Ai Miura

Comparison of functional properties of two fungal antifreeze proteins from Antarctomyces psychrotrophicus and Typhula ishikariensis

Antifreeze proteins are structurally diverse polypeptides that have thermal hysteresis activity and have been discovered in many cold‐adapted organisms. Of these, fungal antifreeze protein has been purified and partially characterized only in a species of psychrophilic basidiomycete, Typhula ishikariensis. Here we report a new fungal antifreeze protein from another psychrophile, Antarctomyces psychrotrophicus. We examined its biochemical properties and thermal hysteresis activity, and compared them with those of the T. ishikariensis antifreeze protein. The antifreeze protein from A. psychrotrophicus was purified and identified as an extracellular protein of approximately 28 kDa, which halved in size following digestion with glycosidase. The A. psychrotrophicus antifreeze protein generated bipyramidal ice crystals and exhibited thermal hysteresis activity (for example thermal hysteresis = 0.42 °C for a 0.48 mm solution) similar to that of fish antifreeze proteins, while a unique rugged pattern was created on the facets of the ice bipyramid. The thermal hysteresis activity of the A. psychrotrophicus antifreeze protein was maximized under alkaline conditions, while that of the T. ishikariensis antifreeze protein was greatest under acidic conditions. The T. ishikariensis antifreeze protein exhibited a bursting ice growth normal to the c‐axis of the ice crystal and high thermal hysteresis activity (approximately 2 °C), as in the case of insect hyperactive antifreeze proteins. From these results, we speculate that the A. psychrotrophicus antifreeze protein is very different from the T. ishikariensis antifreeze protein, and that these two psychrophiles have evolved from different genes.

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.

Co‐operative effect of the isoforms of type III antifreeze protein expressed in Notched‐fin eelpout, Zoarces elongatus Kner

We found that Notched‐fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP‐ and QAE‐sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC‐pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 °C for the QAE1 isoform, 0.11 °C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes ‘active’ when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co‐operative manner.

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein

Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched‐fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20 °C·min−1). It also decreased with increasing seed crystal size under a slow cooling rate (0.01 °C·min−1), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4‐ and 2.5‐fold when ice crystals were annealed for 3 h at 0.05 and 0.25 °C below Tm, respectively. After annealing for 2 h at 0.25 °C below Tm, TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2–3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption–inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2–3 h time scale, which results in the TH dependence on the annealing time.

A part of ice nucleation protein exhibits the ice-binding ability

We generated a recombinant 96‐residue polypeptide corresponding to a sequence Tyr176–Gly273 of ice nucleation protein from Pseudomonas syringae (denoted INP96). INP96 exhibited an ability to shape an ice crystal, whose morphology is highly similar to the hexagonal‐bipyramid generally identified for antifreeze protein. INP96 also showed a non‐linear, concentration‐dependent retardation of ice growth. Additionally, circular dichroism and NMR measurements suggested a local structural construction in INP96, which undergoes irreversible thermal denaturation. These data imply that a part of INP constructs a unique structure so as to interact with the ice crystal surfaces.

Hyperactive antifreeze protein from an Antarctic sea ice bacterium Colwellia sp. has a compound ice‐binding site without repetitive sequences

Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4 °C at a concentration of 0.14 mm, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence‐based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 Å resolution revealed an irregular β‐helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice‐binding site (IBS) located at a flat surface of the β‐helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms.

Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein

Type III antifreeze protein is naturally expressed as a mixture of sulfopropyl‐Sephadex (SP) and quaternary aminoethyl‐Sephadex (QAE)‐binding isoforms, whose sequence identity is approximately 55%. We studied the ice‐binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner. The two isoforms possessed ice‐shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity. For example, the ice growth rate for nfeAFP6 was 1000‐fold higher than that for nfeAFP8 when measured for 0.1 mm protein solution at 0.25 °C below the melting point. Nevertheless, nfeAFP6 exhibited full thermal hysteresis activity in the presence of only 1% nfeAFP8 (i.e. [nfeAFP8]/[nfeAFP6] = 0.01), the effectiveness of which was indistinguishable from that of nfeAFP8 alone. We also observed a burst of ice crystal growth from the tip of the ice bipyramid for both isoforms on lowering the temperature. These results suggest that the ice growth inhibitory activity of an antifreeze protein isoform lacking the active component is restored by the addition of a minute amount of the active isoform.

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout ☆

Antifreeze proteins (AFPs) can bind to the surface of ice crystals and have also been suggested to protect cells from hypothermic damage. The present study reports that type III AFPs from notched-fin eelpout, Zoarces elongatus Kner, can protect cells during hypothermic storage. This fish naturally expresses at least 13 isoforms of type III AFP (denoted NfeAFPs), the primary sequences of which were categorized into SP- and QAE-Sephadex binding groups (SP- and QAE-isoforms). We compared the preservation ability between the extracted isoform mixtures (NfeAFPs) and a recombinant single SP-isoform (RcNfeAFP6). Experiments were performed using cultivated mammalian cells (HepG2) exposed to 4 degrees C for 24-72 h. The preserved cells were evaluated by measuring LDH released, intracellular ATP, and WST-8 reduction. It appeared that the protective effect of the 2 samples increases dose-dependently at concentrations between 2 and 10 mg/ml. Under highest soluble amount of the protein (approximately 10 mg/ml), cell viability significantly improved compared with the ordinary preservation fluid (P<0.01). This effect was larger with NfeAFPs than with RcNfeAFP6 at the same concentration. The successful hypothermic preservation of cells using natural NfeAFPs may have a wide range of applications for cell engineering and clinical medical care.

Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation

It is sometimes desirable to preserve mammalian cells by hypothermia rather than freezing during short term transplantation. Here we found an ability of hypothermic (+4°C) preservation of fish antifreeze protein (AFP) against rat insulinoma cells denoted as RIN-5F. The preservation ability was compared between type I–III AFPs and antifreeze glycoprotein (AFGP), which could be recently mass-prepared by a developed technique utilizing the muscle homogenates, but not the blood serum, of cold-adapted fishes. For AFGP, whose molecular weight is distributed in the range from 2.6 to 34 kDa, only the proteins less than 10 kDa were examined. The viability rate was evaluated by counting of the preserved RIN-5F cells unstained with trypan blue. Significantly, either AFPI or AFPIII dissolved into Euro-Collins (EC) solution at a concentration of 10 mg/ml could preserve approximately 60% of the cells for 5 days at +4°C. The 5-day preserved RIN-5F cells retained the ability to secrete insulin. Only 2% of the cells were, however, preserved for 5 days without AFP. Confocal photomicroscopy experiments further showed the significant binding ability of AFP to the cell surface. These results suggest that fish AFP enables 5-day quality storage of the insulinoma cells collected from a donor without freezing.

Genome Sequence of the Mucoromycotina Fungus Umbelopsis isabellina, an Effective Producer of Lipids

ABSTRACT Umbelopsis isabellina is a fungus in the subdivision Mucoromycotina, many members of which have been shown to be oleaginous and have become important organisms for producing oil because of their high level of intracellular lipid accumulation from various feedstocks. The genome sequence of U. isabellina NBRC 7884 was determined and annotated, and this information might provide insights into the oleaginous properties of this fungus.