Biochemistry 2007, 46, 9605-9614
9605
Residue R120 Is Essential for the Quaternary Structure and Functional Integrity of Human RB-Crystallin† Ste´phanie Simon,*,‡ Magalie Michiel,§ Fe´riel Skouri-Panet,| Jean Pierre Lechaire,⊥ Patrick Vicart,‡ and Annette Tardieu§ EA 300, UniVersite´ Paris 7, Case 7136, 75251 Paris Cedex 05, France, FRE2852, CNRS-UniVersite´ Paris 6, case 29, 7 quai St Bernard, 75252 Paris Cedex 05, France, UMR7590, CNRS-IPGP-UniVersite´ s Paris 6 et 7, 140 rue de Lourmel, 75015 Paris, France, and SerVice de Microscopie Electronique IFR 83, UniVersite´ Paris 6, case 25, 7 quai St Bernard, 75252 Paris cedex 05, France ReceiVed February 14, 2007; ReVised Manuscript ReceiVed May 22, 2007
ABSTRACT: The missense mutation Arg-120 to Gly (R120G) in the human RΒ-crystallin sequence has been reported to be associated with autosomal dominant myopathy, cardiomyopathy, and cataract. Previous studies of the mutant showed a significant ability to aggregate in cultured cells and an increased oligomeric size coupled to an important loss of the chaperone-like activity in Vitro. The aim of this study was to further analyze the role of the R120 residue in the structural and functional properties of RΒ-crystallin. The following mutants were generated, Arg-120 to Gly (R120G), Cys (R120C), Lys (R120K), and Asp (R120D). In cellulo, after expression in two cultured cell lines, NIH-3T3 and Cos-7, the capacity of the wild-type and mutant crystallins to aggregate was evaluated and the protein location was determined by immunofluorescence. In Vitro, the wild-type and mutant crystallins were expressed in Escherichia coli cells, purified by size exclusion chromatography, and characterized using dynamic light scattering, electron microscopy, and chaperone-like activity assays. Aggregate sizes in cellulo and in Vitro were analyzed. The whole of the data showed that the preservation of an Arg residue at position 120 of RΒ-crystallin is critical for the structural and functional integrity of the protein and that each mutation results in specific changes in both structural and functional characteristics.
The R-crystallins belong to the small heat shock protein (sHSP)1 family (1). The sHSPs are found in most organisms (archea, bacteria, plants, and animals) where they are generally induced in response to a variety of physiological and environmental stress (2, 3). In humans, 10 sHSPs have been identified (4, 5). Whereas RΑ-crystallin (RΑ) is essentially expressed in lens, RΒ-crystallin (RΒ) is also expressed in other tissues (muscle, heart, lung, etc.), where it is often found to be associated with other sHSPs (6, 7). A number of point mutations leading to pathologies have now been identified (8). The most well-known is the mutation of Arg-120 to Gly in RΒ (R120G), which is responsible for an autosomal dominant myopathy associated with cardiomyo† This work was supported by the French Ministry of Research (S.S. and M.M.), by the Association Franc¸ aise contre les Myopathies (AFM) (S.S. and Grant 11764 to P.V.), by University Paris 7 (P.V.), by University Paris 6 (F.S.-P. and A.T.), and by the Centre National de la Recherche Scientifique (CNRS) (F.S.-P. and A.T.). * To whom correspondence should be addressed: EA 300, Universite´ Paris 7, Case 7136, 75251 Paris Cedex 05, France. Phone: 33 1 44 27 47 36. Fax: 33 1 44 27 36 11. E-mail:
[email protected]. ‡ EA 300, Universite´ Paris 7. § FRE2852, CNRS-Universite´ Paris 6. | UMR7590, CNRS-IPGP-Universite ´ s Paris 6 et 7. ⊥ IFR83, Universite´ Paris 6. 1 Abbreviations: WT, wild-type; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ADH, alcohol dehydrogenase; CS, citrate synthase; DLS, dynamic light scattering; RA and RΒ, RΑ- and RΒ-crystallin, respectively; hRΒ-WT, human wild-type RΒ-crystallin; sHSP, small heat shock protein; PBS, phosphate-buffered saline; 3D, three-dimensional.
pathy and cataract (9). The R120 residue seems particularly interesting. The corresponding mutation in RΑ (R116C) causes congenital cataract (10) and in HSP22 (K141E or K141N) is responsible for distal hereditary motor neuropathy and Charcot Marie Tooth disease (11, 12). In all these inherited diseases, the transmission is autosomal dominant. Moreover, the equivalent mutation in HSP27 (R148G) induces accumulation of aggregates in cells (13). The involvement of this conserved residue in inherited human diseases highlights its important role in the structure and function of sHSPs. The sHSP family is characterized by a highly conserved C-terminal domain of ∼90 amino acids, “the R-crystallin domain” (14), and a variable N-terminal domain. A typical feature of sHSPs is the formation of large oligomeric complexes (12-40 subunits of 12-50 kDa). In plants and archae, sHSPs have a fixed number of subunits, whereas in mammals, they are polydisperse. In mammalian lenses, RA and RB associate in a 3/1 ratio to form polydisperse oligomers of ∼40 ( 5 subunits and of 800 kDa (15), whereas RA and RB alone form polydisperse oligomers smaller in size, ∼30 subunits and 600 kDa (16). So far, only three threedimensional (3D) structures of sHSPs are known, one from a hyperthermophile archae Methanoccus jannaschii, HSP16.5, with 24 subunits (17); one from wheat, Triticum aestiVum, HSP16.9, with 12 subunits (18); and one from a parasitic flatworm, Taenia saginata, Tsp36, with two subunits, each subunit containing two R-crystallin domains (19). A fourth
10.1021/bi7003125 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/27/2007
9606 Biochemistry, Vol. 46, No. 33, 2007 one is in progress, from the bacteria Xanthomonas axonopodis pv. citri (20), whereas the multiple assemblies of a fifth one, HSP26 from the yeast Saccharomyces cereVisiae, have been modeled from cryoelectron microscopy, yet at a lower resolution (21). Sequence comparison coupled with structure analysis revealed that the R120 mutation was located on a highly conserved β-strand (19). The R-crystallins share unique physicochemical properties, including the ability to rapidly exchange their subunits while keeping the same average number of subunits (22, 23). Moreover, they can change their size and oligomeric state with the in Vitro environment (pH, temperature, etc.) through subunit exchange (24). Both in ViVo and in Vitro they function as molecular chaperones (9, 25-31), whose main function is to associate, not refold, a variety of stress-denatured proteins and prevent further aggregation, in an ATPindependent process. Subunit exchange might control the chaperone-like activity, often associated with changes in oligomeric state (32). The R-crystallins therefore exhibit structural and functional properties in the living world that seem closely associated with their dynamic structure, yet the process by which a point mutation may be sufficient to modify these properties and lead to pathologies remains to be determined. To address these questions, we constructed and expressed in different systems several position 120 RΒ mutants: R120G, R120C, R120K, and R120D. The in cellulo and in Vitro properties of wild-type (WT) and mutant crystallins were analyzed. After expression in cultured cell lines, the capacity to aggregate of the WT and mutant crystallins was evaluated and the protein location was determined by immunofluorescence. The soluble assemblies were characterized in Vitro by dynamic light scattering, electron microscopy, and chaperone assays. EXPERIMENTAL PROCEDURES Cloning and Site-Directed Mutagenesis. Cloning of the hRΒ-WT and R120G cDNA into the pcDNA3 vector (Invitrogen) has been reported previously for the in cellulo studies (9). The R120C, R120D, and R120K mutants were generated by site-directed mutagenesis. For the in Vitro studies, each mutant DNA was then subcloned into bacterial expression vector pET16b (Novagen); the hRΒ-WT cDNA used for expression in E. coli was kindly provided by W. W. de Jong (33) and was cloned into vector pET24d (Novagen). Detailed cloning information and primers used are given in Table 1. All the recombinant vectors were sequenced by automated sequencing. Cell Culture, Transfection, and Immunofluorescence. Two different cell lines, adherent and easy to transfect, were chosen. The murine NIH-3T3 cells are devoid of sHSP expression in the absence of stress. The monkey Cos-7 cells are devoid of RB expression but express HSP27 constitutively. The cells were grown in DMEM Glutamax medium (Gibco), supplemented with 10% fetal calf serum (FCS, Gibco) and penicillin/streptomycin (Gibco) in a 5% CO2 humidified atmosphere. One day prior to transfection, cultured cells were trypsinized and plated on glass coverslips placed in a culture dish. Transfection with 3 µg of each pcDNA3 recombinant vector was carried out using Fugene 6 (Roche Diagnostics) according to the manufacturer’s
Simon et al. Table 1: Detailed Information about the Used Constructs construct number 1 2 3 4 5 6 7 8 9 10
construct designation
method of cloning/ source of the cDNA
restriction site used primersa
pcDNA3-RB WT described previouslyb KpnI/XbaI pcDNA3-RB R120G described previouslyb KpnI/XbaI pcDNA3-RB R120C site-directed KpnI/XbaI mutagenesis pcDNA3-RB R120D site-directed KpnI/XbaI mutagenesis pcDNA3-RB R120K site-directed KpnI/XbaI mutagenesis c pET24d-RB WT described previously XhoI/EcoRI pET16b-RB R120G subcloning from 2 NcoI/XhoI pET16b-RB R120C subcloning from 3 NcoI/XhoI pET16b-RB R120D subcloning from 4 NcoI/XhoI pET16b-RB R120K subcloning from 5 NcoI/XhoI
a, b, c, d a, b, e, f a, b, g, h -
a a, 5′-aaaaaaggtaccatggacatcgccatccaccac-3′; b, 5′-aaaaaatcta-gactatttcttgggggctgcggt-3′; c, cagggagttccactgc aaataccggatccca; d, 5′tgggatccggtatttgcagtggaactccctg-3′; e, 5′-tccagggagttccacgataaataccggatccc-3′; f, 5′-gggatccggtatttatcgtggaactccctgga-3′; g, 5′-tccagggagttccacaaaaaataccggatccc-3′; h, 5′-gggatccggtattttttgtggaac tccctgg-3′. b From ref 9. c From ref 33.
instructions; 24 or 48 h later, cells were rinsed twice with phosphate-buffered saline (PBS) before being fixed and permeabilized by incubation in a cold methanol/acetone solution (7/3) for 5 min at 4 °C. After several washes in PBS, cells were probed using monoclonal mouse antibody anti-RΒ (1/200, Stressgen) diluted in PBS containing 2% FCS. The primary antibody was then detected using a goat anti-mouse antibody coupled to Alexa Fluor 11031 (1/1000, Molecular Probes) diluted in PBS and 2% FCS. The samples were mounted on coverslips using Mowiol (Sigma). Cells were examined using a fluorescence microscope (LEITZ) and images collected via an ORCA-ER digital camera (Hamamatsu) and processed using Simple PCI 6.0 (Compix Inc. imaging systems). To test the expression level of each transfected construct, cells were lysed 48 h post-transfection in buffer A [125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 400 mM dithiothreitol, and 0.01% bromophenol blue] prior to the analysis by Western blotting using the monoclonal mouse anti-RΒ-crystallin antibody (1/10000, Stressgen). A monoclonal mouse anti-vimentin antibody (1/2000, DakoCytomation) was used to normalize the amount of total protein loaded. Assessment of the Ability of hRB-WT and Mutants To Aggregate in Cell Cultures. The presence or absence of aggregates in transitory transfected cells was scored for, at least, 100 randomly transfected cells for each construct. Each datum is the mean value of three independent experiments ( the standard error. A one-way ANOVA test was applied to compare results between sample groups. Differences between groups were considered statistically significant if the p value was
