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J. Biol. Chem., Vol. 276, Issue 15, 11582-11589, April 13, 2001

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Isolation and Characterization of Skin-type, Type I Antifreeze Polypeptides from the Longhorn Sculpin, Myoxocephalus octodecemspinosus^*

Woon-Kai Low^§¶, Qingsong Lin, Costas Stathakis, Ming Miao^§, Garth L. Fletcher^**, and Choy L. Hew^§§§

From the  Division of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada, the ^§ Department of Biochemistry, University of Toronto, Toronto, Ontario, M5G 1L5 Canada, the  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5G 1L5, Canada, the ^** Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, A1C 5S7 Canada, and the  Department of Biological Sciences, National University of Singapore, Singapore, 119 260, Singapore

The antifreeze polypeptides (AFPs) are found in several marine fish and have been grouped into four distinct biochemical classes (type I-IV). Recently, the new subclass of skin-type, type I AFPs that are produced intracellularly as mature polypeptides have been identified in the winter flounder (Pleuronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). This study demonstrates the presence of skin-type AFPs in the longhorn sculpin (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Using polymerase chain reaction-based methods, a clone that encoded for a type I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol. A recombinant protein was produced in Escherichia coli and antifreeze activity was characterized. Four individual Ala-rich polypeptides with antifreeze activity were isolated from the skin tissue. One polypeptide was completely sequenced by tandem MS. This study provides the first evidence of a fish species that produces two different biochemical classes of antifreeze proteins (type I and type IV), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF306348.

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				W.-K. Low, Q. Lin, and C. L. Hew The Role of N and C Termini in the Antifreeze Activity of Winter Flounder (Pleuronectes americanus) Antifreeze Proteins J. Biol. Chem., March 14, 2003; 278(12): 10334 - 10343. [Abstract] [Full Text] [PDF]