Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions

<div><p>Alanine-rich α-helical (type I) antifreeze proteins (AFPs) are produced by a variety of fish species from three different orders to protect against freezing in icy seawater. Interspersed amongst and within these orders are fishes making AFPs that are completely different in both sequence and structure. The origin of this variety of types I, II, III and antifreeze glycoproteins (AFGPs) has been attributed to adaptation following sea-level glaciations that occurred after the divergence of most of the extant families of fish. The presence of similar types of AFPs in distantly related fishes has been ascribed to lateral gene transfer in the case of the structurally complex globular type II lectin-like AFPs and to convergent evolution for the AFGPs, which consist of a well-conserved tripeptide repeat. In this paper, we examine the genesis of the type I AFPs, which are intermediate in complexity. These predominantly α-helical peptides share many features, such as putative capping structures, Ala-richness and amphipathic character. We have added to the type I repertoire by cloning additional sequences from sculpin and have found that the similarities between the type I AFPs of the four distinct groups of fishes are not borne out at the nucleotide level. Both the non-coding sequences and the codon usage patterns are strikingly different. We propose that these AFPs arose via convergence from different progenitor helices with a weak affinity for ice and that their similarity is dictated by the propensity of specific amino acids to form helices and to align water on one side of the helix into an ice-like pattern.</p></div

Properties of the type I AFPs from the four groups of fish.

a<p>The sequence of a larger circulating isoform is unknown but an accurate mass and rough composition were determined from an enriched sample <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Evans4" target="_blank">[66]</a>.</p>b<p>Longhorn sculpin skin and circulating isoforms are highly similar, but one shorthorn sculpin cDNA from skin encodes a distinct isoform.</p

Evolutionary relationships between AFP-producing fishes and the similarities between type I AFPs.

<p>A) Phylogenetic relationships (not to scale) among AFP-producing fish from analysis of complete mitochondrial genomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Miya1" target="_blank">[67]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Lavoue1" target="_blank">[69]</a> or selected nuclear and mitochondrial sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Matschiner1" target="_blank">[45]</a>. Estimated divergence times (Ma, some with 95% highest posterior density limits) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Matschiner1" target="_blank">[45]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Steinke1" target="_blank">[47]</a> are shown at some nodes. Species names are colored by AF(G)P type as indicated on the right. Representative ribbon structures are shown for types II, III, and I AFPs (PDB 2PY2, 1HG7, 1WFA from top to bottom, red = helix, green = strand, gray = coil). The colored bars at the bottom indicate climate differences marked by the presence (blue) or absence (red) of large ice sheets. Common names of representative AFP-producing fish are indicated but their scientific names are as follows; herring (<i>Clupea harengus</i>), Arctic cod (<i>Boreogadus saida</i>), cunner (<i>Tautogolabrus adspersus</i>), ocean pout (<i>Zoarces americanus</i>), Atlantic snailfish (<i>Liparis atlanticus</i>), dusky snailfish (<i>Liparis gibbus</i>), sea raven (<i>Hemitripterus americanus</i>), longhorn sculpin (<i>Myoxocephalus octodecemspinosus</i>), shorthorn sculpin (<i>Myoxocephalus scorpius</i>), Antarctic toothfish (<i>Dissostichus mawsoni</i>), winter flounder (<i>Pseudopleuronectes americanus</i>) and rainbow smelt (<i>Osmerus mordax</i>). B) Alignment of representative type I skin AFPs from three fishes from three separate orders (winter flounder (M63478.1), longhorn sculpin (AF306348.1) and cunner (JF937681.2). Potential or known ice-binding residues within the 11-aa repeat that show an <i>i</i>, <i>i</i>+4, <i>i</i>+8 spacing pattern are indicated with plus symbols (Ala) and number symbols (Thr) with asterisks denoting residues that are identical in all sequences. Acidic and basic residues are in red and blue font respectively, with Ala highlighted yellow and Thr in white font with black highlighting. Potential helix-stabilizing salt bridges consisting of basic and acidic residues with the more effective <i>i</i>, <i>i</i>+4 separation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Marqusee1" target="_blank">[70]</a> are double underlined. The cunner isoform is also found in blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285-Evans2" target="_blank">[42]</a>.</p

Sculpin AFPs.

<p>A) Alignment of Sculpin AFPs. New sequences from shorthorn sculpin cDNAs from liver (Liv), larvae (Lar) or genomic DNA (G) are compared to known shorthorn skin (Skin) and longhorn skin (LHS) sequences. As the deduced peptide sequences are low complexity, they were aligned based on the DNA sequence alignment, which is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285.s001" target="_blank">Fig. S1</a> along with the accession numbers. Thr is highlighted light green and other polar residues are highlighted dark green with white font. Basic residues are highlighted cyan (Lys) or blue (Arg), acidic are highlighted red in black (Asp) or white (Glu) font, hydrophobic residues (except Ala) are highlighted gray and exceptional residues (Pro and Gly) are highlighted yellow. B) Dot matrix comparisons of selected sculpin isoforms. A line indicates a match of at least 9 out of 10 bases. Coding regions are denoted by blue bars.</p

Amino acids encoded in alternative reading frames by adjacent Ala codons.

<p>Amino acids encoded in alternative reading frames by adjacent Ala codons.</p

Dot matrix comparisons of type I cDNAs from the four different groups of fishes.

<p>A line indicates a match of at least 9 out of 12 bases with red indicating a sense/antisense match. The blue bars denote the coding region (signal peptides excluded). These sequences correspond to those shown in Fig. 3.</p

Ala codon usage in type I AFPs.

<p>The height of the color bars represents the fraction of each of the Ala codons in each dataset or sequence. The cDNAs used encoded the following AFPs; WF–hyp, LHS, SHS–skin, Atlantic snailfish AFP and cunner AFP. The number of non-AFP sequences used for each group is as follows: winter flounder, 70; longhorn sculpin, 10; cunner, 10 and four species in the snailfish family (Liparidae), 8. The accession numbers for these sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081285#pone.0081285.s005" target="_blank">Text S1</a>.</p

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization

Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

<div><p>A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, <em>Marinomonas primoryensis</em>. <em>Mp</em>AFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide <em>Mp</em>AFP into five distinct regions, all of which require mM Ca²⁺ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca²⁺-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of <em>Mp</em>AFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.</p> </div