Structures of shorthorn sculpin antifreeze polypeptides

Abstract

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly a-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The a-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.To avoid freezing, many species ofmarine fishes inhabiting the Newfoundland and Labrador coastal waters produce antifreeze polypeptides (AFP) in the winter months [I -31. These species include the winter flounder [4-61, shorthorn sculpin [7], sea raven [8] and ocean pout [9]. Isolation and characterization of these AFP have indicated three distinct types of polypeptides based on the differences in amino acid composition and secondary structure [4-91. The AFP from flounder and sculpin comprise one such class.The AFP from the winter flounder, Pseudopleuronectus americunus can be fractionated into a t least seven active components by reverse-phase high-performance liquid chromatography (HPLC) [6]. The two major components, each with a relative molecular mass of about 3300, are closely related. Both consist of eight or nine different amino acids of which alanine accounts for 60% of the residues [4-61. The amino acid sequence of these two components have been elucidated [lo -121. The 37-residue-long polypeptide chain in each component has been found to contain three repeating sequences of 11 amino acid residues, Thr-(X)2-Y-(X)7-, where X represents a non-polar amino acid (mainly alanine) and Y is a polar amino acid [lo-121. The AFP from the shorthorn sculpin, Myoxocephulus scorpius, are similar to those of the winter flounder with respect to the abundance of alanine in their amino acid compositions and their high cr-helical contents [7, 131. However, they contain 12 different amino acids, and have slightly larger molecular masses withCorrespondence to

Type I Shorthorn Sculpin Antifreeze Protein

“…The wild-type protein isolated from blood serum (XSS3) has been reported to exhibit measurable thermal hysteresis (3). Although no raw data were presented, XSS3 was reported to give 0.39 degrees hysteresis (ϳ10 mg/ml, 0.02 M NH 4 HCO 3 ), whereas the second major protein isolated from the fish, XSS8, gave a higher hysteresis value of 0.67°C.…”

“…In contrast to HPLC6, type I AFPs from the grubby sculpin (Myoxocephalus aenaeus) (31), the shorthorn sculpin (Myoxocephalus scorpius) (3,32), and the Arctic sculpin (Myoxocephalus scorpioides) (33) have been much less studied. These proteins differ from both HPLC6 and sequences from the right-eye flounders in the N-terminal region of the sequence.…”

“…Two proteins, XSS3 and XSS8 (Table I), have been isolated from the blood serum of the shorthorn sculpin (3). It has been estimated using CD spectroscopy that XSS8, the major component, is more helical than XSS3, and it has been proposed that there are two different structural and possibly functional do-mains, the nine-residue N terminus and the alanine-rich helical region between residues 8 and 42 (3).…”

“…It has been estimated using CD spectroscopy that XSS8, the major component, is more helical than XSS3, and it has been proposed that there are two different structural and possibly functional do-mains, the nine-residue N terminus and the alanine-rich helical region between residues 8 and 42 (3). Vacuum phase molecular dynamics studies on SS8, a closely related sequence to XSS8, concluded that the polypeptide conformation resembles an idealized ␣-helix, except for a short section of the N-terminal region (34).…”

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

“…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. The region of highest variability in type I AFPs lies in the C-terminal regions (7,9).…”