Biological Characterization of Antifreeze Proteins from Antarctic Marine Diatom and its Application

Abstract

Antifreeze proteins (AFPs) have an ability to bind ice crystals and cause the thermal hysteresis (TH), which lowers the freezing temperature. The sequence, physiological property, gene and protein expression, localization, structure, ice-binding site, and in vivo and in vitro applications of antifreeze proteins from Chaetoceros neogracile (Cn-AFPs), which is Antarctic marine diatom, were analyzed. The amino acid sequences of Cn-AFPs showed a high sequence similarity and were closely related with psychrophilic microorganisms AFPs or IBPs. In silico analysis of the Cn-AFP promoter revealed that it contained various stress-responsive motifs and the expression level of Cn-AFP gene, and proteins were dramatically increased under those stress conditions. The intracellular localization of Cn-AFP was identified by immunocytochemistry and immuno-gold labeling. Also, Cn-AFP clearly showed a thermal hysteresis activity and ice crystal morphology. The maximum TH values of recombinant mature Cn-AFPs are approximately 1.2°C at 10 mg/ml protein concentration. The ice crystals formed by Cn-AFP showed burst or snowflake form. In silico 3D-structure analysis exhibit that the Cn-AFP consists seven right-handed β-helical loops with a triangular cross section. Simulation of the 3D-structure of mutant forms of Cn-AFP in silico predicted an ice-binding site located on the b-face of Cn-AFP. Unexpectedly, two of mutant Cn-AFP increased its antifreeze activity to suggest ice-binding site of this protein. For in vitro application of Cn-AFPs, we developed a coating method of AFPs to aluminum plate using aluminum binding peptide and trehalose. Indeed, the Cn-AFP transgenic plants and diatoms were invented to develop the cold resistance organisms and identify the physiological roles of Cn-AFPs in other organisms.