Identification and validation of differentially expressed transcripts in a hepatocyte model of cold-induced glycerol production in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) avoid freezing by producing antifreeze protein (AFP) and accumulating glycerol. Glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature. Hepatocytes were isolated from the livers of fish acclimated to 8°C. Cells were incubated at warm (8°C; nonglycerol accumulating) or cold (0.4°C; glycerol accumulating) temperature over a 72-h time course. Reciprocal suppression subtractive hybridization libraries enriched for cold-responsive transcripts were constructed at 72 h. Microarray analyses using a 16K salmonid cDNA array were performed at 24, 48, and 72 h. Expression of type II AFP and 21 carbohydrate, amino acid, or lipid metabolism-related transcripts were validated using quantitative RT-PCR. Type II AFP transcript levels were not directly temperature related. In cold cells, levels of the glucose synthesis transcript were transiently higher. Increased glycerol production was not associated with increased phosphofructokinase or cytosolic glycerol-3-phosphate dehydrogenase transcript levels. Levels of transcripts (phosphoenolpyruvate carboxykinase, mitochondrial malate dehydrogenase, alanine aminotransferase, glutamate dehydrogenase, and aquaglyceroporin 9) associated with mobilization of amino acids to fuel glycerol accumulation were all transiently higher, suggesting a common regulatory mechanism. In cold compared with warm cells, pyruvate dehydrogenase kinase [an inhibitor of pyruvate dehydrogenase (PDH)] transcript levels were 20-fold higher. Potent inhibition of PDH would direct pyruvate and oxaloacetate derived from amino acids to glycerol, as opposed to oxidation via the citric acid cycle. Levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were higher following cold incubation. Finally, this study also presents the novel finding of increased glutamine synthetase transcript levels in response to low temperature.

Calcium-Binding Generates the Semi-Clathrate Waters on a Type II Antifreeze Protein to Adsorb onto an Ice Crystal Surface.

Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca2+-dependent species of AFP, however, it has not been clarified how 1 mol of Ca2+-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94-E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein.

Calcium ion implicitly modulates the adsorption ability of ion-dependent type II antifreeze proteins on an ice/water interface: a structural insight.

Ion dependent type II antifreeze proteins (AFPs) are an unusual design of natural evolution for cold-acclimatization of fishes in the Antarctic region. This class of proteins requires Ca2+ to perform an unusual biological recognition, binding to a specific ice plane. However, an ice-protein complex is yet to be characterized at the molecular scale. Here, using equilibrium simulations, free energy calculations and metadynamics, we have elucidated this unusual ice recognition phenomenon at the atomistic level. The origin of ion selectivity has been critically investigated to identify the role of different ions in the dynamics and ice binding ability of the protein. We have demonstrated that within the type II protein matrix, the preferred coordination number of Ca2+ is seven involving five protein atoms and two water molecules. Due to this coordination geometry, the ion binding loop adopts a flat solvent exposed conformation which helps the AFP to efficiently adsorb on the prism plane. The ice binding surface (IBS) adsorbs on the ice surface mediated by a layer of ordered water. Structural synergy between the ice/water interface of the prism plane and the water structure around the IBS makes the adsorption highly favorable. On the other hand, the preferred geometry of the Zn2+ coordination sphere within the AFP matrix is tetrahedral. Both the coordination number and the coordination bond length are smaller for Zn2+ in comparison to Ca2+. Thus to optimize the coordination sphere for Zn2+ within the protein matrix, a kink is introduced in the ion binding loop, a part of the IBS. Therefore, the IBS and ice surface complementarity is greatly perturbed which leads to less effective adsorption.

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short beta-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and beta-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca(2+)-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.

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.

Analysis of ice-binding sites in fish type II antifreeze protein by quantum mechanics.

Many organisms living in cold environments can survive subzero temperatures by producing antifreeze proteins (AFPs) or antifreeze glycoproteins. In this paper we investigate the ice-binding surface of type II AFP by quantum mechanical methods, which, to the best of our knowledge, represents the first time that molecular orbital computational approaches have been applied to AFPs. Molecular mechanical approaches, including molecular docking, energy minimization, and molecular dynamics simulation, were used to obtain optimal systems for subsequent quantum mechanical analysis. We selected 17 surface patches covering the entire surface of the type II AFP and evaluated the interaction energy between each of these patches and two different ice planes using semi-empirical quantum mechanical methods. We have demonstrated the weak orbital overlay phenomenon and the change of bond orders in ice. These results consistently indicate that a surface patch containing 19 residues (K37, L38, Y20, E22, Y21, I19, L57, T56, F53, M127, T128, F129, R17, C7, N6, P5, G10, Q1, and W11) is the most favorable ice-binding site for both a regular ice plane and an ice plane where water O atoms are randomly positioned. Furthermore, for the first time the computation results provide new insights into the weakening of the ice lattice upon AFP binding, which may well be a primary factor leading to AFP-induced ice growth inhibition.

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.

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis).

A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.

Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris.

Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.