LEA proteins prevent protein aggregation due to water stress

Abstract

LEA (late embryogenesis abundant) proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock. However, although various functions of LEA proteins have been proposed, their precise role has not been defined. Recent bioinformatics studies suggest that LEA proteins might behave as molecular chaperones, and the current study was undertaken to test this hypothesis. Recombinant forms of AavLEA1, a group 3 LEA protein from the anhydrobiotic nematode Aphelenchus avenae, and Em, a group 1 LEA protein from wheat, have been subjected to functional analysis. Heat-stress experiments with citrate synthase, which is susceptible to aggregation at high temperatures, suggest that LEA proteins do not behave as classical molecular chaperones, but they do exhibit a protective, synergistic effect in the presence of the so-called chemical chaperone, trehalose. In contrast, both LEA proteins can independently protect citrate synthase from aggregation due to desiccation and freezing, in keeping with a role in water-stress tolerance; similar results were obtained with lactate dehydrogenase. This is the first evidence of anti-aggregation activity of LEA proteins due to water stress. Again, a synergistic effect of LEA and trehalose was observed, which is significant given that non-reducing disaccharides are known to accumulate during dehydration in plants and nematodes. A model is proposed whereby LEA proteins might act as a novel form of molecular chaperone, or 'molecular shield', to help prevent the formation of damaging protein aggregates during water stress.

Figure 1. LEA proteins alone do not prevent heat-induced aggregation of CS

Aggregation kinetics of CS at 43 °C in the presence or absence of LEA proteins and a small heat-shock protein p26 are shown, as determined by light scattering (in arbitrary units ‘au’ on the y-axis) assayed by a fluorescence spectrophotometer. Key to symbols: CS (final concentration 75 nM) alone (●), CS plus group 1 LEA (1 μM, △), CS plus group 3 LEA (1 μM, ▲), CS plus p26 (75 nM, □) or CS plus p26 (150 nM, ■).

Figure 2. Synergistic effect of trehalose and LEA protein on heat-induced aggregation of CS

(A) The aggregation kinetics of CS at 43 °C in the presence or absence of LEA proteins and trehalose were determined as in Figure 1. CS alone (final concentration 75 nM, ●), in the presence of 100 mM (□) or 400 mM trehalose (■), 1 μM group 1 LEA protein and 100 mM trehalose (△) or 1 μM group 3 LEA protein and 100 mM trehalose (▲). (B) Aggregation kinetics of CS (final concentration 75 nM) alone (●), in the presence of 100 mM trehalose (□), 100 mM trehalose and 100 nM group 3 LEA protein (△), 100 mM trehalose and 500 nM group 3 LEA protein (▲) or 100 mM trehalose and 1 μM group 3 LEA protein (■).

Figure 3. Effect of desiccation on CS aggregation and activity

(A) Aggregation and (B) activity of 0.12 mg of CS after desiccation (open bar), in the presence of 0.24 mg of group 1 LEA protein (black bar), 0.24 mg of group 3 LEA protein (grey bar) or 0.24 mg of BSA (dark grey bar). Aggregation is measured by the effect of light scattering giving an apparent A₃₄₀ in the spectrophotometer; enzyme activity is assayed according to standard methods and results are expressed as percentage of control activity. One drying cycle corresponds to vacuum drying (without freezing) for 1 h in a modified tray freeze-dryer followed by immediate rehydration in water to the original volume. Results after two and four drying cycles are shown. *P<0.05 and **P<0.01 shown above the bar represent results significantly different from those for CS alone. Statistical tests were not performed for the four drying cycle data in (A) due to the anomalous aggregation of CS, noted in the text.

Figure 4. A synergistic effect of LEA proteins and trehalose on protection of LDH during drying

Residual activity of LDH after vacuum drying in the presence of group 3 LEA protein (▲), BSA (△), group 3 LEA protein and 100 mM trehalose (□) or BSA and 100 mM trehalose (●); the dotted line refers to the residual activity due to protection by 100 mM trehalose alone. Trehalose shows a synergistic protective effect with LEA protein, but not with BSA, where an additive effect is seen.

Figure 5. Protection of CS by protein protectants from aggregation and inactivation by freezing and thawing

(A) Aggregation and (B) activity of 0.25 mg/ml CS only after freezing in liquid N₂ and thawing at ambient temperature (open bar), in the presence of 0.5 mg/ml group 1 LEA protein (black bar), 0.5 mg/ml group 3 LEA protein (grey bar) or 0.5 mg/ml BSA (dark grey bar). Aggregation is measured by apparent A₃₄₀ as previously; enzyme activity is assayed according to standard methods and results are expressed as percentage of control activity. One freeze–thaw cycle corresponds to snap freezing in liquid N₂ followed immediately by thawing at ambient temperature. Results after two and four cycles are shown. *P<0.05 and **P<0.01 shown above the bar represent results significantly different from those for CS alone.

Figure 6. Protection of LDH by protein protectants from aggregation due to freezing and thawing

LDH aggregation on repeated freezing in liquid N₂ and thawing at ambient temperature is indicated by light scattering at A₃₄₀. Results are shown for 12.5 μM LDH alone (open bar), for LDH in the presence of 25 μM group 1 LEA protein (black bar), for LDH in the presence of 25 μM group 3 LEA protein (grey bar) and for LDH together with 25 μM BSA (dark grey bar). *P<0.05 and **P<0.01 shown above the bar represent results significantly different from those for LDH alone.

Figure 7. Effect of different molar ratios of LEA proteins and BSA on CS aggregation and activity after desiccation

(A) Aggregation and (B) activity of CS, measured as previously, after two cycles of desiccation in the presence of varying concentrations of group 1 LEA protein (■), group 3 LEA protein (●) or BSA (▲). Although BSA is not able to prevent CS aggregation due to drying, even at a 10:1 molar ratio, the LEA proteins partially or completely abolish aggregation at 5:1 and 10:1 molar ratios respectively.