Structural variations in the alanine‐rich antifreeze proteins of the pleuronectinae

Abstract

The sequence and activity of antifreeze proteins from two righteye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 1 1-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.The current model for the mechanism of action of winter flounder antifreeze protein (AFP) dates back to the first sequence study done on these proteins [I] and has been reviewed recently [2, 31. These alanine-rich AFP are amphiphilic ahelices. The major components are 37 amino acids long and are built up of three 1 I-amino-acid repeats of the consensus sequence Thr-(Xaa)2-Asx-(Xaa)7 where Xaa is predominantly alanine. Each repeat represents almost three complete turns of a-helix organized such that the hydrophilic Thr and Asx side chains project on the same side of the helix from the first and second turns of each repeat. The model proposes that these hydrophilic groups hydrogen bond to the surface of microscopic ice crystals and thereby inhibit their growth along the preferred a axes [3]. In support of this model the 0.45-nm spacing between the Thr and Asx side chains matches the spacing between oxygen atoms in the ice lattice along the a axis.There are a number of approaches than can be employed to identify the key structural features of these antifreezes. Covalent modification of the AFPs and replacement of select amino acids are methods currently being used to test various facets of the model (Hew, C. L., personal communication).An alternative approach to the study of structure and function relationships in proteins is to examine the sequences of naturally occurring variants. In the context of the a-helical alaninerich AFPs, intraspecies variants have been sequenced in the winter flounder [4,5]. In this report we examine the AFP from ...

Cloning and characterization of a cold-and ABA-inducible Arabidopsis gene

“…The amount of identical amino acids is 28 ~o and that of biochemically similar amino acids 41~o. Similarities of the same order were found to some other flounder AFP sequences [8,22,36]. All these AFPs are classified in the same group and are highly homologous to each other.…”

Structure, function and evolution of antifreeze proteins

“…The lower activity of the yellowtail flounder AFP appears to result from its lack of certain ice-binding residues [13,20]. The yellowtail flounder AFP is also encoded by a multigene family [20]. It seems likely that the flounder have evolved antifreezes with different repeat numbers by unequal crossing over [21].…”

“…In yellowtail flounder (P. ferrugineus), the primary serum antifreeze has four repeats, although the sequence is distinct from those of flounder, and this protein has lower activity than the three-or four-repeat winter flounder AFPs [19,20]. The lower activity of the yellowtail flounder AFP appears to result from its lack of certain ice-binding residues [13,20]. The yellowtail flounder AFP is also encoded by a multigene family [20].…”

“…The ribbon diagram of HPLC 6 image was generated using RasMol. References are: winter flounder serum HPLC6 [79], winter flounder serum AFP9 [19], winter flounder skin SAFP2 [27], yellowtail flounder YTAFP [20], shorthorn sculpin serum SS3 [25], shorthorn sculpin skin SSAFP [28], and HPLC6 structure [13].…”

Applications of Type I Antifreeze Proteins: Studies with Model Membranes & Cryoprotectant Properties

“…There are two subclasses of type I AFPs ( Table 2): (i) those isolated from the blood serum of the winter flounder (Pseudopleuronectes americanus) [28], yellowtail flounder (Limanda ferruginea) [29] and Alaskan plaice (Pleuronectes quadritaberulatus) [30], which comprise typically three or four ThrX2AsxX7 subunits and minor sequence variations, and (ii) those derived from the grubby (Myoxocephalus aenaeus) [31], shorthorn [32] and Arctic shorthorn [33] (Myoxocephalus scorpius) sculpins, that contain an unusual N-terminal sequence lacking the helical structure present in the rest of the sequence (e.g., SS3, SS8, Table 2). A characteristic of all sequences is the presence of charged residues that form salt bridges (i, i+4), presumably to assist aqueous solubility.…”