Group 3 Late Embryogenesis Abundant Research Articles

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides.

Sphingobacterium sp. is a yellowish Gram-negative bacterium that is usually characterized by high concentrations of sphingophospholipids as lipid components. As microbial enzymes have been in high demand in industrial fields in the past few decades, this study hopes to provide significant information on lipase activities of Sphingobacterium sp., since limited studies have been conducted on the Sphingobacterium sp. lipase. A microbe from one collected Artic soil sample, ARC4, was identified as psychrotolerant Sphingobacterium sp., and it could grow in temperatures ranging from 0°C to 24°C. The expression of Sphingobacterium sp. lipase was successfully performed through an efficient approach of utilizing mutated group 3 late embryogenesis abundant (G3LEA) proteins developed from Polypedilum vanderplanki. Purified enzyme was characterized using a few parameters, such as temperature, pH, metal ion cofactors, organic solvents, and detergents. The expressed enzyme is reported to be cold adapted and has the capability to work efficiently under neutral pH (pH 5.0 to 7.0), cofactors like Na+ ion, and the water-like solvent methanol. Addition of nonionic detergents greatly enhanced the activity of purified enzyme. IMPORTANCE The mechanism of action of LEA proteins has remained unknown to many; in this study we reveal their presence and improved protein expression due to the molecular shielding effect reported by others. This paper should be regarded as a useful example of using such proteins to influence an existing expression system to produce difficult-to-express proteins.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Protective Functions of Group 3 Late Embryogenesis Abundant (G3LEA) Proteins in Enterococcus faecium During Vancomycin Treatment.

Late embryogenesis abundant (LEA) proteins protect organisms from various environmental stresses; however, the underlying mechanism of LEA mediated therapeutic evasion is still unclear in both eukaryotes and prokaryotes. In this study, group 3 LEA protein (G3LEA) of vancomycin-resistant Enterococcus faecium under sublethal concentration of vancomycin stress was evaluated and shown to have two functions: the first is the reduction of reactive oxygen species (ROS) content, preventing apoptosis by suppressing apoptotic proteins Cas3 and MAOB, and the second is activating specific drug efflux pumps. Sublethal vancomycin model was established with using Propidium Iodide (PI) stain. Real-time PCR was conducted to evaluate the expression of G3lea. Flow cytometry and confocal microscope using Anti- G3LEA, anti- MAOB, and anti- Cas3 were performed to assess the expression of G3LEA. Under sublethal vancomycin stress, G3LEA is upregulated, suppressing the expression of apoptotic markers and increasing specific efflux markers. These results suggest that G3LEA protein suppresses antibiotic mediated apoptosis in prokaryotic cells and plays a key role in understanding and preventing antibiotic resistance.

The effect of salt and temperature on the conformational changes of P1LEA-22, a repeat unit of plant Late Embryogenesis Abundant proteins.

The effect of choline chloride on the conformational dynamics of the 11-mer repeat unit P1LEA-22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA-22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl3 or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α-helix and β-sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl3 and choline chloride salts on P1LEA-22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage-preventing measure of Late Embryogenesis Abundant proteins.

Replica exchange molecular dynamics simulation study on the mechanism of desiccation-induced structuralization of an intrinsically disordered peptide as a model of LEA proteins.

Group 3 late embryogenesis abundant (G3LEA) proteins, which act as a well-characterized desiccation protectant in anhydrobiotic organisms, are structurally disordered in solution, but they acquire a predominantly α-helical structure during drying. Thus, G3LEA proteins are now accepted as intrinsically disordered proteins (IDPs). Their functional regions involve characteristic 11-mer repeating motifs. In the present study, to elucidate the origin of the IDP property of G3LEA proteins, we applied replica exchange molecular dynamics (REMD) simulation to a model peptide composed of two tandem repeats of an 11-mer motif and its counterpart peptide whose amino acid sequence was randomized with the same amino acid composition as that of the 11-mer motif. REMD simulations were performed for a single α-helical chain of each peptide and its double-bundled strand in a wide water content ranging from 5 to 78.3 wt%. In the latter case, we tested different types of arrangement: 1) the dipole moments of the two helices were parallel or anti-parallel and 2) due to the amphiphilic nature of the α-helix of the 11-mer motif, two types of the side-to-side contact were tested: hydrophilic-hydrophilic facing or hydrophobic-hydrophobic facing. Here, we revealed that the single chain alone exhibits no IDP-like properties, even if it involves the 11-mer motif, and the hydrophilic interaction of the two chains leads to the formation of a left-handed α-helical coiled coil in the dry state. These results support the cytoskeleton hypothesis that has been proposed as a mechanism by which G3LEA proteins work as a desiccation protectant.

A LEA model peptide protects the function of a red fluorescent protein in the dry state

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide.

The effect of phosphorylation on the salt-tolerance-related functions of the soybean protein PM18, a member of the group-3 LEA protein family

Enzymatically driven post-translated modifications (PTMs) usually happen within the intrinsically disordered regions of a target protein and can modulate variety of protein functions. Late embryogenesis abundant (LEA) proteins are a family of the plant intrinsically disordered proteins (IDPs). Despite their important roles in plant stress response, there is currently limited knowledge on the presence and functional and structural effects of phosphorylation on LEA proteins. In this study, we identified three phosphorylation sites (Ser90, Tyr136, and Thr266) in the soybean PM18 protein that belongs to the group-3 LEA proteins. In yeast expression system, PM18 protein increased the salt tolerance of yeast, and the phosphorylation of this protein further enhanced its protective function. Further analysis revealed that Ser90 and Tyr136 are more important than Thr266, and these two sites might work cooperatively in regulating the salt resistance function of PM18. The circular dichroism analysis showed that PM18 protein was disordered in aqueous media, and phosphorylation did not affect the disordered status of this protein. However, phosphorylation promoted formation of more helical structure in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE). Furthermore, in dedicated in vitro experiments, phosphorylated PM18 protein was able to better protect lactate dehydrogenase (LDH) from the inactivation induced by the freeze-thaw cycles than its un- or dephosphorylated forms. All these data indicate that phosphorylation may have regulatory effects on the stress-tolerance-related function of LEA proteins. Therefore, further studies are needed to shed more light on functional and structural roles of phosphorylation in LEA proteins.

Group 3 LEA protein model peptides protect enzymes against desiccation stress

We tested whether model peptides for group 3 late embryogenesis abundant (G3LEA) proteins, which we developed previously, are capable of maintaining the catalytic activities of enzymes dried in their presence. Three different peptides were compared: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif found in G3LEA proteins from an African sleeping chironomid; 2) PvLEA-44, which is made of four tandem repeats of the same 11-mer motif; and 3) a peptide whose amino acid composition is the same as that of PvLEA-22, but whose sequence is scrambled. We selected two enzymes, lactate dehydrogenase (LDH) and β-d-galactosidase (BDG), as targets because they have different isoelectric point (pI) values, in the alkaline and acidic range, respectively. While these enzymes were almost inactivated when dried alone, their catalytic activity was preserved at ≥70% of native levels in the presence of any of the above three peptides. This degree of protection is comparable to that conferred by several full-length G3LEA proteins, as reported previously for LDH. Interestingly, the protective activity of the peptides was enhanced slightly when they were mixed with trehalose, especially when the molar content of the peptides was low. On the basis of these results, the G3LEA model peptides show promise as protectants for the dry preservation of enzymes/proteins with a wide range of pI values.

DISTINCTIVE LOCALIZATION OF GROUP 3 LATE EMBRYOGENESIS ABUNDANT SYNTHESIZING CELLS DURING BRINE SHRIMP DEVELOPMENT.

Despite numerous studies on late embryogenesis abundant (LEA) proteins, their functions, roles, and localizations during developmental stages in arthropods remain unknown. LEA proteins protect crucial proteins against osmotic stress during the development and growth of various organisms. Thus, in this study, fluorescence in situ hybridization was used to determine the crucial regions protected against osmotic stress as well as the distinctive localization of group 3 (G3) LEA(+) cells during brine shrimp development. Several cell types were found to synthesize G3 LEA RNA, including neurons, muscular cells, APH-1(+) cells, and renal cells. The G3 LEA(+) neuronal cell bodies outside of the mushroom body projected their axonal bundles to the central body, but those inside the mushroom body projected their axonal bundles toward the deutocerebrum without innervating the central body. The cell bodies inside the mushroom body received axons of the G3 LEA(+) sensory cells at the medial ventral cup of the nauplius eye. Several glands were found to synthesize G3 LEA RNA during the nauplius stages of brine shrimp, including the sinus, antennal I and II, salt, and three ectodermal glands. This study provides the first demonstration of the formation of G3 LEA(+) sinus glands at the emergence stages of brine shrimp. These results suggest that G3 LEA protein is synthesized in several cell types. In particular, specific glands play crucial roles during the emergence and nauplius stages of brine shrimp.

Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation

Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect (Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.

Salt Effects on the Structural and Thermodynamic Properties of a Group 3 LEA Protein Model Peptide

To sequestrate or scavenge ionic species in desiccated cells is one of the putative functions of group 3 late embryogenesis abundant (G3LEA) proteins. We still lack direct physicochemical information on how G3LEA proteins and their characteristic primary amino acid sequences, i.e., 11-mer motif repeats, behave in the presence of salts under water-deficit conditions. In the current study, we investigated salt effects as a function of water content on the structural and thermodynamic properties of the 22-mer peptide (PvLEA-22), consisting of two tandem repeats of the consensus 11-mer motif of G3LEA proteins from the larvae of P. vanderplanki. The results of circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopic measurements indicate four main points as follows: (1) PvLEA-22 is in random coils in the aqueous solutions with or without a salt. (2) Dried PvLEA-22, whether salt-free or mixed with NaCl or KCl, is largely folded as α-helix. (3) When dried with MgCl(2) or CaCl(2), PvLEA-22 adopts β-sheet structure as well as random coil. (4) PvLEA-22 faithfully reproduces the conformational changes of the native LEA protein in response to added salts. Furthermore, through temperature-modulated differential scanning calorimetry (TMDSC) measurements, dried PvLEA-22 is found to be in the glassy state at ambient temperatures, independent of which salt is present. On the basis of these results, we discuss the intrinsic nature and putative functional roles of G3LEA proteins under salt-rich conditions.

Desiccation-Induced Structuralization and Glass Formation of Group 3 Late Embryogenesis Abundant Protein Model Peptides

Anhydrobiotic (i.e., life without water) organisms are known to produce group 3 late embryogenesis abundant (G3LEA) proteins during adaptation to severely water-deficient conditions. Their primary amino acid sequences are composed largely of loosely conserved 11-mer repeat units. However, little information has been obtained for the structural and functional roles of these repeat units. In this study, we first explore the consensus sequences of the 11-mer repeat units for several native G3LEA proteins originating from anhydrobiotic organisms among insects (Polypedilum vanderplanki), nematodes, and plants. Next, we synthesize four kinds of model peptides (LEA models), each of which consists of four or two repeats of the 11-mer consensus sequences for each of the three organisms. The structural and thermodynamic properties of the LEA models were examined in solution, in dehydrated and rehydrated states, and furthermore in the presence of trehalose, since a great quantity of this sugar is known to be produced in the dried cells of most anhydrobiotic organisms. The results of Fourier transform infrared (FTIR) spectroscopic measurements indicate that all of the LEA models transform from random coils to alpha-helical coiled coils on dehydration and return to random coils again on rehydration, both with and without trehalose. In contrast, such structural changes were never observed for a control peptide with a randomized amino acid sequence. Furthermore, our differential scanning calorimetry (DSC) measurements provide the first evidence that the above 11-mer motif-containing peptides themselves vitrify with a high glass transition temperature (>100 degrees C) and a low enthalpy relaxation rate. In addition, they play a role in reinforcing the glassy matrix of the coexisting trehalose. On the basis of these results, we discuss the underlying mechanism of G3LEA proteins as desiccation stress protectants.

1P-068 グループ3LEAタンパク質のモデルペプチドがタンパク質凝集に与える効果(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

1P-068 グループ3LEAタンパク質のモデルペプチドがタンパク質凝集に与える効果(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

25. Model study of the desiccation induced structural transformations of Group-3 Late Embryogenesis Abundant (G3LEA) proteins expressed in anhydrobiotic organisms

25. Model study of the desiccation induced structural transformations of Group-3 Late Embryogenesis Abundant (G3LEA) proteins expressed in anhydrobiotic organisms

3P024 モデル系を用いたグループ3LEAタンパク質(G3LEA)の乾燥誘導構造変化に関する研究(ヘム蛋白質、電子状態、蛋白質(構造・構造機能相関,口頭発表,第45回日本生物物理学会年会)

3P024 モデル系を用いたグループ3LEAタンパク質(G3LEA)の乾燥誘導構造変化に関する研究(ヘム蛋白質、電子状態、蛋白質(構造・構造機能相関,口頭発表,第45回日本生物物理学会年会)

Model study of the Desiccation-Induced Structural Transformations of Group-3 Late Embryogenesis Abundant (G3LEA) Proteins(Papers presented at the 53rd Annual Meeting)

Model study of the Desiccation-Induced Structural Transformations of Group-3 Late Embryogenesis Abundant (G3LEA) Proteins(Papers presented at the 53rd Annual Meeting)