Isolation and characterization of skin-type, type I antifreeze polypeptides from the longhorn sculpin, Myoxocephalus octodecemspinosus

The antifreeze polypeptides (AFPs) are found in several marine fish and hav e been grouped into four distinct biochemical classes (type I-IV), Recently , the new subclass of skin-type, type I AFPs that are produced intracellula rly as mature polypeptides have been identified in the winter flounder (Ple uronectes americanus) and the shorthorn sculpin (Myoxocephalus scorpius). T his study demonstrates the presence of skin-type AFPs in the longhorn sculp in (Myoxocephalus octodecemspinosus), which produces type IV serum AFPs. Us ing polymerase chain reaction-based methods, a clone that encoded for a typ e I AFP was identified. The clone lacked a signal sequence, indicating that the mature polypeptide is produced in the cytosol, A recombinant protein w as produced in Escherichia coli and antifreeze activity was characterized, Four individual Ala-rich polypeptides with antifreeze activity mere isolate d 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 I V), and enforces the notion that skin-type AFPs are a widespread biological phenomenon in fish.