Simultaneous Recording of Remote Domain Dynamics in Membrane Proteins Using the Double-Labeled DXB/DXT Technique.

Protein dynamics play important roles in biological functions, which accompany allosteric structure changes. Diffracted X-ray blinking (DXB) uses monochromatic X-rays and nanocrystal probes. The intramolecular motion of target proteins is analyzed from the intensity changes in detector signals at the diffraction rings. In contrast, diffracted X-ray tracking (DXT) elucidates molecular dynamics by analyzing the trajectories of Laue spots. In this study, we have developed a dual-labeling technique for DXB and DXT, allowing the simultaneous observation of motions at different domains in proteins. We identified zinc oxide (ZnO) crystals as promising candidates for the second labeling probes due to their excellent diffraction patterns, high chemical stability, and favorable binding properties with proteins. The diffraction spots from the ZnO crystals are sufficiently separated from those of gold, enabling independent motion analysis at different domains. Dual-labeling DXB was employed for the motion analysis of the 5-HT2A receptor in living cells. Simultaneous motion recording of the N-terminus and the second extracellular loop demonstrated ligand-induced motion suppression at both domains. The dual-labeling DXT technique demonstrated a capsaicin-induced peak shift in the two-dimensional motion maps at the N-terminus of the TRPV1 protein, but the peak shift was not obvious in the C-terminus. The capsaicin-induced motion modulation was recovered by the addition of the competitive inhibitor AMG9810.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

The Blinking of Small-Angle X-ray Scattering Reveals the Degradation Process of Protein Crystals at Microsecond Timescale.

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal's domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.

Visualization of the Dynamics of Photoinduced Crawling Motion of 4-(Methylamino)Azobenzene Crystals via Diffracted X-ray Tracking.

The photoinduced crawling motion of crystals is a continuous motion that azobenzene molecular crystals exhibit under light irradiation. Such motion enables object manipulation at the microscale with a simple setup of fixed LED light sources. Transportation of nano-/micromaterials using photoinduced crawling motion has recently been reported. However, the details of the motion mechanism have not been revealed so far. Herein, we report visualization of the dynamics of fine particles in 4-(methylamino)azobenzene (4-MAAB) crystals under light irradiation via diffracted X-ray tracking (DXT). Continuously repeated melting and recrystallization of 4-MAAB crystals under light irradiation results in the flow of liquid 4-MAAB. Zinc oxide (ZnO) particles were introduced inside the 4-MAAB crystals to detect diffracted X-rays. The ZnO particles rotate with the flow of liquid 4-MAAB. By using white X-rays with a wide energy width, the rotation of each zinc oxide nanoparticle was detected as the movement of a bright spot in the X-ray diffraction pattern. It was clearly shown that the ZnO particles rotated increasingly as the irradiation light intensity increased. Furthermore, we also found anisotropy in the rotational direction of ZnO particles that occurred during the crawling motion of 4-MAAB crystals. It has become clear that the flow perpendicular to the supporting film of 4-MAAB crystals is enhanced inside the crystal during the crawling motion. DXT provides a unique means to elucidate the mechanism of photoinduced crawling motion of crystals.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

Real-Time Observation of Capsaicin-Induced Intracellular Domain Dynamics of TRPV1 Using the Diffracted X-ray Tracking Method.

The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 µs/frame and 12.5 ms/frame. The data from the 100 µs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 µs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel.

The effect of ice-binding proteins on the cryopreservation of Caenorhabditis elegans.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth. IBPs have also been reported to stabilize cell membranes under non-freezing conditions. The effects of IBPs help to reduce cold- and freezing-induced damage to cells and tissues in cryopreservation. Here, we examined whether certain IBPs, namely, fish NfeIBP6 and NfeIBP8 and fungal AnpIBP1a N55D (AnpIBP), improve the recovery rate of the nematode Caenorhabditis elegans after a deep cryopreservation at -80°C. The expression of fungus-derived AnpIBP in C. elegans significantly improved its recovery rate. This result provides useful information to establish a cryopreservation technique for long-term storage using IBP molecules.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.

A mutation to a fish ice-binding protein synthesized in transgenic Caenorhabditis elegans modulates its cold tolerance.

A cryoprotectant known as ice-binding protein (IBP) is thought to facilitate the cold survival of plants, insects, and fungi. Here, we prepared a genetically modified Caenorhabditis elegans strain to synthesize fish-derived IBPs in its body wall muscles and examined whether the antifreeze activity modification of this IBP by point mutation affects the cold tolerance of this worm. We chose a 65-residue IBP identified from notched-fin eelpout, for which the replacement of the 20th alanine residue (A20) modifies its antifreeze activity. These mutant proteins are denoted A20L, A20G, A20T, A20V, and A20I along with the wild-type (WT) protein. We evaluated the survival rate (%) of the transgenic C. elegans that synthesized each IBP mutant following 24 h of preservation at -5, +2, and +5 °C. Significantly, a dramatic improvement in the survival rate was detected for the worms synthesizing the activity-enhanced mutants (A20T and A20I), especially at +2 °C. In contrast, the rate was not improved by the expression of the defective mutants (A20L, A20G, WT and A20V). The survival rate (%) probably correlates with the antifreeze activity of the IBP. These data suggest that IBP protects the cell membrane by employing its ice-binding mechanism, which ultimately improves the cold tolerance of an IBP-containing animal.

Adsorption of ice-binding proteins onto whole ice crystal surfaces does not necessarily confer a high thermal hysteresis activity.

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called "thermal hysteresis (TH)", and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called "ice recrystallization inhibition (IRI)". To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity.

Dynamic observations of various oligomers in amyloid ß isoforms using laboratory diffracted X-ray blinking.

Acceleration of societal ageing has increased the global incidence of geriatric diseases such as Alzheimer's disease (AD), and the demands for proper diagnosis and monitoring of those diseases are also increasing daily. We utilized diffracted X-ray blinking (DXB) for amyloid ß (Aß) isoforms, which are thought to be closely related to AD, to discriminate among the dynamics of individual particles in early and long-term oligomerisation and aggregation inhibiting environments. Among the various Aß isoforms, the dynamics of Aß (1-42), which is known to be the most toxic form, were the slowest (the dynamics were lower by 78% com-pared with short-term incubation), and the dynamics were restored (the dynamics increased by 105% compared with normal aggregation) in an environment that suppressed oligomerisation of Aß (1-42). It has been confirmed that the use of DXB allows measurements of dynamics related to the functional states of the target molecules.

Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma.

Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, using a mouse model of obesity-induced hepatocellular carcinoma (HCC), we identified the release mechanism of SASP factors, which include interleukin-1ß (IL-1ß)- and IL-1ß-dependent IL-33, from senescent hepatic stellate cells (HSCs) via gasdermin D (GSDMD) amino-terminal-mediated pore. We found that IL-33 was highly induced in senescent HSCs in an IL-1ß-dependent manner in the tumor microenvironment. The release of both IL-33 and IL-1ß was triggered by lipoteichoic acid (LTA), a cell wall component of gut microbiota that was transferred and accumulated in the liver tissue of high-fat diet-fed mice, and the release of these factors was mediated through cell membrane pores formed by the GSDMD amino terminus, which was cleaved by LTA-induced caspase-11. We demonstrated that IL-33 release from HSCs promoted HCC development via the activation of ST2-positive Treg cells in the liver tumor microenvironment. The accumulation of GSDMD amino terminus was also detected in HSCs from human NASH-associated HCC patients, suggesting that similar mechanism could be involved in a certain type of human HCC. These results uncover a release mechanism for SASP factors from sensitized senescent HSCs in the tumor microenvironment, thereby facilitating obesity-associated HCC progression. Furthermore, our findings highlight the therapeutic potential of inhibitors of GSDMD-mediated pore formation for HCC treatment.

Dynamic motions of ice-binding proteins in living Caenorhabditis elegans using diffracted X-ray blinking and tracking.

The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to -10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at -10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at -10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.

Persistent viral shedding of severe acute respiratory syndrome coronavirus 2 after treatment with bendamustine and rituximab: A case report.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is detectable in nasopharyngeal specimens for up to 12-20 days regardless of the presence of chronic diseases in patients. We report a case of prolonged SARS-CoV-2 infection that lasted for more than eight weeks. The patient had persistent lymphopenia after receiving six cycles of bendamustine and rituximab (BR) therapy for follicular lymphoma; the last chemotherapy session was completed nine months before admission. The first nasopharyngeal specimen (NPS) for the SARS-CoV-2 polymerase chain reaction assay tested positive for the N501Y variant five weeks before admission. The patient's general and respiratory conditions gradually worsened; therefore, he was admitted to our hospital, and the same SARS-CoV-2 variant was subsequently identified on admission. Treatment for coronavirus disease was initiated, and the patient's condition improved; however, the NPS tested positive on day 15. The patient was discharged on day 28 and was instructed to isolate at home for a month. Hence, possible prolonged SARS-CoV-2 shedding should be considered in patients who receive BR therapy.

Diffracted X-ray Tracking Method for Measuring Intramolecular Dynamics of Membrane Proteins.

Membrane proteins change their conformations in response to chemical and physical stimuli and transmit extracellular signals inside cells. Several approaches have been developed for solving the structures of proteins. However, few techniques can monitor real-time protein dynamics. The diffracted X-ray tracking method (DXT) is an X-ray-based single-molecule technique that monitors the internal motion of biomolecules in an aqueous solution. DXT analyzes trajectories of Laue spots generated from the attached gold nanocrystals with a two-dimensional axis by tilting (θ) and twisting (χ). Furthermore, high-intensity X-rays from synchrotron radiation facilities enable measurements with microsecond-timescale and picometer-spatial-scale intramolecular information. The technique has been applied to various membrane proteins due to its superior spatiotemporal resolution. In this review, we introduce basic principles of DXT, reviewing its recent and extended applications to membrane proteins and living cells, respectively.

Corynebacterium pseudodiphtheriticum as a Pathogen in Bacterial Co-Infection in COVID-19 Patients on Mechanical Ventilation.

Many studies have been conducted on ventilator-associated complications (VACs) in patients with coronavirus 2019 (COVID-19). However, in these studies, the causative organisms were similar, and there were no reports on VAC corresponding with Corynebacteria. Coryneforms are frequently cultured in cases of polymicrobial infections and are usually considered contaminants in respiratory specimens. However, Corynebacterium pseudodiphtheriticum or C. striatum is known to be a pathogen in lower respiratory tract infections. We report three cases of VAC, probably due to C. pseudodiphtheriticum, in patients with COVID-19. If purulent lower respiratory tract specimens showed coryneform predominantly upon Gram staining, empirical therapy should be started. Furthermore, species identification and drug susceptibility testing should be performed.

Prone positioning redistributes gravitational stress in the lung in normal conditions and in simulations of oedema.

NEW FINDINGS: What is the central question of this study? How does the interaction between posture and gravity affect the stresses on the lung, particularly in highly inflated gravitationally non-dependent regions, which are potentially vulnerable to increased mechanical stress and injury? What is the main finding and its importance? Changes in stress attributable to gravity are not well characterized between postures. Using a new metric of gravitational stress, we show that regions of the lung near maximal inflation have the greatest gravitational stresses while supine, but not while prone. In simulations of increased lung weight consistent with severe pulmonary oedema, the prone lung has lower gravitational stress in vulnerable, non-dependent regions, potentially protecting them from overinflation and injury. ABSTRACT: Prone posture changes the gravitational vector, and potentially the stress induced by tissue deformation, because a larger lung volume is gravitationally dependent when supine, but non-dependent when prone. To evaluate this, 10 normal subjects (six male and four female; age, means ± SD = 27 ± 6 years; height, 171 ± 9 cm; weight, 69 ± 13 kg; forced expiratory volume in the first second/forced expiratory volume as a percentage of predicted, 93 ± 6%) were imaged at functional residual capacity, supine and prone, using magnetic resonance imaging, to quantify regional lung density. We defined regional gravitational stress as the cumulative weight, per unit area, of the column of lung tissue below each point. Gravitational stress was compared between regions of differing inflation to evaluate differences between highly stretched, and thus potentially vulnerable, regions and less stretched lung. Using reference density values for normal lungs at total lung capacity (0.10 ± 0.03 g/ml), regions were classified as highly inflated (density < 0.13 g/ml, i.e., close to total lung capacity), intermediate (0.13 ≤ density < 0.16 g/ml) or normally inflated (density ≥ 0.16 g/ml). Gravitational stress differed between inflation categories while supine (-1.6 ± 0.3 cmH2 O highly inflated; -1.4 ± 0.3 cmH2 O intermediate; -1.1 ± 0.1 cmH2 O normally inflated; P = 0.05) but not while prone (-1.4 ± 0.2 cmH2 O highly inflated; -1.3 ± 0.2 cmH2 O intermediate; -1.3 ± 0.1 cmH2 O normally inflated; P = 0.39), and increased more with height from dependent lung while supine (-0.24 ± 0.02 cmH2 O/cm supine; -0.18 ± 0.04 cmH2 O/cm prone; P = 0.05). In simulated severe pulmonary oedema, the gradient in gravitational stress increased in both postures (all P < 0.0001), was greater in the supine posture than when prone (-0.57 ± 0.21 cmH2 O/cm supine; -0.34 ± 0.16 cmH2 O/cm prone; P = 0.0004) and was similar to the gradient calculated from supine computed tomography images in a patient with acute respiratory distress syndrome (-0.51 cmH2 O/cm). The non-dependent lung has greater gravitational stress while supine and might be protected while prone, particularly in the presence of oedema.

Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells.

Cells for therapeutic use are often preserved at +4 °C, and the storage period is generally limited to 2-3 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10-20 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into -5 °C-, -2 °C-, or +4 °C-chilled "unfrozen" solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the -5 °C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at -5 °C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane-water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.

Laboratory diffracted x-ray blinking to monitor picometer motions of protein molecules and application to crystalline materials.

In recent years, real-time observations of molecules have been required to understand their behavior and function. To date, we have reported two different time-resolved observation methods: diffracted x-ray tracking and diffracted x-ray blinking (DXB). The former monitors the motion of diffracted spots derived from nanocrystals labeled onto target molecules, and the latter measures the fluctuation of the diffraction intensity that is highly correlated with the target molecular motion. However, these reports use a synchrotron x-ray source because of its high average flux, resulting in a high time resolution. Here, we used a laboratory x-ray source and DXB to measure the internal molecular dynamics of three different systems. The samples studied were bovine serum albumin (BSA) pinned onto a substrate, antifreeze protein (AFP) crystallized as a single crystal, and poly{2-(perfluorooctyl)ethyl acrylate} (PC8FA) polymer between polyimide sheets. It was found that not only BSA but also AFP and PC8FA molecules move in the systems. In addition, the molecular motion of AFP molecules was observed to increase with decreasing temperature. The rotational diffusion coefficients (DR) of BSA, AFP, and PC8FA were estimated to be 0.73 pm2/s, 0.65 pm2/s, and 3.29 pm2/s, respectively. Surprisingly, the DR of the PC8FA polymer was found to be the highest among the three samples. This is the first report that measures the molecular motion of a single protein crystal and polymer by using DXB with a laboratory x-ray source. This technique can be applied to any kind of crystal and crystalline polymer and provides atomic-order molecular information.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6-8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.