MAY/JUNE 1999 Volume 10, Number 3 © Copyright 1999 by the American Chemical Society
COMMUNICATIONS Molecular Chaperone-Like Activity of Hydrogel Nanoparticles of Hydrophobized Pullulan: Thermal Stabilization with Refolding of Carbonic Anhydrase B Kazunari Akiyoshi,* Yoshihiro Sasaki, and Junzo Sunamoto* Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan. Received October 26, 1998
We have been studying the formation of hydrogel nanoparticles by the self-aggregation of hydrophobized polysaccharide and the effective complexation between these nanoparticles as a host and various globular soluble proteins as a guest. This paper describes a new finding that refolding of the heatdenatured enzyme effectively occurs with the nanoparticles and β-cyclodextrin according to a mechanism similar to that of a molecular chaperone. In particular, the irreversible aggregation of carbonic anhydrase B (CAB) upon heating was completely prevented by complexation between the heat-denatured enzyme and hydrogel nanoparticles formed by the self-aggregation of cholesteryl groupbearing pullulan (CHP). The complexed CAB was released by dissociation of the self-aggregate upon the addition of β-cyclodextrin. The released CAB refolded to the native form, and almost 100% recovery of the activity was achieved. The thermal stability of CAB was drastically improved by capture of the unfolded form which was then released to undergo refolding.
Most proteins irreversibly aggregate upon heating. Molecular chaperones selectively trap heat-denatured proteins (1-3) or their intermediates in refolding (4, 5) to prevent their irreversible aggregation. The host chaperone releases the protein in a refolded form through a change in the conformation of the complex with the aid of ATP and another co-chaperone. Chaperonins, which have multiple subunits and a cage-like structure, are a well-investigated family of molecular chaperones (6). For example, GroEL acts as a host in macromolecular selfassembly by enclosing a folding intermediate protein as a guest in its central hydrophobic cavity (6). This is an * To whom correspondence should be addressed.
interesting example of host-guest chemistry in a macromolecular system. Surfactants (7, 8) and cyclodextrins (9) are known to prevent the aggregation of the partly intermediate protein that is formed in the refolding process and in heat denaturation. Gellman et al. demonstrated an interesting system for protein refolding via the sequential use of low molecular weight surfactants and cyclodextrins as a stripping agent (10). In their system, however, it is sometimes difficult to completely remove the surfactant after treatment. Water-soluble polymers such as poly(ethylene oxide) (11) and polyamino acids (12) also prevent protein aggregation, although their efficiency is low. The amphiphilic nature of proteins, especially their
10.1021/bc9801272 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/10/1999
322 Bioconjugate Chem., Vol. 10, No. 3, 1999
hydrophobicity, is important in the complexation between a chaperone and denatured protein. To mimic or simulate the function of molecular chaperones, we newly designed an amphiphilic hydrogel nanoparticle that behaves as a host for a guest protein. Hydrophobized polysaccharides such as cholesteryl group-bearing pullulan (CHP) spontaneously form hydrogel nanoparticles in water by intraand/or intermolecular self-aggregation (13, 14). Furthermore, these hydrogel nanoparticles spontaneously complex with various soluble proteins or enzymes in water (15-17). In these cases, we have observed several different types of complexation, as follows. Type I: Enzymes such as R-chymotrypsin or horseradish peroxidase are complexed by polysaccharide nanoparticles at room temperature or physiological temperature and lose their enzyme activity in the complex (15). Type II: Enzymes are complexed in the native form and retain their activity even in the complex, for example, lipase (unpublished result). In both cases, the complexed proteins gain substantial thermal stabilization due to fixation of the proteins or enzymes in the hydrogel matrix of the nanoparticles. In this work, we newly report another interesting type of complexation: Type III. In this case, carbonic anhydrase B (CAB) was complexed quantitatively by the nanoparticles in a heat-denatured form above its denaturation temperature (55 °C). Another interesting and important finding is that complexed CAB was effectively (almost quantitatively) released in its refolded native form in the presence of β-cyclodextrin. This is similar to the two-step mechanism of a molecular chaperone, that is, capture of a heat-denatured protein and release of the refolded protein. In addition, this system provided effective thermal stabilization of CAB. In this paper, we describe this unique behavior of hydrogel nanoparticles of CHP self-aggregate, which is different from those previously reported for the immobilization and stabilization of enzymes or proteins in an ordinary polymer matrix. The preparation and characterization of CHP nanoparticles have been reported elsewhere (13, 14). Cholesteryl group-bearing pullulan (CHP-108-0.9, where the molecular weight of the parent pullulan was 108 000 and the degree of substitution of the cholesteryl moiety per 100 glucose units was 0.9) was used in this study. The average hydrodynamic radius (RH) of the nanoparticles was approximately 18 nm by dynamic light scattering. The apparent molecular weight (8.2 × 105) and the aggregation number of the self-aggregate (approximately 7) were determined by the static light-scattering method. The root-mean-square radius of gyration (RG) was 15.1 nm. The association of cholesteryl moieties provides noncovalently cross-linked points in hydrogel nanoparticles (14). The heat-induced aggregation of CAB (molecular weight 30 000, 0.06 mg/mL, 2 µM) was investigated by the lightscattering method at 400 nm in the absence and presence of CHP. When CAB was heated above its denaturation temperature (55 °C) to 63 °C, aggregation started within 5 min (Figure 1). The hydrophobic portions of globular protein are exposed upon partial unfolding of the protein under heating. This results in aggregation of the protein. This aggregation of CAB was significantly prevented in the presence of CHP nanoparticles. When the molar ratio of CHP nanoparticles to CAB was above 0.2, the solution was optically clear even after heating at 63 °C for 40 min. In contrast, the parent pullulan did not show the same behavior even at a rather high concentration of pullulan. Thus, CHP nanoparticles effectively prevented the ag-
Akiyoshi et al.
Figure 1. Self-aggregation of CAB in the presence of CHP nanoparticles and pullulan at 63 °C: (a) free CAB (0.06 mg/ mL, 2 µM). (b) Pullulan (molecular weight 108 000), 0.15 mg/ mL. (c) CHP nanoparticles (0.082 mg/mL, 0.1 µM as CHP nanoparticles concentration): CAB (0.06 mg/mL, 2 µM) ) 0.05:1 (as a molar ratio). (d) CHP nanoparticles: CAB ) 0.1:1. (e) CHP nanoparticles: CAB ) 0.2:1; and (f) CHP nanoparticles, CAB ) 1:1.
gregation and sedimentation of heat-denatured CAB. This effect is similar to the behavior of a molecular chaperone in nature. A mixture of the CHP nanoparticles (2 µM, as a molar per nanoparticle) and CAB (2 µM) was incubated at 70 °C for 10 min and then cooled to 25 °C. Quantitative complexation between CAB and the CHP nanoparticles was confirmed by high-performance size-exclusion chromatography (HPSEC, Superdex 200HR column, Pharmacia, UV detection at 280 nm) (data not shown). No spontaneous dissociation of CAB was observed even after storage for a week at room temperature. The CHP nanoparticles did not complex with native CAB at 25 °C even after incubation for 32 h. The secondary structure of native CAB consists of 6.6% R-helical and 30.9% β-forms (17). With the CHP-CAB complex, the proportions of the helical and β-forms were computationally estimated to be 24.6% and 12.0%, respectively, on the basis of the CD spectrum. The heat denaturation and subsequent complexation of CAB with CHP nanoparticles induced a large conformational change in its secondary structure. Goto and co-workers previously pointed out the possibility that the R-helical intermediate of denatured protein is a nonhierarchical intermediate of β-sheet proteins such as CAB (18). CHP nanoparticles may capture the heat-denatured intermediate of CAB. The change in the mean residual ellipticity of CAB was investigated in the presence of CHP as a function of temperature at 222 nm. The mixture of CAB and CHP nanoparticles was gradually heated from 25 to 70 °C at 0.17 °C/min and then cooled to 25 °C at the same rate (Figure 2). No significant change was observed in the secondary structure of CAB upon heating to 40 °C. At around 50 °C, however, the ellipticity showed a large sigmoidal decrease. This temperature coincides with the denaturation temperature of CAB. CHP nanoparticles themselves do not show any structural change even after heating to 70 °C. The nanoparticles selectively and effectively complex only with heat-denatured CAB. The increased hydrophobicity of the denatured unfolded protein may prefer complexation with amphiphilic hydrogel nanoparticles compared to the completely folded native protein. The dynamics of a chaperone function are controlled by suitable effectors (6). ATP interacts with the chaperone-protein complex and induces a conformational change in the chaperone to release the bound protein, accompanied by complete refolding, and the original protein
Communications
Figure 2. Mean residual ellipticity (θ) at 222 nm as a function of temperature in 50 mM Tris-sulfate (pH 7.5). A solution mixture of CAB and CHP nanoparticles was heated from 25 to 70 °C at 10 °C/h and then cooled to 25 °C: [CHP nanoparticles] ) [CAB] ) 15 µM.
Bioconjugate Chem., Vol. 10, No. 3, 1999 323
Figure 4. Stability of CAB in the presence of CHP nanoparticles at various temperatures. The mixture of CAB (2 µM) and CHP nanoparticles (2 µM) was treated for 10 min at various temperatures. The p-nitrophenyl acetate hydrolysis by CAB was monitored at 25 °C in 50 mM Tris-sulfate buffer, pH 7.5, after the addition of β-cyclodextrin.
Figure 3. Recovery of enzyme activity of complexed CAB in the prescence of β-cyclodextrin (3 mM) at 25 °C in 50 mM Trissulfate buffer (pH 7.5).
activity is fully recovered. CHP self-aggregates undergo dissociation with the addition of β-cyclodextrin (19). The cross-linking domains provided by the hydrophobic cholesteryl groups are destructed upon encapsulation of the cholesteryl moiety by the β-cyclodextrin cavity. Destruction of the nanoparticles subsequently induces the release of complexed CAB. To the CHP-CAB complex, which was prepared by mixing the CHP nanoparticles (2 µM) and CAB (2 µM) at 70 °C for 10 min, was added β-cyclodextrin (3 mM), and the mixture was kept for 24 h at 25 °C. HPSEC analysis of the reaction mixture showed a complete release of free CAB (data not shown) from the complex under these conditions. Figure 3 shows the recovery of the enzyme activity of CAB with the addition of β-cyclodextrin as a function of incubation time.1 Complexed CAB (2 µM) did not show any enzyme activity in the absence of β-cyclodextrin. However, with the addition of β-cyclodextrin (3 mM) to the complex (CHP, 2 µM), 90% of the enzyme activity recovered after 6 h, and recovery was almost 100% after 24 h. The mixture of CAB and CHP nanoparticles was heated for 10 min at various temperatures and then cooled to 25 °C. The enzyme activity that remained after the addition 1 An aliquot containing CAB was heated at 70 °C for 10 min in 50 mM Tris, pH 7.5, and then chilled to room temperature for 10 min. A dry acetonitrile solution (0.2 mL) of 10 mM p-nitrophenylacetate (pNPA) was added to 1.8 mL of the sample solution (CAB, 1.8 µM in 50 mM Tris-sulfate, pH 7.5). After 20 s of mixing, an increase in the p-nitrophenolate concentration was monitored by the absorbance at 400 nm as a function of time. The percent recovery of enzyme activity was calculated on the basis of the initial velocity of the activity of native CAB (20).
Figure 5. Schematic representation of controlled association between CHP nanoparticles and CAB.
of β-cyclodextrin was investigated (Figure 4). The irreversible heat denaturation of CAB was effectively prevented by complexation with CHP nanoparticles even at higher temperatures. As a result, the thermal stability of CAB was drastically enhanced in this system. In conclusion, in this study, we mimicked the complex function of a molecular chaperone using a relatively simple system consisting of CHP self-aggregate nanoparticles and β-cyclodextrin. Figure 5 shows a schematic two-step mechanism, that is, the capture of heatdenatured unfolded protein and the release of the refolded form. Through this processes, the enzyme gains significant thermal stabilization. This is a novel method for the thermal stabilization of enzymes using the wellcontrolled association with a simple macromolecule. Protein refolding in urea or GuHCl-induced denaturation system is under investigation using a similar hydrophobized polysaccharide system. ACKNOWLEDGMENT
This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. Y.S. gratefully acknowledges a predoctoral fellowship from the Japan Society for the Promotion of Science. LITERATURE CITED (1) Ho¨ll Neugebauer, B., Rudolph, R., Schmidt, M., and Buchner, J. (1991) Reconstitution of a heat shock effect in vitro:
324 Bioconjugate Chem., Vol. 10, No. 3, 1999 influence of GroE on the thermal aggregation of R-glucosidase from yeast. Biochemistry 30, 11609-11614. (2) Taguchi, H., and Yoshida, M. (1993) Chaperonin from Thermus thermophilus can protect several enzymes from irreversible heat denaturation by capturing denaturation intermediate. J. Biol. Chem. 268, 5371-5375. (3) Kawata, Y., Nosaka, K., Hongo, K., Mizobata, T., and Nagai, J. (1994) Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation. FEBS Lett. 345, 229-232. (4) Weissman, J. S., Hohl, C. M., Kovalenko, O., Kashi, Y., Chen, S., Braig, K., Saibil, H. R., Fenton, W. A., and Horwich, A. L. (1995) Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES. Cell 83, 577-587. (5) Mayhew, M., da Silva, A. C., Martin, J., Erdjument Bromage, H., Tempst, P., and Hartl, F. U. (1996) Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature 379, 420-426. (6) Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B. (1994) The crystal structure of the bacterial chaperonin GroEL at 2.8 A [see comments]. Nature 371, 578-586. (7) Tandon, S., and Horowitz, P. M. (1986) Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of lauryl maltoside. J. Biol. Chem. 261, 15615-15681. (8) Tandon, S., and Horowitz, P. M. (1987) Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent. J. Biol. Chem. 262, 4486-4491. (9) Karuppiah, N., and Sharma, A. (1995) Cyclodextrins as protein folding aids. Biochem. Biophys. Res. Commun. 211, 60-66. (10) Rozema, D., and Gellman, S. H. (1995) Artificial chaperones: Protein refolding via sequential use of detergent and cyclodextrin. J. Am. Chem. Soc. 117, 2373-2374; (1996) Artificial chaperone-assisted refolding of carbonic anhydrase B. J. Biol. Chem. 271, 3478-3487. (11) Cleland, J. L., Hedgepeth, C., and Wang, D. I. (1992) Polyethylene glycol enhanced refolding of bovine carbonic
Akiyoshi et al. anhydrase B. Reaction stoichiometry and refolding model. J. Biol. Chem. 267, 13327-13334. (12) Cosolvent effects on refolding and aggregation. Cleland, J. L., and Wang, D. I. (1993) in Biocatalist design for stability and specificity, ACS Symposium Series, pp 151-166. (13) Akiyoshi, K., and Sunamoto, J. (1996) Supramolecular assemblies of hydrophobized polysaccharide. Supramol. Sci. 3, 157-163. (14) Akiyoshi, K., Deguchi, S., Tajima, H., NIshikawa, T., and Sunamoto, J. (1997) Microscopic structure and thermoresponsiveness of a hydrogel nanoparticle by self-assembly of a hydrophobized polysaccharide. Macromolecules 30, 857861. (15) Nishikawa, T., Akiyoshi, K., and Sunamoto, J. (1994) Supramolecular assembly between nanoparticles of hydrophobized polysaccharide and soluble protein complexation between the self-aggregate of cholesterol-bearing pullulan and alpha-chymotrypsin. Macromolecules 27, 7654-7659. (16) Nishikawa, T., Akiyoshi, K., and Sunamoto, J. (1996) Macromolecular complexation between bovine serum albumin and the self-assembled hydrogel nanoparticle of hydrophobized polysaccharides. J. Am. Chem. Soc. 118, 6110-6115. (17) Akiyoshi, K., Nishikawa, T., Shichibe, S., and Sunamoto, J. (1995) Stablization of insulin upon supramolecular complexation with hydrophobized polysaccharide nanoparticle. Chem. Lett., 707-708. (18) Shiraki, K., Nishikawa, K., and Goto, Y. (1995) Trifluoroethanol-induced stabilization of the alpha-helical structure of beta-lactoglobulin: implication for non-hierarchical protein folding. J. Mol. Biol 245, 180-194. (19) Akiyoshi, K., Sasaki, Y., Kuroda, K., and Sunamoto, J. (1998) Controlled association of hydrophobized polysaccharide by cyclodextrin. Chem. Lett., 93-94. (20) Pocker, Y., and Guilbert, L. J. (1972) Catalytic versatility of erythrocyte carbonic anhydrase. Kinetic studies of the enzyme-catalyzed hydrolysis of methylpyridyl carbonates. Biochemistry 11, 180-190.
BC9801272
