Phosphorylation of αB-Crystallin Alters Chaperone Function through Loss of Dimeric Substructure

It is shown that phosphorylation of αB-crystallin at Ser-45 results in uncontrolled aggregation, and an innovative tandem mass spectrometry approach is demonstrated how this alteration in behavior stems from disruption of dimeric substructure within the polydisperseαB- Crystallin assembly.

Abstract

Phosphorylation is the most common posttranslational modification of the α-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of αB-crystallin. Here we show that phosphorylation of αB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse αB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of αB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue. Phosphorylation is the most common posttranslational modification of the α-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of αB-crystallin. Here we show that phosphorylation of αB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse αB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of αB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue. The mammalian small heat shock protein (sHSP) 1The abbreviations used are: sHSP, small heat shock protein; MS, mass spectrometry; MS/MS, tandem mass spectrometry; 1P-αB, αB-crystallin with S19D mutation; 2P-αB, αB-crystallin with S19D/S45D double mutation; WT-αB, wild-type αB-crystallin; SEC, size-exclusion chromatography; MALS, multiangle laser light scattering. 1The abbreviations used are: sHSP, small heat shock protein; MS, mass spectrometry; MS/MS, tandem mass spectrometry; 1P-αB, αB-crystallin with S19D mutation; 2P-αB, αB-crystallin with S19D/S45D double mutation; WT-αB, wild-type αB-crystallin; SEC, size-exclusion chromatography; MALS, multiangle laser light scattering. αB-crystallin, although systemically expressed (1Bhat S.P. Nagineni C.N. Biochem. Biophys. Res. Commun. 1989; 158: 319-325Crossref PubMed Scopus (495) Google Scholar), is found primarily in the eye lens where it associates with the closely related αA-crystallin into large hetero-oligomers. While its lenticular function is also structural, both αB- and αA-crystallin and the hetero-oligomer α-crystallin (2Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1744) Google Scholar) have been shown to display molecular chaperone activity in vitro and to arrest the aggregation of the β- and γ-crystallins in the lens (3Horwitz J. Semin. Cell Dev. Biol. 2000; 11: 53-60Crossref PubMed Scopus (174) Google Scholar). Unlike ATP-dependent chaperones, the α-crystallins and other sHSPs are not thought to actively refold non-native proteins but rather to incorporate them into large complexes, thereby preventing their nonspecific aggregation (4Haslbeck M. Buchner J. Arrigo A.-P. Muller W.E.G. Small Stress Proteins. Springer-Verlag New York Inc., New York2002: 37-59Google Scholar, 5Stromer T. Ehrnsperger M. Gaestel M. Buchner J. J. Biol. Chem. 2003; 278: 18015-18021Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 6Friedrich K.L. Giese K.C. Buan N.R. Vierling E. J. Biol. Chem. 2004; 279: 1080-1089Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This property of the α-crystallins is regarded as crucial in the maintenance of lens transparency. As there is no protein turnover in the central part of the lens, the exceptionally long lifetime of lenticular proteins means that the α-crystallins are susceptible to the accumulation of a variety of posttranslational modifications that are thought to disrupt their structure (7van Kleef F.S. De Jong W.W. Hoenders H.J. Nature. 1975; 258: 264-266Crossref PubMed Scopus (124) Google Scholar, 8Lampi K.J. Ma Z. Hanson S.R. Azuma M. Shih M. Shearer T.R. Smith D.L. Smith J.B. David L.L. Exp. Eye Res. 1998; 67: 31-43Crossref PubMed Scopus (230) Google Scholar, 9Ma Z. Hanson S.R. Lampi K.J. David L.L. Smith D.L. Smith J.B. Exp. Eye Res. 1998; 67: 21-30Crossref PubMed Scopus (163) Google Scholar). For αB-crystallin in the lens, the major modifications have been identified as phosphorylation at serine residues 19, 45, and 59 (10Miesbauer L.R. Zhou X. Yang Z. Sun Y. Smith D.L. Smith J.B. J. Biol. Chem. 1994; 269: 12494-12502Abstract Full Text PDF PubMed Google Scholar). Despite phosphorylation being a common feature of αB-crystallin both inside and outside the lens (11Mann E. McDermott M.J. Goldman J. Chiesa R. Spector A. FEBS Lett. 1991; 294: 133-136Crossref PubMed Scopus (31) Google Scholar), the in vivo significance of these modifications and their effect on chaperone-like activity remain unclear (12Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (573) Google Scholar). Phosphorylation of α-crystallin has been reported variously to have no effect on chaperone activity (13Nicholl I.D. Quinlan R.A. EMBO J. 1994; 13: 945-953Crossref PubMed Scopus (396) Google Scholar, 14Carver J.A. Nicholls K.A. Aquilina J.A. Truscott R.J. Exp. Eye Res. 1996; 63: 639-647Crossref PubMed Scopus (67) Google Scholar), to reduce chaperone activity (15Kamei A. Hamaguchi T. Matsuura N. Masuda K. Biol. Pharm. Bull. 2001; 24: 96-99Crossref PubMed Scopus (37) Google Scholar), and to cause a decrease in oligomeric size (16Moroni M. Garland D. Biochim. Biophys. Acta. 2001; 1546: 282-290Crossref PubMed Scopus (11) Google Scholar). Furthermore, it has been shown that in vitro phosphorylation of αB-crystallin compromises its inhibitory activity toward actin polymerization (17Wieske M. Benndorf R. Behlke J. Dolling R. Grelle G. Bielka H. Lutsch G. Eur. J. Biochem. 2001; 268: 2083-2090Crossref PubMed Scopus (68) Google Scholar). The use of site-directed mutagenic substitution of aspartate for serine residues to mimic phosphorylated αB-crystallin led to the report of smaller oligomers with significantly reduced chaperone efficacy (18Ito H. Kamei K. Iwamoto I. Inaguma Y. Nohara D. Kato K. J. Biol. Chem. 2001; 276: 5346-5352Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). αB-Crystallin, like many mammalian sHSPs, is inherently polydisperse, and this has presented many difficulties for structural biologists (12Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (573) Google Scholar). Nanoelectrospray ionization mass spectrometry (19Wilm M.S. Mann M. Int. J. Mass Spectrom. Ion Process. 1994; 136: 167-180Crossref Scopus (872) Google Scholar) is capable of providing unique insight into the subunit composition and dynamics of large noncovalent complexes such as sHSPs (20Sobott F. Benesch J.L.P. Vierling E. Robinson C.V. J. Biol. Chem. 2002; 277: 38921-38929Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 21Benesch J.L.P. Sobott F. Robinson C.V. Anal. Chem. 2003; 75: 2208-2214Crossref PubMed Scopus (138) Google Scholar). Although the homogeneity of many of the non-mammalian sHSPs permits a relatively simple interpretation of subunit stoichiometry by means of this technique, it has not been possible, however, to interpret data from polydisperse proteins such as αB-crystallin and other mammalian sHSPs. The large number of oligomeric species involved leads to a substantial overlap of peaks, and therefore the spectra obtained cannot be interpreted unambiguously. We have recently shown, however, that tandem mass spectrometry (MS/MS), in which discrete packets of ions are isolated and subjected to collisions, is capable of defining the relative populations of different oligomers within the polydisperse assembly of αB-crystallin (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). Here we apply this approach to investigate the effect of phosphorylation upon the quaternary structure of αB-crystallin. We used aspartate mutations of serine residues known to be phosphorylated in vivo (10Miesbauer L.R. Zhou X. Yang Z. Sun Y. Smith D.L. Smith J.B. J. Biol. Chem. 1994; 269: 12494-12502Abstract Full Text PDF PubMed Google Scholar) (S19D (1P-αB) and S19D/S45D (2P-αB)) and compared their properties with wild-type αB-crystallin (WT-αB). We show here that these proteins differ substantially in their ability to reduce protein aggregation and that these differences are directly linked to alterations in their quaternary substructure, as determined by nanoelectrospray ionization mass spectrometry. Because sHSPs, including αB-crystallin, are up-regulated in a number of neurodegenerative diseases, cancers, and desmin-related myopathy (23Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google Scholar), this structure-function relationship has implications not only for the mechanism of the chaperone-like behavior of sHSPs but also for elucidating the reasons for their accumulation associated with such diseases. Preparation of αB-Crystallins—PCR technology was used for generation of human αB-crystallin with serine-to-aspartate mutations at Ser-19 and Ser-45. For generation of the point mutation S19D, DNA of wild-type αB-crystallin was used as a template for the PCR amplification, and the following oligonucleotide primers were used: forward, 5′-TTTCCACGACCCCAGCCGCCTCT-3′, and reverse, 5′-CGGCTGGGGTCGTGGAAAGAAAAGAA-3′. Two restriction sites, at the 5′ NdeI site and at the 3′ XhoI site, were added to the PCR product. The expression vector PET 21b(+) was linearized with NdeI and XhoI and subsequently ligated with the gel-purified PCR product using the same cohesive ends. The ligation product was then transformed into Escherichia coli One Shot cells by standard methods. After amplification of the construct plasmid, DNA was extracted by using the QIAprep Spin system. The DNA was digested with NdeI and XhoI and analyzed by gel electrophoresis. Clones that contained the DNA insert of mutated αB-crystallin were sequenced to confirm the sequence of the insert. A clone that contained the point mutation S19D was used as a template for the PCR amplification to generate the point mutation S45D. The following oligonucleotide primers were used: forward, 5′-TTCCCTGGATCCCTTCTACCTTCGGCCA-3′, and reverse, 5′-AGAAGGGATCCAGGGAAGTAGACGTC-3′. The proteins were expressed and purified as described previously (24Horwitz J. Huang Q.L. Ding L. Bova M.P. Methods Enzymol. 1998; 290: 365-383Crossref PubMed Scopus (194) Google Scholar). Chaperone Assay—The chaperone efficiency of αB-crystallin and the phosphorylation mimics toward reduced α-lactalbumin were assessed by using a modified assay (25Farahbakhsh Z.T. Huang Q.L. Ding L.L. Altenbach C. Steinhoff H.J. Horwitz J. Hubbell W.L. Biochemistry. 1995; 34: 509-516Crossref PubMed Scopus (203) Google Scholar). Briefly, apo-α-lactalbumin (0.5 mg/ml final) was mixed with αB-crystallin and each of the mimics (0.235 mg/ml final) in the presence of 20 mm dithiothreitol. The final sample volumes were 100 μl in 200 mm ammonium acetate. The mixtures, as well as a control of apo-α-lactalbumin, were placed in identical cuvettes in a heated (37 °C) multicell block. Their apparent absorbance at 360 nm was monitored in parallel over a 30-min period in a Cary 400 Scan spectrophotometer. Size-Exclusion Chromatography of αB-Crystallin·Substrate Complexes—Samples were prepared as for the chaperone assay with the following exceptions. 1) After 15 min at 37 °C, the reaction was quenched by placing the mixtures on ice, and 2) the final concentration of αB-crystallin was 0.6 mg/ml. The entire volume of each sample was loaded onto a Superdex 200HR 10/30 size-exclusion column (Amersham Biosciences) and eluted at 0.4 ml/min with 200 mm ammonium acetate at 8 °C. The column was calibrated with Sigma gel filtration markers. Circular Dichroism—Far UV circular dichroism spectra of the proteins were obtained by using a Jasco J-810 spectropolarimeter. Measurements were performed at 25 °C in a cylindrical 0.1 mm path-length cell and represent the sum of 15 accumulations/sample. Protein concentrations were carefully standardized to 1.2 mg/ml in 200 mm ammonium acetate buffer. Size-Exclusion Chromatography and Light Scattering—The native proteins were analyzed by using size-exclusion chromatography with on-line light scattering, absorbance, and refractive index detectors (26Wen J. Arakawa T. Philo J.S. Anal. Biochem. 1996; 240: 155-166Crossref PubMed Scopus (442) Google Scholar). A Superose 6HR 10/30 column (Amersham Biosciences) was connected in-line to a UV detector (Amersham Biosciences UV-900), a DAWN EOS (Wyatt Technology) multiangle laser light-scattering detector, and an Optilab-DSP (Wyatt Technology) refractive index detector. Samples were loaded onto the column at a concentration of 1.5 mg/ml and eluted with 200 mm ammonium acetate, 1 mm dithiothreitol, pH 7.0. Mass Spectrometry Analysis—Approximately 2 mg of the purified protein was buffer-exchanged by loading onto a Superdex 200HR 10/30 size-exclusion column (Amersham Biosciences) and eluting at 0.4 ml/min with 200 mm ammonium acetate at 8 °C. Fractions corresponding to the peak elution volume were pooled and concentrated to 1 mg/ml using a Millipore Biomax centrifugal filtration device. Nanoelectrospray mass spectrometry experiments were performed on a Q-Tof II instrument (Micromass UK Ltd.) that has been modified for high mass operation (27Sobott F. Hernández H. McCammon M.G. Tito M.A. Robinson C.V. Anal. Chem. 2002; 74: 1402-1407Crossref PubMed Scopus (436) Google Scholar). Conditions were carefully chosen to allow the ionization and detection of the αB-crystallins without disrupting the noncovalent interactions that maintain the quaternary structure. To dissociate the oligomers, ions of the selected m/z range were collided with argon atoms in the collision cell of the mass spectrometer. The asymmetric distribution of charge between the products allowed the unambiguous identification of the products. The experimental for the of these and spectra are the same as described previously (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). of Phosphorylation Chaperone investigate chaperone function we of the αB-crystallins with an reduced and monitored their apparent absorbance to light The results demonstrate that and the of aggregation which an in apparent absorbance min both a to that of the control of α-lactalbumin in the of αB-crystallin. By the caused a in apparent absorbance from that of the control at and To investigate the species these we used size-exclusion chromatography to the 15 min of and elution with major and of 25 and min 1 and corresponding to molecular of and 1 is αB-crystallin, which as a peak in the of We 2 to species both chaperone and These are in the elution of in the elution of and The elution for the same for and as well as an peak of molecular mass A eluting min was to These results demonstrate chaperone-like properties for and but the of and chaperone to molecular species in the of these experiments show that there is a in the chaperone behavior of compared with that of and We found that and aggregation by the of large complexes with the 2P-αB, by with the as by a apparent absorbance the control in the chaperone assay This leads to the of complexes of Phosphorylation on the of protein function is related to we have examined these αB-crystallins for structural differences associated with this in chaperone The effect of phosphorylation on structure was examined by using UV circular dichroism proteins at to the of this was toward for and for phosphorylation at serine residues in the of αB-crystallin results in an structure. The effects of these structural on the of αB-crystallin were by using to a multiangle laser light-scattering detector to the oligomeric distribution of the αB-crystallins This molecular mass that is of the interactions between the proteins and protein eluted as a peak at and this peak with The data show that species within the range of with an molecular mass of This mass range was and for and 2P-αB, with both an molecular mass to These results that phosphorylation not the molecular mass of αB-crystallin but rather an in Although an mass of the oligomers in the volume being it is not capable of the different species We have recently however, that a tandem mass spectrometry approach such relative of the oligomers in a polydisperse protein (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). We obtained mass spectra of the αB-crystallin proteins between and with the peak at m/z the most peak in each These spectra however, not directly because of the large number of charge To this we used of the oligomers in the (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). The mechanism of such an approach, as to oligomeric be described as The peak corresponding to of the oligomeric each with and m/z for WT-αB, 1P-αB, and 2P-αB, was isolated and subjected to with argon atoms in the collision cell of the mass (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). the we obtained from such an of from the oligomers that have known as were in the range of the corresponding to mass spectrometry of of the peak in the of and in of the oligomers into at m/z and oligomers at high m/z (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). to be from the oligomers in this the of the to the oligomers, the are to allow identification of the oligomers that the polydisperse assembly of This is To the spectra for the we have a to oligomers from which have been These spectra demonstrate that the are to allow to the species the polydisperse assembly. we also differences between the relative populations of the different corresponding to oligomers of from oligomers of are common to the spectra of the of the in the entire of the to oligomers we the relative of the oligomeric species (22Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (211) Google Scholar). This allowed to the oligomeric size of the αB-crystallins to be and for WT-αB, 1P-αB, and 2P-αB, in with the molecular mass obtained by for The in the spectra and the the that forms oligomers with an number of in to with an this is for and is not for these it is that human αB-crystallin dimeric substructure and that this is by We have examined the effects of serine phosphorylation on the and chaperone-like function of αB-crystallin by aspartate mimics at sites found in has been reported previously that the oligomeric of is significantly reduced upon phosphorylation (18Ito H. Kamei K. Iwamoto I. Inaguma Y. Nohara D. Kato K. J. Biol. Chem. 2001; 276: 5346-5352Abstract Full Text Full Text PDF PubMed Scopus (177) Google however, were between the molecular of the proteins was this that the oligomeric distribution upon a of the phosphorylation was in the spectra a for an number of a also for sHSPs from E. K.L. Slingsby C. Vierling E. Struct. Biol. 2001; PubMed Scopus Google Scholar), M. S. T. Ehrnsperger M. S. Buchner J. EMBO J. PubMed Scopus Google Scholar, M. N. T. N. S. Buchner J. EMBO J. 2004; PubMed Scopus Google Scholar), and R. Nature. 1998; PubMed Scopus Google Scholar), which the of of oligomeric species an number of also the presence of quaternary These that the R. Slingsby C. Vierling E. Protein Chem. 2002; Scopus Google Scholar) of the sHSPs be within αB-crystallin and other mammalian sHSPs. We by the aspartate that phosphorylation at Ser-19 has effect on the chaperone activity distribution of oligomers in the assembly. By by 2P-αB, we show that phosphorylation at Ser-45 in differences in the distribution of oligomers by this protein and a alteration in chaperone-like This be with the sequence to the in the of the sHSPs R. Slingsby C. Vierling E. Protein Chem. 2002; Scopus Google Scholar). Although no structure for αB-crystallin, the related from has been shown to be of α-crystallin into of the are the other have E. K.L. Slingsby C. Vierling E. Struct. Biol. 2001; PubMed Scopus Google Scholar). The α-crystallin interactions a for oligomeric assembly. and by the and the to the E. K.L. Slingsby C. Vierling E. Struct. Biol. 2001; PubMed Scopus Google Scholar, R. Slingsby C. Vierling E. Protein Chem. 2002; Scopus Google Scholar). αB-crystallin interactions to quaternary substructure, the that only of the these the presence of a of oligomers in of the αB-crystallin phosphorylation at Ser-45 in the disruption of by the to a of dimeric with the related structure therefore for the of with of in proteins as well as the in in The for chaperone activity an between a and in which sites (4Haslbeck M. Buchner J. Arrigo A.-P. Muller W.E.G. Small Stress Proteins. Springer-Verlag New York Inc., New York2002: 37-59Google Scholar, R. Slingsby C. Vierling E. Protein Chem. 2002; Scopus Google Scholar). We that the disruption of dimeric caused by phosphorylation this thereby such that phosphorylated αB-crystallin actively with αB-crystallin has been found in patients with degenerative brain disease (11Mann E. McDermott M.J. Goldman J. Chiesa R. Spector A. FEBS Lett. 1991; 294: 133-136Crossref PubMed Scopus (31) Google Scholar), and high of at Ser-45 of αB-crystallin, in human lenses (10Miesbauer L.R. Zhou X. Yang Z. Sun Y. Smith D.L. Smith J.B. J. Biol. Chem. 1994; 269: 12494-12502Abstract Full Text PDF PubMed Google Scholar) to the of The of phosphorylated αB-crystallin in non-lenticular for the most M. J. Int. J. Biol. 1998; PubMed Scopus Google Scholar). that αB-crystallin is up-regulated in a number of neurodegenerative diseases, cancers, and desmin-related myopathy (23Clark J.I. Muchowski P.J. Curr. Opin. Struct. Biol. 2000; 10: 52-59Crossref PubMed Scopus (220) Google Scholar), this structure-function relationship has implications not only for the mechanism of their chaperone-like activity but also for the accumulation of α-crystallins associated with such diseases.