Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying
Graphical abstract
Introduction
Anhydrobiosis or “life without water” is a phenomenon that has received much attention and although mechanisms responsible for cellular desiccation tolerance have been proposed (e.g. [1], [2]), many functional aspects are still unresolved [3]. There is, however, widespread consensus that sugars and Late Embryogenesis Abundant (LEA) proteins can be major contributors to cell stability in the dry state, even in cells that naturally do not contain LEA proteins [4]. In addition, some organisms can achieve desiccation tolerance without the accumulation of sugars [5], [6]. LEA proteins have been first identified in plant seeds during maturation, when the seeds attain desiccation tolerance [7], but were later also found in vegetative plant organs, in bacteria and various anhydrobiotic invertebrates [8], [9].
The precise in vivo function of most LEA proteins remains unresolved, which may at least in part be due to their unstructured nature in solution, which has made functional predictions impossible. However, many of these proteins fold mainly into α-helices during drying [10]. Results from various in vitro assays suggest that some LEA proteins are involved in the stabilization of cellular constituents such as proteins and membranes, but other functions have also been proposed [8], [9]. Only for the cold induced Arabidopsis thaliana LEA proteins COR15A and COR15B, membrane stabilization during freezing could be clearly established as their in vivo function, while enzyme stabilization could be excluded [11]. However, many in vitro investigations have shown that LEA proteins can effectively prevent inactivation of sensitive enzymes such as lactate dehydrogenase (LDH) during freezing or drying [9], [12]. Under the appropriate drying conditions such enzymes aggregate, which may contribute to their inactivation. Aggregation can be prevented by many LEA proteins that are thought to function as “molecular shields” by preventing direct contact between enzyme molecules [13], [14], [15].
In addition, as an adaptive response to water loss, most organisms accumulate compatible solutes, such as sugars [16]. During drying, most sugars do not crystallize, but rather form a glass. Due to the low molecular mobility, the glassy state immobilizes macromolecules, thus providing protection e.g. to cells in dry plant seeds [17], [18]. LEA proteins can be embedded in such a glassy matrix, increasing the glass transition temperature [19]. It has been proposed that H-bonding interactions between sugars and proteins may enhance the stability of cytoplasmic glasses in seeds and pollen, thereby contributing to the exceptional stability of these structures in the dry state [2], [18], [20].
The aim of the present study was to investigate the functional activity of the structurally disordered protein LEA7 from A. thaliana with respect to dehydration and freezing stress. LEA7 is located in the cytosolic compartment of plant cells [21] and increases the desiccation tolerance of transgenic yeast cells [22]. In a previous study we presented evidence that LEA7 is able to interact with lipid membranes in the fully hydrated and in the dry state [23], suggesting a function of the protein in membrane stabilization. Here, we show that LEA7 is not able to protect liposomes during dehydration, but rather has protective activity for the labile enzyme LDH and enzymes found in the total soluble proteome of Arabidopsis leaves. We present evidence from Fourier-transform infrared (FTIR) spectroscopy for interactions between LEA7 and LDH and also between LEA7 and the Arabidopsis proteome, thus decreasing the degree of protein aggregation and preservation of enzyme activity. In the presence of LEA7 the average strength of H-bonding interactions in dry sugar (sucrose and verbascose) glasses was increased, indicating that protein and sugars interact to form a more tightly packed matrix in comparison to pure sugars.
Section snippets
Materials
Lactate dehydrogenase from rabbit muscle (LDH), β-lactoglobulin (LG) from bovine milk and sucrose were obtained from Sigma (St. Louis, MO), verbascose from Megazyme (Wicklow, Ireland). RNaseA from bovine pancreas (RNaseA) was from Roche (Basel, Switzerland). D2O (99.98%) was purchased from Deutero GmbH (Kastellaun, Germany) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was obtained from Avanti Polar Lipids (Alabaster, AL).
Expression and purification of recombinant LEA7
The LEA7 gene (At1g52690) was cloned into the pDEST17
Liposomes are not stabilized by LEA7 during drying
Our previous investigation indicated that LEA7 interacts with liposomes in the dry state [23]. Based on these data we hypothesized that LEA7 may stabilize membranes against the stresses associated with drying and rehydration. Here, we tested this hypothesis by monitoring leakage of the fluorescent dye CF from liposomes (Fig. 1). The results indicate that LEA7 only had a marginal effect on liposome stability. CF leakage was reduced by about 7%, from 98% in the absence of additives to 91% in the
Discussion
Two main functions have been attributed to LEA proteins, namely membrane and enzyme protection during freezing and drying [9], [49]. Although our knowledge about the structural requirements for LEA proteins to perform either function is not sufficient yet to propose that they are mutually exclusive, evidence in favor of such a hypothesis is accumulating. Examples include two LEA proteins from a desiccation-tolerant rotifer, of which one shows enzyme protection and one membrane interaction [50],
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Cited by (46)
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2023, Protein Expression and PurificationThe Group 3 LEA proteins of Artemia franciscana for cryopreservation
2022, CryobiologyTarget enzymes are stabilized by AfrLEA6 and a gain of α-helix coincides with protection by a group 3 LEA protein during incremental drying
2021, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :For example, LEA proteins are thought to interact with trehalose to stabilize vitrified sugar glasses by increasing the glass transition temperature (Tg) [22,23]. Additionally, LEA proteins preserve the activity of desiccation-sensitive enzymes [24–27], may act as a molecular shield to sterically reduce harmful protein aggregation [25,28–32], protect lipid bilayers of various composition during freezing and drying [33–40], may form structural networks to reduce physical stress [41], and may sequester divalent ions [5]. An important yet unresolved issue regarding protection by LEA proteins is whether gain of secondary structure during drying is a prerequisite for functions like stabilization of macromolecular targets (cf. [5–7,31,32,42–44]).
Overexpression of 14-3-3 proteins enhances cold tolerance and increases levels of stress-responsive proteins of Arabidopsis plants
2019, Plant ScienceCitation Excerpt :Considering proteins specifically down-represented exclusively in WT plants, worth mentioning is LEA7, a member of the LEA protein family, whose primary function is to confer desiccation tolerance during seed maturation but that is also involved in the tolerance to stress. Recently, LEA7 has been demonstrated to preserve in vitro enzyme activity upon freezing [78]. In WT plants, abundance of LEA7 was greatly reduced by cold, whereas it was statistically unaffected in 14-3-3ε-overexpressing plants.
- 1
Permanent address: Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
- 2
Present address: Siemens AG, Siemensdamm 50, D-13629 Berlin, Germany.
- 3
Present address: Vilmorin SA, Rotue du Manoir, 49250 La Ménitré, France.
- 4
Present address: UMR 1332, Biologie du Fruit et Pathologie, INRA Bordeaux-Aquitaine, 33882 Villeneuve d'Ornon Cedex, France.