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

Multiple ice‐binding proteins of probable prokaryotic origin in an Antarctic lake alga, Chlamydomonas sp. ICE‐MDV (Chlorophyceae)

“…), originally identified as Chlamydomonas but possibly is a species of Chloromonas (Jung et al. ). The amino acid sequences of the type II isoforms have no relation to the type I sequences, although one type II IBP, like the type I IBPs, is predicted to have an ice‐binding face that is rich in threonine residues (Jung et al.…”

“…The amino acid sequences of the type II isoforms have no relation to the type I sequences, although one type II IBP, like the type I IBPs, is predicted to have an ice‐binding face that is rich in threonine residues (Jung et al. ). No other algal type II IBPs exist in any of the databases, while one homolog was found in a bacterium that experiences severe water stress, in which a water‐binding protein could be an advantage (Raymond et al.…”

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

“…Cn-AFP, an antifreeze protein from an Antarctic marine diatom, has been studied from a physiological and structural perspective [ 25 , 26 ]. Like other AFPs which have a multiple gene family [ 12 , 13 , 14 , 15 , 16 , 17 , 18 ], it was expected that C. neogracile would possess additional isoform AFPs. In this study, we reported a new isoform of C. neogracile AFP gene ( Cn-isoAFP ) and characterized the protein based on biochemical and physiological analysis.…”

“…[ 14 ], Chloromonas sp. [ 15 ], and Fragilariopsis sp. [ 16 ], have also been studied using diverse analytic approaches.…”

Ice-Binding Proteins in Plants

“…Interestingly, an IBP from the Antarctic microalga, Chloromonas sp. , has recently been identified that has remarkably similar properties to that seen with plant IBPs, with a low TH (∼ 0.4°C at 5 mg/mL) and hexagonal ice-shaping ( Jung et al, 2016 ). We suggest that IBPs from algal species, such as that described above, may also be good candidates for transgenics.…”