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 a mino acids (aa) long (14 kDa) and is derived from an initial 163 aa tr anslation product that is synthesised in the liver. Signal peptide cle avage algorithms, as well as transgenic expression studies in fall arm yworm cells, predict the formation of a 146 aa (16 kDa) proprotein int ermediate. A protein of this size that cross-reacted with anti-sea rav en AFP antibody was detected in sea raven serum using phosphate/urea S DS-PAGE, and was purified by size-exclusion chromatography and reverse -phase HPLC. N-terminal sequencing and mass spectrometry identified th e protein as the predicted proAFP, and immunoblotting suggested that i t is the predominant form present in liver. These results are consiste nt with production and storage of a proAFP intermediate in the liver, and its subsequent processing to mature AFP during or soon after its r elease into the circulation.