Partially folded aggregation intermediates of human gammaD-, gammaC-, and gammaS-crystallin are recognized and bound by human alphaB-crystallin chaperone

Abstract

Human gamma-crystallins are long-lived, unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone alpha-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins have made it difficult to identify the conformation of the human gamma-crystallin substrate species recognized by human alpha-crystallin. The three major human lens monomeric gamma-crystallins, gammaD, gammaC, and gammaS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human alphaB-crystallin chaperone during refolding suppressed the aggregation pathways of the three human gamma-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The alphaB-crystallin oligomers formed long-lived stable complexes with their gammaD-crystallin substrates. Using alpha-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone-substrate complex. Binding of substrate gamma-crystallins with two or three of the four buried tryptophans replaced by phenylalanines showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with alpha-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood but becoming saturated with advancing age.

Fig. 1

Purification and structural characterization of recombinant Human αB-Crys. Human WT αB-Crys was expressed and purified from E. coli cells (see Experimental Procedures). A) Representative size-exclusion chromatogram showing the final, polishing step of the purification procedure. WT αB-Crys eluted as a single peak (from 11 to 14.5 ml) with apparent molecular weight between 500-600 kDa. B) Fractions corresponding to the αB-Crys elution peak were run in a 14% SDS-PAGE gel. C) Far-UV CD spectra (198-250 nm) of native αB-Crys at pH 7.0 and 37°C. D) Fluorescence emission spectra of native () and unfolded (●) αB-Crys at 37°C. Protein concentration for far-UV CD and fluorescence emission measurements was 100 μg/ml.

Fig. 2

Suppression of the aggregation of partially folded WT γD-, γS- and γC-crystallins by αB-Crys chaperone. A) γD-Crys, B) γS-Crys, and C) γC-Crys aggregate during refolding out of high concentrations of GdnHCl at a protein concentration of 100 μg/ml and a final GdnHCl of 0.5 M. The suppression reactions were done at 1 γ:1 αB and 1 γ:5 αB ratios.

Fig. 3

Size-exclusion chromatograms for αB-Crys and γD-, γC-, or γS-Crys suppression reactions. A) 1 γD:5 αB suppression reaction () and 1 γD:5 αB mixture of native proteins (—) chromatograms are shown. B) 1 γC:5 αB suppression reaction () and 1 γC:5 αB mixture of native proteins (—) chromatograms are shown. C) 1 γS:5 αB suppression reaction () and 1 γS:5 αB mixture of native proteins (—) chromatograms are shown. The suppression reactions shown in Fig. 2 were applied to a Superose 6 size-exclusion column (for the native mixture samples’ preparation see Materials and Methods) Arrows correspond to elution volume of standards. Numbers represent standards’ molecular weights in kDa.

Fig. 4

Aggregation of partially unfolded γD-Crys during refolding. 1 γD:0 αB reactions for γD unfolded at GdnHCl concentrations encompassing the intermediate to unfolded protein transition. γD was unfolded at 2.49, 2.82, 3.02, or 5.03 M GdnHCl for 24 h at 37°C to ensure equilibrium conditions. Protein was refolded by diluting the GdnHCl concentration to 0.5 M at 37°C. The final concentration protein concentration was 50 μg/ml for all samples.

Fig. 5

Tryptophan fluorescence of γD-Crys in complex with αB-Crys chaperone. Tryptophan residues at positions 9 and 60 in αB-Crys were substituted to phenylalanines. A) Fluorescence emission spectra for native WT αB-Crys (●) and W9F/W60F αB-Crys () at 25°C. λ_EX was set to 300 nm. B) W9F/W60F αB-Crys suppressed the aggregation of γD-Crys under the same refolding and aggregation described previously (see Materials and Methods). Final γD-Crys concentration was 100 μg/ml and final GdnHCl concentration was 0.5 M at 37°C. C) A high molecular weight complex could be isolated from the 1γD:5 noTrp αB suppression reactions during SEC. The volume highlighted by the gray bar corresponds to the fraction collected for fluorescence measurements. D) Relative fluorescence emission spectrum of the fraction containing the γD—αB complex (). For comparison purposes, the fluorescence emission spectrum of native (○) and fully unfolded (●) WT γD-Crys (20 μg/ml) are shown. The λ_EX was set to 300 nm for all samples. Traces were normalized to the fluorescence intensity of the maximum λ_EM for unfolded γD-Crys.

Fig. 6

Tryptophan fluorescence spectra from double-Trp γD-Crys mutants in complex with αB-Crys chaperone. A) Crystal structure of γD-Crys (PDB: 1HK0) showing Trp pairs conserved within the N-terminal domain (W130F/W156F γD-Crys) or the C-terminal domain (W42F/W68F γD-Crys). B) Relative fluorescence emission spectra of W130F/W156F γD-Crys unfolded at different concentrations of GdnHCl. W130F/W156F γD-Crys (20 μg/ml) was incubated in Refolding buffer with 0, 1.6, 2.0, 2.6 and 5.1 M GdnHCl for 24 h at 37°C. Fluorescence emission spectra were recorded using a λ_EX set to 300 nm. All traces were normalized to the fluorescence intensity of the max. λ_EM for W130F/W156F γD-Crys unfolded at 5.1 M GdnHCl. C) Relative fluorescence emission spectra of W42F/W68F γD-Crys unfolded at different concentrations of GdnHCl. W42F/W68F γD-Crys (20 μg/ml) was incubated in Refolding buffer with 0, 2.3, 2.6, 3.1 and 5.2 M GdnHCl for 24 h at 37°C. Fluorescence emission spectra were recorded using an λ_EX set to 300 nm. All traces were normalized to the fluorescence intensity of the max. λ_EM for W42F/W68F γD-Crys unfolded at 5.2 M GdnHCl. D) SEC chromatograms for 1 γD:5 noTrp αB suppression reactions using W130F/W156F () or W42F/W68F () γD-Crys as substrates. The shaded area on the trace indicates the fraction used for fluorescence measurements. E) Relative fluorescence emission spectrum of the fraction containing the γD—αB complex from 1 W130F/W156F γD: 5 noTrp αB suppression reaction (). For comparison purposes, the fluorescence emission spectrum of native (○) and fully unfolded (●) W130F/W156F γD-Crys (50 μg/ml) are shown. The λ_EX was set to 300 nm for all samples. Traces were normalized to the fluorescence intensity of the maximum λ_EM for unfolded γD-Crys. F) Relative fluorescence emission spectrum of the fraction containing the γD—αB complex isolated from 1 W42F/W68F γD: 5 noTrp αB suppression reaction (). The λ_EX was set to 300 nm for all samples and the protein concentration for the native (○) and fully unfolded (●) W42F/W68F γD-Crys protein samples was 50 μg/ml. Traces were normalized as described above.

Fig. 7

Fluorescence emission spectra of triple-Trp γD-Crys mutants in complex with αB-Crys. A) Relative fluorescence emission spectra for W42only γD-Crys in complex with noTrp αB-Crys (). B) Fluorescence emission spectra for W130only γD-Crys in complex with noTrp αB-Crys (). Spectra for their respective native (○) and unfolded (●) controls is also shown (protein concentration for W42only γD-Crys was 50 μg/ml and for W130only γD-Crys was 25 μg/ml). The λ_EX was 300 nm and the traces were normalized as described previously in Figure 6. Complexes were isolated from 1γD:5αB suppression reactions in which the final concentration of γD was 50 μg/ml.

Fig. 8

SEC chromatograms of αB—γD complexes at different time points after suppression reaction. SEC chromatograms of 0.5 ml aliquots removed from a 1 γD:5 αB suppression reaction incubated at 37°C with continuous agitation. Time points at which aliquots were collected are shown on the chromatogram. The peak that eluted at 7.6 ml is the designated high molecular weight complex and eluted in the void of the column (V₀= 8 ml). The peak that eluted at 13.5 ml corresponded to wild-type αB-Crys. Inset shows the high molecular weight peak that eluted at 7.6 ml in detail.

Fig. 9

Addition of αB-Crys at very early time points after initiation of refolding and aggregation of partially folded WT γD-Crys. A) O.D. _350nm data for no addition of αB-Crys (1 γD:0 αB ratio) and addition 0, 2, 6, and 10 sec after WT γD-Crys was added to Refolding buffer (1 γD:5 αB ratio). Buffer was constantly stirred with a magnetic spin bar and γD-Crys was added 60 sec after light scattering data collection was initiated. Samples were allowed to mix for the first 30 sec after γD-Crys addition. B) Light scattering data presented in (A) showing traces from 50-100 sec after recordings were initiated. C) SEC traces for suppression reactions shown in (A). Samples were loaded onto a Superose 6 SEC column. The fraction corresponding to the γD—αB complex was collected for fluorescence emission measurements (gray bar). D) Raw fluorescence emission spectra for fractions highlighted in (C) and containing the γD—αB complex formed during addition of αB-Crys 0, 2, 6, and 10 sec after addition and initiation of γD-Crys refolding and aggregation. λ_EX was set to 300 nm.

Fig. 10

A molecular model for age-related cataract formation in the lens. Environmental stresses could lead to covalent damage, such as deamidation or photo-oxidation, to the crystallins. This damage could destabilize γ-crystallins, which are otherwise very stable, populating aggregation-prone species (I*). α-crystallin would sequester such species in younger individuals (green arrow). But as we age, the finite levels of free α-crystallin in the mature lens fibers will be diminished leading to aggregation (orange arrows).