Molecular Cloning and Characterization of Antifreeze Proteins from an Arctic Yeast, Two Antarctic Fishes and Olive Flounder

Abstract

Antifreeze proteins (AFPs) are proteins that depress the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals. The difference between the freezing and melting points is called thermal hysteresis (TH). They also have ice recrystallization inhibition (RI) activity which prevents the growth of larger grains of ice at the expense of smaller grains. Since DeVries first isolated antifreeze proteins from Antarctic teleost fishes, a great number of AFPs and other IBPs with a variety of sequences and structures have been discovered from insects, plants, bacteria, diatoms, fungi, and algae from cold environments. The culture supernatant of a psychrophilic yeast isolated from an Arctic pond showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP (yIBP) was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of ~26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yIBP was determined and was found to encode a 261 amino acid protein with molecular mass of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria. In a phylogenetic analysis, yIBP distinctively clusters together with other fungal proteins and was separated from diatom and bacterial IBPs. Enzymatic deglycosylation using PNGase F in denatured condition and its PAS staining on SDS-PAGE indicated that this yIBP had one N-linked glycans. In order to examine the effect of the glycosylation on TH activity and RI activity, yIBP was deglycosylated using PNGase F in native condition. However the complete removal of glycnas was not successful even in excess of PNGase F. Meanwhile, it was observed that the partial deglycosylated yIBP showed the declined TH activity than untreated one, and the recombinant yIBP showed lower activity than nature yIBP, suggesting that the glycosylation may be involved in TH activity. However the RI activity was not affected. CD analysis showed that the secondary structure and denaturation profile of nature yIBP was different with those of recombinant IBPs suggesting that the glycosylation may affect the secondary or tertiary structure of yIBP to enhance the TH activity of this protein.

Preliminary EST analysis was performed in liver and muscle of olive flounder (Paralichthys olivaceus) cultured in Korea to obtain expression profiling. 69 clones of liver and 80 clones of muscle which had relative large insert DNA were sequence. This relative small size experiment indicated that these two libraries were useful for mass EST analysis. Among the ESTs of liver analyzed, a 5’-deleted cDNA sequence similar to type IV AFP gene of longhorn sculpin was identified and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP showed 77% similarity with longhorn sculpin AFP and 74% similarity with shorthorn sculpin AFP. The genome structure of this gene composed of 4 exons. The recombinant AFP of flounder showed TH activity and star-shaped ice crystal growth at lower concentration. Type IV AFP genes were cloned and sequenced from two Antarctic fishes. The deduced amino acid sequences of both fishes showed high identity with type IV AFP of longhorn sculpin and shorthorn sculpin. These putative AFPs were expressed using a bacterial expression system and the recombinant AFPs showed a weak TH activity and their ice crystal grew like a star-shaped morphology. The existence and conservation of apolipoprotein-like type IV AFPs among several fishes indicated that these proteins might have another function to bind a ligand such as lipid.