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. 2001 Apr;6(2):126-35.
doi: 10.1379/1466-1268(2001)006<0126:iototm>2.0.co;2.

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein

R I Viner  1 , J S Clegg
Affiliations

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein

R I Viner et al. Cell Stress Chaperones. 2001 Apr.

Abstract

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.

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Figures

Fig. 1.
Fig. 1.
Inactivation kinetics of CS in the presence and absence of p26. Inactivation of CS (75 nM) at 43°C in the presence of 188 nM p26 (0.109 mg/mL, •) or 0.1 mg/mL of BSA (♦) was monitored by measuring CS activity at the times indicated. CS activity is shown as a percentage of enzyme activity before heating, which was set to 100%. Data points are means ± standard errors for n = 5
Fig. 2.
Fig. 2.
Influence of p26 on the thermal aggregation of CS. CS (75 nM) was incubated at 43°C alone or in the presence of p26 (37.5–188 nM) with BSA (0.1 mg/mL) or with trehalose (Tre). Aggregation was measured by light scattering at 500 nm. This experiment is 1 of 4 that produced comparable results
Fig. 3.
Fig. 3.
Detection of p26-CS complexes by SEC. (A) p26 (150 nM) was incubated with 75 nM CS at 43°C for the indicated times, then analyzed on a Toso Haas G 4000 SWXL column as described in the Materials and Methods section. (B) A total of 150 nM p26 and 75 nM CS were incubated alone for 60 minutes at 43°C then analyzed similarly. Proteins were monitored by absorbance at 220 nm. The retention times of protein molecular mass standards (×10−3) are shown above part A. Blue dextran (2000 kDa) indicates the void volume (Vo)
Fig. 4.
Fig. 4.
Effects of p26 and trehalose on the thermal unfolding of CS. CS (75 nM) was incubated at 43°C for 60 minutes alone (lane 2) or in the presence of 188 nM p26 (lanes 3 and 7) or with 188 nM p26 and 0.8 M trehalose (lanes 4, 5, and 8). After heating, samples were immediately separated on 4–10% native gels, blotted to nitrocellulose, and stained with antibody to CS (lanes 1–5) or p26 (lanes 6–9). Lane 1: CS was incubated for 60 minutes at 25°C. Lane 5: CS was incubated at 43°C for 30 minutes in the presence of p26, then 0.8 M trehalose was added and incubation continued another 30 minutes. Stds are molecular mass markers (×10−3 kDa). Each lane for native gels received 3.8 μg of CS and 13.7 μg of p26
Fig. 5.
Fig. 5.
Effects of p26 and trehalose on the protection and reactivation of thermally inactivated CS. CS (75 nM) was first incubated for 15 minutes at 43°C in the presence of 188 nM p26 or with 0.1 mg/mL of BSA, with or without trehalose as shown (open bars) and CS activity determined immediately after heating. This treatment is referred to as protection. Comparable reaction mixtures were then incubated for an additional 60 minutes at 25°C to enable recovery of enzyme activity (filled bars), a treatment we call reactivation. CS activity before the initial heating was assigned a value of 100%, and other data were normalized to this value (bars are means ± standard errors for n = 4)
Fig. 6.
Fig. 6.
Influence of trehalose on the refolding of heat-denaturated CS. CS was either heated alone (lanes 2 and 3) or with 188 nM p26 (lanes 4, 5, 9, and 10) as described in Figure 4 and then incubated for another 60 minutes at 25°C. Proteins were separated on 4–10% native gels, blotted to nitrocellulose, and stained with antibody to CS (lanes 1–5) or p26 (lanes 6–10). Lane 1: CS incubated for 2 hours at 25°C. Lanes 2 and 3: CS after heating was incubated for 60 minutes at 25°C alone (2) or with 0.8 M trehalose (3). Lanes 4, 5, 9, and 10: CS was heated with 188 nM p26 then incubated for 60 minutes at 25°C in the presence (5 and 10) or absence of 0.8 M trehalose (4 and 9). Lane 6: p26 was incubated for 2 hours at 25°C. Lanes 7 and 8: p26 was heated for 60 minutes at 43°C and then incubated for another 60 minutes at 25°C, either alone (7) or with 0.8 M trehalose (8). Stds are protein standards (×10−3 kDa). Each lane received 3.8 μg of CS and 13.7 μg of p26
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