Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity

Abstract

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.

Fig. 1.

Expression and purification of Hsp30C recombinant protein from E coli. Total bacterial protein from E coli BL21(DE3) cells containing either the expression vector (pRSETB) or the vector with a DNA insert containing the entire Hsp30C gene open reading frame (30C) were collected either before or after 4 hours of IPTG addition. Protein was analyzed by SDS-PAGE and visualized by Coommassie blue staining. Total bacterial lysates containing 30C were passed over a nickel affinity column and 30C was purified from residual bacterial contaminants as described in Experimental Procedures. Five μg of purified 30C was loaded onto the gel. The asteriks indicate the location of 30C. Molecular mass markers in kDa are indicated on the left side of the figure

Fig. 2.

Production of polyclonal anti-30C antibody. Recombinant 30C was injected into rabbits to produce a polyclonal anti-30C antibody. The antibody was affinity purified from crude sera using a nickel affinity column and tested for specificity by immunoblot analysis with protein from control (22°C; lane 1, 15 μg) and heat shocked (33°C for 2 hours; lane 2, 5 μg; lane 3, 15 μg) Xenopus A6 tissue culture cells. Molecular mass markers in kDa are indicated on the left side of the figure

Fig. 3.

Effect of thermal stress on the survival of E coli cells overexpressing 30C. E coli BL21 (DE3) cells were transformed with pRSETB expression vector or pRSETB with a DNA insert containing the open reading frame for Hsp30C. Thermotolerance assays were performed as described in Experimental Procedures. Similar results were obtained for 3 trials and the data were expressed as a percentage of the number of colony forming units at t = 0 hours. ▵: BL21(DE3), □: BL21(DE3) + pRSETB, ○: BL21(DE3) + pRSETB/Hsp30C

Fig. 4.

Elution profile of 30C from a Sepharose CL-4B column. (A) 30C heated at 42°C for 50 minutes was applied to a Sepharose CL-4B column maintained at 22°C. Eluant fractions were measured by spectrophotometry at 280 nm. Calibration standards are indicated above the curve: thyroglobin (669 kDa), ferritin (440 kDa), aldolase (158 kDa). Void volume (V_o) was determined using blue dextran. (B) an equal amount of eluant from each fraction was analyzed by immunoblot analysis. The presence of 30C in the different fractions was detected using a polyclonal anti-30C antibody

Fig. 5.

Prevention of heat-induced aggregation of CS by 30C. Effect of various molar quantities of 30C (□, 0.1 μM; ▵, 0.2 μM; ○, 0.5 μM) and IgG (•, 0.5 μM) and BSA (▴, 0.5 μM) on heat-induced aggregation of CS (x, 0.1 μM) at 42°C was determined by means of a light scattering assay. Data are representative of 4–6 trials and were calculated as a percentage of the maximum aggregation of CS after 60 minutes and were expressed as the mean ±SD

Fig. 6.

30C has a minimal effect on the prevention of thermal inactivation of CS. CS was heated at 42°C either alone (X) or with 30C in a 1:1 (○) or 5:1 (•)30C:CS molar ratio as described in Experimental Procedures. The enzymatic activity of CS from each mixture was expressed as a percentage of initial CS activity. The data are representative of 4–6 trials and are shown as the mean ±SD

Fig. 7.

Production of 30C terminal deletion mutants. Recombinant 30C mutants missing either the first 17 amino acids (N-30C) or the last 25 amino acids (C-30C) were expressed and purified as for 30C and described in Experimental Procedures. The 30C, N-30C and C-30C proteins were separated using SDS-PAGE and stained with Commassie blue. Molecular mass markers in kDa are indicated on the vertical axis

Fig. 8.

The carboxyl end of 30C is involved in the prevention of heat-induced CS aggregation. CS was incubated alone (+) or with 30C (○), N-30C (□) or C-30C (▵) at a 5:1 molar ratio (Hsp (0.5 μM): CS (0.1 μM)) at 42°C and assayed by light scattering as outlined in Experimental Procedures. Data were calculated as a percentage of the maximum aggregation of CS after 60 minutes and was expressed as the mean ±SD

Fig. 9.

The carboxyl region of 30C is required for solubility. The extent of heat-induced aggregation of 30C, N-30C and C-30C at 42°C was examined using light scattering assays as detailed in Experimental Procedures. •, 0.8 μM 30C; ○ 300 μM 30C; ▪, 0.8 μM of N-30C; □, 300 μM of N-30C; ▴, 0.8 μM of C-30C; ▵ 300 μM of C-30C

Fig. 10.

30C maintains heat-treated LUC in a soluble state. (A) LUC was incubated alone (+) or with 30C (○), N-30C (□, or C-30C (▵) in a 5:1 molar ratio (Hsp(0.5 μM): LUC(0.1 μM)) at 42°C. Data were calculated as a percentage of the maximum aggregation of LUC after 60 minutes and were expressed as the mean ±SD. (B) Immunoblot analysis of LUC in the total, T, supernatant, S, and pellet, P, fractions of control and heat-treated mixtures from panel A as described in Experimental Procedures

Fig. 11.

30C binds to LUC during heat treatment. (A) LUC was heated at 42°C with 30C in a 5:1 (30C:LUC) molar ratio as described in Experimental Procedures. Size exclusion chromatography was used to characterize the formation of 30C and LUC into large oligomeric complexes. V_o indicates the position of the void volume as determined by blue dextran. ○: non-heat-treated 30C, □: non-heat-treated luciferase, ▴: heat-treated 30C and LUC. (B) 30C is present in each eluant fraction containing LUC. An equal amount from selected eluant fractions obtained from size exclusion chromatography of heat-treated 30C and LUC as indicated above was immunoblotted using an anti-LUC or an anti-30C antibody as described in Experimental Procedures

Fig. 12.

The 30C carboxyl region is necessary for association with heat-treated LUC. LUC was mixed with 30C, N-30C or C-30C in a 5:1 (30C: LUC) molar ratio and heated at 42°C (hs) or kept at 22°C (con) for 30 minutes. Coimmunoprecipitation with anti-30C antibody was performed as described in Experimental Procedures. The presence of LUC (upper panel) and 30C (lower panel) as determined by immunoblot analysis is indicated by arrows A and B, respectively. Location of IgG is indicated by the asterisk.