| Several species of marine teleosts that inhabit ice-laden seawaters produce antifreeze (glyco)proteins (AF(G)Ps), which have been classified into five classes (AFPs type I to IV, and AFGPs) based on biochemical characteristics. The type I AFPs are partially amphipathic, single α-helices that are rich in Ala. Almost all previously characterized AF(G)Ps are extracellular proteins that were isolated from the blood plasma, and are, or are assumed to be, liver-derived proteins (liver-type AFPs). However, in the winter flounder, Pleuronectes americanus, which produces type I, liver-type AFPs (wflAFPs), a new sub-class of type I AFPs, the skin-type AFPs (wfsAFPs), has been identified. The wfsAFPs are widely expressed as mature intracellular polypeptides, and have approximately half the activity of wfIAFPs. Previously, it was proposed that the incomplete IBMs of wfsAFPs, ATAAA, versus complete IBMs of wfIAFPs, LTAAN resulted in the lower activity of wfsAFPs. This study has determined that the shorthorn sculpin, Myoxocephalus scorpius, and the longhorn sculpin, Myoxocephalus octodecemspinosus, also produce intracellular type I AFP, skin-type AFPs. Skin-type AFPs were isolated and cloned from both species and were found to be homologues due to similarities in the untranslated regions, although the shorthorn sculpin skin-type AFP (sssAFP-2) was atypical in both length and sequence. At 92 residues, sssAFP-2 would be the largest known naturally occurring type I AFP and it appears to lack the repeated 11-residue motifs commonly found in type I AFPs. Assay of a recombinant protein based on sssAFP-2 determined that it was a highly active AFP. The longhorn sculpin skin-type AFP, IssAFP, was a more typical type I AFP, and investigation of a recombinant protein determined that it has activity levels comparable to wfsAFPs. It was further determined that for winter flounder AFPs, the helix termini are the major factors leading to the different levels of antifreeze activity between wflAFPs and wfsAFPs. The termini of type I AFPs are critical to activity because of their individual contributions to activity and their ability to impart α-helical character to other regions of the polypeptide. Furthermore, the N-terminus possesses the greatest influence on overall activity. |
