the rapid detection of both the concentration-dependent activity and stability of two AFPs with high sensitivity, where the detection range was 100-fold lower than that of the TH-based method. We suggest that our newly developed method is very suitable for simple and high-throughput measurement of AFP activity.