Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

抗冻蛋白研究进展(英文)

很多越冬的生物会产生抗冻蛋白,这些抗冻蛋白能够吸附到冰晶的表面改变冰晶形态并抑制冰晶的生长.抗冻蛋白在很多生物体内都被发现,不同的抗冻蛋白结构差异非常大.目前的一些研究揭示了几种抗冻蛋白的结构,并提出了抗冻蛋白与冰晶的结合模型,但是还没有一种机制能解释所有抗冻蛋白的作用机理.抗冻蛋白能被广泛的应用到农业、水产业和低温储藏器官、组织和细胞,利用转基因技术提高植物的抗冻性具有重要应用价值.而抗冻蛋白基因的表达调控则有待进一步阐明.     

Ice nucleation and antinucleation in nature

Plants and ectothermic animals use a variety of substances and mechanisms to survive exposure to subfreezing temperatures. Proteinaceous ice nucleators trigger freezing at high subzero temperatures, either to provide cold protection from released heat of fusion or to establish a protective extracellular freezing in freeze-tolerant species. Freeze-avoiding species increase their supercooling potential by removing ice nucleators and accumulating polyols. Terrestrial invertebrates and polar marine fish stabilize their supercooled state by means of noncolligatively acting antifreeze proteins. Some organisms also depress their body fluid melting point to ambient temperature by evaporation and/or solute accumulation.     

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.     

昆虫抗冻蛋白的分离纯化及特性分析

昆虫抗冻蛋白具有很高的热滞活性,可保护机体免受结冰引起的伤害。昆虫抗冻蛋白的分离纯化多采用凝胶过滤层析、离子交换层析及HPLC等技术,已用于鱼类抗冻蛋白纯化的冰亲和纯化(IAP)技术也可考虑应用于昆虫抗冻蛋白的分离提纯。昆虫抗冻蛋白具有高活性,规则的一级结构及类似的冰晶结合表面等特性。     

Partitioning of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity

Antifreeze proteins (AFPs) inhibit the growth of ice by binding to the surface of ice crystals, preventing the addition of water molecules to cause a local depression of the freezing point. AFPs from insects are much more effective at depressing the freezing point than fish AFPs. Here, we have investigated the possibility that insect AFPs bind more avidly to ice than fish AFPs. Because it is not possible to directly measure the affinity of an AFP for ice, we have assessed binding indirectly by examining the partitioning of proteins into a slowly growing ice hemisphere. AFP molecules adsorbed to the surface and became incorporated into the ice as they were overgrown. Solutes, including non-AFPs, were very efficiently excluded from ice, whereas AFPs became incorporated into ice at a concentration roughly equal to that of the original solution, and this was independent of the AFP concentration in the range (submillimolar) tested. Despite their >10-fold difference in antifreeze activity, fish and insect AFPs partitioned into ice to a similar degree, suggesting that insect AFPs do not bind to ice with appreciably higher affinity. Additionally, we have demonstrated that steric mutations on the ice binding surface that decrease the antifreeze activity of an AFP also reduce its inclusion into ice, supporting the validity of using partitioning measurements to assess a protein's affinity for ice.     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus

We purified many kinds of antifreeze proteins with high activity from the leaves of Ammopiptanthus mongolicus by several biochemical techniques. The antifreeze activities of these AFPs were measured by both osmometry and differential scanning calorimetry, and the inhibition of growth of ice crystals by the AFPs was obvious. Additionally, the antifreeze proteins were analyzed by sequencing, glycosylation reaction, mass spectroscopy, and circular dichroism spectroscopy. Both samples have some other unique structures different from those of fishes and of insects. It was suggested that plant AFPs might have a particular antifreeze mechanism in comparison with that of fish and insects.     

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Structure and evolutionary origin of Ca(2+)-dependent herring type II antifreeze protein

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.     

利用伪氨基酸组分和支持向量机预测抗冻蛋白

抗冻蛋白是一类具有提高生物抗冻能力的蛋白质。抗冻蛋白能够特异性的与冰晶相结合,进而阻止体液内冰核的形成与生长。因此,对抗冻蛋白的生物信息学研究对生物工程发展。提高作物抗冻性有重要的推动作用。本文采用由400条抗冻蛋白序列和400条非抗冻蛋白序列构成数据集,以伪氨基酸组分为特征,利用支持向量机分类算法预测抗冻蛋白,对训练集预测精度达到91．3％,对测试集预测精度达到78．8％。该结果证明伪氨基酸组分能够很好的反映抗冻蛋白特性,并能够用于预测抗冻蛋白。     

Why does insect antifreeze protein from Tenebrio molitor produce pyramidal ice crystallites?

The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the orientations and molecular compositions of the primary ice surfaces that undergo growth rate reduction by the insect AFPs are obtained from first principles. Besides the basal face and primary prism, the ice surfaces engaged by insect AFPs include the specific ice pyramids produced by the insect AFP Tenebrio molitor (TmAFP). TmAFP-induced pyramids differ fundamentally from the ice pyramids produced by fish AFPs and antifreeze protein glycoproteins (AFPGs) as regards the ice surface configurations and the mode of interaction with the protein IBS. The molecular compositions of the TmAFP-induced pyramids are strongly bonded in two dimensions and have the constant face indices (101). In contrast, the molecular composition of the ice pyramids produced by fish AFPs and AFPGs are strongly bonded in only one direction and have variable face indices (h 0 l), none of which equal (101). The thus far puzzling behavior of the TmAFP in producing pyramidal crystallites is fully explained in agreement with experiment.     

Identification of antifreeze proteins and their functional residues by support vector machine and genetic algorithms based on n-peptide compositions

For the first time, multiple sets of n-peptide compositions from antifreeze protein (AFP) sequences of various cold-adapted fish and insects were analyzed using support vector machine and genetic algorithms. The identification of AFPs is difficult because they exist as evolutionarily divergent types, and because their sequences and structures are present in limited numbers in currently available databases. Our results reveal that it is feasible to identify the shared sequential features among the various structural types of AFPs. Moreover, we were able to identify residues involved in ice binding without requiring knowledge of the three-dimensional structures of these AFPs. This approach should be useful for genomic and proteomic studies involving cold-adapted organisms.     

Purification and Structure Analysis of Antifreeze Proteins from Ammopiptanthus mongolicus

Abstract

We purified many kinds of antifreeze proteins with high activity from the leaves of Ammopiptanthus mongolicus by several biochemical techniques. The antifreeze activities of these AFPs were measured by both osmometry and differential scanning calorimetry, and the inhibition of growth of ice crystals by the AFPs was obvious. Additionally, the antifreeze proteins were analyzed by sequencing, glycosylation reaction, mass spectroscopy, and circular dichroism spectroscopy. Both samples have some other unique structures different from those of fishes and of insects. It was suggested that plant AFPs might have a particular antifreeze mechanism in comparison with that of fish and insects.     

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves,Crowns, and Roots by Tissue Printing

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.     

昆虫抗冻蛋白的研究进展

热滞效应(Tberm Hysteresis Action)最早在昆虫研究中发现,后来研究表明,它是抗冻蛋白(Antifreeze Proteins,AFPs)的一种基本性质。和鱼类,植物AFPs相比,昆虫AFPs具有更高 的热滞活性和独特的化学结构特征。昆虫AFPs在昆虫抗冻生理过程中起着相当重要的作用,表现在以下三个方面：①抑制一些冰晶形成;②提高冰冻耐受性;③可能参与水分平衡过程。光周期,气温和湿度是调控AFPs生物合成与降解的三种外部因子,而体内激素的变化可能是直接调节脂肪体合成AFPs的内部因子。     

Ordered surface carbons distinguish antifreeze proteins and their ice-binding regions

Antifreeze proteins (AFPs) are found in cold-adapted organisms and have the unusual ability to bind to and inhibit the growth of ice crystals. However, the underlying molecular basis of their ice-binding activity is unclear because of the difficulty of studying the AFP-ice interaction directly and the lack of a common motif, domain or fold among different AFPs. We have formulated a generic ice-binding model and incorporated it into a physicochemical pattern-recognition algorithm. It successfully recognizes ice-binding surfaces for a diverse range of AFPs, and clearly discriminates AFPs from other structures in the Protein Data Bank. The algorithm was used to identify a novel AFP from winter rye, and the antifreeze activity of this protein was subsequently confirmed. The presence of a common and distinct physicochemical pattern provides a structural basis for unifying AFPs from fish, insects and plants.     

Maintenance of the supercooled state in the gut fluid of overwintering pyrochroid beetle larvae, Dendroides canadensis : role of ice nucleators and antifreeze proteins

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Antifreeze protein-induced morphological modification mechanisms linked to ice binding surface

The mechanisms by which the antifreeze protein (AFP) modifies the ice morphology are identified precisely as surface poisoning by the ice binding surface (IBS) of insect AFPs and as bridge-induced surface reconstruction by the IBS of fish AFPs and antifreeze glycoproteins. The primary surfaces of hexagonal ice have predetermined face indices. The "two-dimensional" insect type IBS has regularly spaced binding intervals in two directions. It causes surface poisoning by matching and reinforcing simultaneously intersecting strong bonding directions on the primary ice surfaces. The secondary ice surfaces have variable face indices. The "one-dimensional" and "irregular" IBS variants of fish AFPs and antifreeze glycoproteins are either linearly extended with regular ice binding intervals or have ice binding sites lacking spacing regularity. These variants can bridge transversely lattice periods or shorter oxygen-oxygen distances between parallel adjacent strong bonding directions that do not intersect. Thus, one-dimensional and irregular IBS variants induce supplementary bridges cross-wise on selected secondary surfaces by mimicking strong bonding directions that are not present in the ice structure. These proteins cause surfaces with variable face indices, which in the absence of the AFPs would not grow flat, to appear in the morphology. Whereas for the primary ice surfaces it is only the morphological importance that is determined by the experimental conditions, for the secondary ice surfaces it is the face indices themselves that become adjusted in the process of maximizing the AFP-substrate interaction through attainment of the best structural match. The growth morphology of the AFP-ice system is derived from various factors, including the face indices, surface molecular compositions, relative growth rates, and the mechanisms responsible for that morphology. The theoretical formulation agrees with experiments over a wide range and resolves these, to date, unexplained phenomena.