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Review Structure, Function and Evolution of Antifreeze Proteins CMLS, Cell. Mol. Life Sci. 55 (1999) 271–283 1420-682X/99/020271-13 $ 1.50+0.20/0 © Birkha¨user Verlag, Basel, 1999 Review Structure, function and evolution of antifreeze proteins K. V. Ewarta,Q.Linb and C. L. Hewb,* aNRC Institute for Marine Biosciences, 1411 Oxford St., Halifax (Nova Scotia B3H 3Z1, Canada), e-mail: [email protected] bResearch Institute, Hospital for Sick Children and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto (Ontario M5G 1L5, Canada), Fax +1 416 978 8802, e-mail: [email protected] Received 2 September 1998; received after revision 21 October 1998; accepted 2 November 1998 Abstract. Antifreeze proteins bind to ice crystals and structures and mechanisms of action have provided new modify their growth. These proteins show great diver- understanding of these proteins in different contexts. sity in structure, and they have been found in a variety The purpose of this review is to present the develop- of organisms. The ice-binding mechanisms of antifreeze ments in contrasting research areas and unite them in proteins are not completely understood. Recent findings order to gain further insight into the structure and on the evolution of antifreeze proteins and on their function of the antifreeze proteins. Key words. Antifreeze; ice; protein structure; protein function; evolution; cell membranes. Introduction has been made in the study of antifreeze-ice interac- tions. However, a precise definition of the interactions The antifreeze proteins and glycoproteins [AF(G)P] between AF(G)Ps and ice that would give rise to an- were first identified in Antarctic teleost fishes as the tifreeze activity has not yet been obtained. It would causative agents of serum freezing point depression [1]. In Antarctic fish species, the antifreeze glycoproteins seem obvious that functional groups positioned to (AFGP) were found to lower the freezing point by more match the ice lattice on a particular plane would lead to than 1 °C to match that of seawater, and their name ice binding and antifreeze activity. Yet in this context reflects this biological function. The antifreeze glyco- we have not succeeded in defining what, exactly, distin- proteins were immediately recognised as unusual be- guishes an antifreeze protein from a non-antifreeze cause they caused a freezing point depression far greater protein. This is a fascinating question in protein struc- than would be predicted from colligative properties ture and function and in protein evolution. It is also of alone [1]. Later studies demonstrated that the an- interest in biotechnology because the design of new tifreezes lower the freezing point by interacting directly peptides or of peptidomimetics with antifreeze activity with the ice surface and causing a thermal hysteresis [2], will require a priori knowledge of the requisite struc- and this would account for their highly efficient noncol- tures for ice binding and growth inhibition. ligative freezing point depression. Excellent progress Among teleost fishes, five unrelated types of AF(G)Ps have been characterised, and each has a very narrow taxonomic distribution [3]. Since their discovery in fish, * Corresponding author. numerous different AFPs have been found in plants, 272 K. V. Ewart, Q. Lin and C. L. Hew Developments in antifreeze protein research Table 1. Overview of antifreeze protein characteristics. Protein Species Structural type Protein homology Ice binding site References Fish type I AFP righteye flounders, single h-helix, many undetected Thr and resid- [19, 20, 25, 79, 80] sculpins with sequence repeat ues associated Fish type II AFP sea raven, rainbow smelt, globular; smelt and galactose-binding; with it corresp- [31, 33, 81] Atlantic herring herring are Ca2+- C-type lectins in herring onds to Ca2+ dependent binding site Fish type III AFP eel pouts such as ocean globular with one undetected residues on and [82, 83] pout (Macrozoarces flattened surface flanking flat sur- americanus) face Fish type IV AFP longhorn sculpin antiparallel helix low-density lipoprotein unknown [84] (Myoxocephalus octo- bundle receptor-binding domain decimspinosis) of apolipoprotein E3 AFGP cods and Antarctic polymer of Ala-Ala- none – de novo evol- Gal-GalNAc reviewed in [4, 49] nototheniids Thr and variants, di- ution saccharide on each Thr Chitinase AFP winter rye endochitinase endochitinase unknown [58] Glucanase AFP winter rye endoglucanase endoglucanase unknown [58] Thaumatin AFP winter rye thaumatin-like thaumatin unknown [58] Budworm AFP spruce budworm (Chor- Thr- and Cys-rich undetected unknown [61] istoneura fumiferana) (non-repeating) Beetle AFP Dendroides canadensis, Thr- and Cys-rich undetected unknown [62, 63] mealworm beetle with repeating struc- ture animals, fungi and bacteria [4]. However, their distribu- proteins are summarised in table 1. The diversity of the tion appears strictly limited to species that are exposed antifreezes has been covered in recent reviews [3, 4, 10]. to cold or freezing temperatures, and in many cases Evidence for even greater variety among antifreezes is their occurrence within those species is limited to peri- accumulating. Antifreeze activity is found in many spe- ods of cold exposure. The diversity and specificity of the cies of plants, but with the exception of the winter rye AF(G)Ps is complicated by the recent discovery of (Secale cereale) AFPs, the active proteins remain to be activities in these proteins that are distinct from ice characterised. The antifreezes of bacteria are also not binding and freezing point depression [5]. They appear yet well understood, although investigation is progress- to protect mammalian cell membranes from damage at ing. For example, the AFP of Pseudomonas putida,a temperatures above 0 °C [6, 7]. Thus, our perspectives common soil bacterium, has been purified, and it has an on the distribution, diversity and roles of AF(G)Ps are amino acid composition and other compositional prop- in transition. erties similar to those of the bacterial ice nucleating This review will focus on some of the current develop- proteins. This protein differs from other known an- ments in our knowledge of AF(G)Ps. Recent reviews tifreezes in having both ice nucleating and antifreeze have addressed many aspects of AF(G)Ps [3, 5, 8–10]. activities [11]. Sequencing and structural studies on this However, in various areas of study, our understanding protein will be of great interest. of AF(G)Ps is progressing rapidly. Therefore, in this The different AF(G)Ps have two unusual and contrast- review, recent progress in our understanding of AF(G)P ing origins. Some are rogue proteins that have emerged evolution will be meshed with information on their from protein families with completely unrelated func- structure and function. By integrating different areas of tions. Others are genuinely novel proteins whose genes antifreeze research, we intend to gain new insights into came to exist through relatively recent genetic events. their activity and their role in nature. The surprising diversity of the AF(G)Ps together with their very narrow distributions has led to the hypothesis that they have each evolved recently and independently, well after present animal and plant orders or species Overview of AFP diversity had attained their current forms [12]. During geologi- AFPs from several species have been isolated and char- cally recent cooling and glaciation events, it is likely acterised. The proteins for which sequence or structural that species underwent tremendous selective pressure to information has been obtained include the five types of avoid freezing under progressively cooler conditions AFPs found in fish, two types from insects and the three [12]. This situation would have favoured any means of AFPs of a plant, winter rye. Characteristics of these lowering the freezing point that would not cause physi- CMLS, Cell. Mol. Life Sci. Vol. 55, 1999 Review Article 273 Figure 1. Protein sequences and a structure of representative fish type I AFPs. The sequences are aligned by eye for ease of comparison. 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]. ological disturbances, and it could well have led to the rugineus), the primary serum antifreeze has four re- present multitude of noncolligative antifreezes. peats, 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 An alanine theme with variations lower activity of the yellowtail flounder AFP appears to result from its lack of certain ice-binding residues [13, The type I fish AFPs are alanine-rich h-helical proteins, 20]. The yellowtail flounder AFP is also encoded by a and they are found in the righteye flounders and in multigene family [20]. It seems likely that the flounder certain sculpins (fig. 1). Most are small proteins that have evolved antifreezes with different repeat numbers form single amphiphilic or partly amphiphilic h-helices [13]. The prototype and most carefully studied type I by unequal crossing over [21]. It is interesting that no AFP (isoform HPLC6) is the major serum isoform of two-repeat or five-repeat AFPs have been identified in winter flounder (Pleuronectes americanus) [13, 14]. This flounder. Studies have suggested structural and func- AFP and another abundant isoform (HPLC8) found in tional constraints on repeat numbers in these an- flounder serum are both 37-residue proteins that con- tifreezes. Analyses of synthetic or chimeric antifreeze tain 11-residue sequence repeats consisting of Thr-X variants have suggested that two-repeat antifreezes are 10 / [15]. The Thr residues of winter flounder type I AFP unstable and or inactive, and the five-repeat form may (HPLC6) are regularly spaced along one side of the have limited solubility in aqueous solutions [22–24].
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