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Abstract: 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. [Copyright &y& Elsevier]

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 ototif 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 followsc (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 NaCI or KCI, is largely folded as α-helix. (3) When dried with MgCl2 or CaCl2, 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-lid, conditions. [ABSTRACT FROM AUTHOR]

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 1l-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 (Polypedilurn vanderplank,), 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 a-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 °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. [ABSTRACT FROM AUTHOR]

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha ; preculture treatment of these cells with 0.5 M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation–rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a “molecular shield”. Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins. [ABSTRACT FROM AUTHOR]