Evidence for a proprotein intermediate during maturation of type II antifreeze protein in sea raven, Hemitripterus americanus

The circulating Type II antifreeze protein (AFP) in sea raven is 129 amino acids (aa) long (14 kDa) and is derived from an initial 163 aa translation product that is synthesised in the liver. Signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kDa) proprotein intermediate. A protein of this size that cross-reacted with anti-sea raven AFP antibody was detected in sea raven serum using phosphate/urea SDS-PAGE, and was purified by size-exclusion chromatography and reverse-phase HPLC. N-terminal sequencing and mass spectrometry identified the protein as the predicted proAFP, and immunoblotting suggested that it is the predominant form present in liver. These results are consistent with production and storage of a proAFP intermediate in the liver, and its subsequent processing to mature AFP during or soon after its release into the circulation.