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 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.
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Division of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada.
Lin
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Stathakis
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Miao
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Fletcher
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GL
Hew
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005399:Fishes; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012867:Skin / Q000737:chemistry*
D021301:Antifreeze Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins