Skin Antifreeze Protein Genes of the Winter Flounder, Pleuronectes americanus, Encode Distinct and Active Polypeptides without the Secretory Signal and Prosequences

Abstract

Distinct antifreeze polypeptides (AFP) were isolated from the skin of the winter flounder, Pleuronectes americanus, by gel filtration and reverse phase high performance liquid chromatography. In parallel, several cDNA clones were isolated from a skin cDNA library using a liver AFP cDNA probe. Both protein and DNA sequence analyses indicate that flounder skin contains several distinct but homologous alanine-rich AFPs. Although the skin type AFPs contain 11 similar amino acid repeats found in the secretory liver type AFPs, the skin type AFPs are mature polypeptides lacking both the signal and prosequences, indicating that they may function intracellularly. The skin type AFP is significantly less active in thermal hysteretic activity than the liver type AFP. Genomic Southern analysis indicates that like the liver type AFP genes, there are multiple copies (30-40 copies) of skin type AFP. Although the liver type AFP genes are specifically expressed in the liver and to a lesser extent in intestine, the skin type AFP genes are expressed in all tissues examined including the liver and abundantly in exterior tissues, i.e. skin, scales, fin, and gills, suggesting an important protecting role in these exterior tissues.

The Role of N and C Termini in the Antifreeze Activity of Winter Flounder (Pleuronectes americanus) Antifreeze Proteins

“…AFPs lies in the C-terminal regions (7,9). This study examines the contributions of the N-and Cterminal sequences to the antifreeze activity of the liver-and skin-type AFPs.…”

“…1). Most notably, wflAFP-6 has the N-terminal sequence DTASDA, whereas all wfsAFPs begin with MDAP (9). Furthermore, the MDAP sequence can be found in the longhorn sculpin skin-type AFP (22), and in the minor serum AFPs of the shorthorn and grubby sculpins (23,24), all of which have ϳ50% activity of wflAFP-6.…”

“…wflAFP-6 (formerly known as HPLC-6) is the major winter flounder plasma AFP and is produced in the liver as a prepro-precursor, and then secreted into circulation, where it is then processed into the mature polypeptide of 37 residues (10,11). Conversely, the wfsAFPs have a wider tissue distribution and are produced as mature intracellular polypeptides (9).…”

“…The wflAFPs and wfsAFPs share a high level of structural identity even though the wfsAFPs have approximately half the activity of wflAFP-6 (9). Both wflAFPs and wfsAFPs possess two 11-residue motifs of the structure TaaXAXXAAXX (where the first Thr is always conserved, uppercase A is a conserved Ala, lowercase a is almost always Ala, and X can be one of several amino acids), with wflAFP-6 having a third full motif at the N terminus (see Fig.…”

“…The winter flounder (Pleuronectes americanus) produces two subclasses of type I AFPs, the liver-type (wflAFPs) and the skin-type AFPs (wfsAFPs), which are encoded by distinct gene families (9). wflAFP-6 (formerly known as HPLC-6) is the major winter flounder plasma AFP and is produced in the liver as a prepro-precursor, and then secreted into circulation, where it is then processed into the mature polypeptide of 37 residues (10,11).…”