Molecular Vision 2003; 9:315-322 <http://www.molvis.org/molvis/v9/a44/> Received 12 February 2003 | Accepted 5 July 2003 | Published 7 July 2003

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Characterization of αA-crystallin from high molecular weight aggregates in the normal human lens

Noriko Fujii,¹ Minori Awakura,¹ Larry Takemoto,² Mitsushi Inomata,³ Takumi Takata,¹ Norihiko Fujii,¹ Takeshi Saito¹
 
 

¹Research Reactor Institute, Kyoto University, Osaka, Japan; ²Division of Biology, Kansas State University, Manhattan, KS; ³Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Correspondence to: Noriko Fujii, Ph.D., Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan; Phone: +81-724-51-2496; FAX: +81-724-51-2630; email: nfujii@hl.rri.kyoto-u.ac.jp

Abstract

Purpose: Lens α-crystallins, composed of two subunits of αA- and αB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the α-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare αA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization.

Methods: HMW and LMW αA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in αA-crystallin from HMW and LMW fractions.

Results: The chaperone activity of HMW αA-crystallin was lower than that of LMW αA-crystallin. Fluorescence spectra indicated that HMW αA-crystallin was more hydrophobic than that of LMW. Isomerization of normal α-linkage to abnormal β-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW αA-crystallin compared with that of LMW αA-crystallin. The D/L ratio of β-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW αA-crystallin was significantly lower than that of the LMW form.

Conclusions: These results were qualitatively the same as those previously found in αA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of αA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.

Introduction

α-Crystallin, a major protein of the ocular lens, is thought to play a role in maintaining lens transparency. In its native state, α-crystallin is a large, water-soluble low molecular weight aggregate (LMW) with a molecular weight of approximately 800 kDa comprised of two kinds of polypeptides, αA- and αB-crystallins, containing 173 and 175 amino acid residues, respectively. Since the αA and αB monomers are approximately 20 kDa, the aggregate molecules contain approximately 40 subunits. As aggregation of α-crystallin progresses, it becomes part of high molecular weight (HMW) aggregates (more than 1,000 kDa) and insoluble proteins. In aged and cataractous lenses, a large amount of HMW, aggregated α-crystallin is observed. The mechanisms of aggregation and insolublization of α-crystallin are not known, but may be related to post-translational modifications such as deamidation [1-3], racemization and isomerization [4-13], truncation [2,9,14-16], phosphorylation [2,17], oxidation [2,18-20], an increase in intramolecular disulfide bonding [2,21], and glycation [22]. Racemization and isomerization of amino acids in proteins can cause major changes in their structure, since different side chain orientations can induce an abnormal peptide backbone. Truncation of α-crystallin subunits, an increase of intramolecular disulfide bonding, and advanced glycation end products (AGEs) also may perturb the normal close-packing structure of crystallins. Therefore, these post-translational modifications can induce a partial unfolding of α-crystallin aggregates, resulting in a reduction of chaperone-like activity, followed by the eventual formation of cataracts. Previous studies indicated that oxidation and isomerization of αA-crystallin decrease its chaperone activity [23,24]. In the present study we characterized the LMW and HMW forms of αA-crystallin in order to better understand the effects of posttranslational modifications such as truncation, isomerization, and racemization of select amino acids upon the aggregation behavior and chaperone activity of the molecule.

Methods

Purification of HMW and LMW αA-crystallin from normal lenses of human donors

Normal human lenses from 50 year old and approximately 2 year old donors (3-4 in each group) were obtained from the National Disease Research Interchange (Philadelphia, PA). HMW and LMW forms of α-A crystallin were prepared as previously described [3]. The lenses were homogenized in phosphate buffer (0.06 M sodium phosphate, 0.1 M sodium sulfate, pH 7.0) at 4 °C. The homogenate was centrifuged at 16,000x g for 15 min at 4 °C, and the supernatant was applied to a Biosep-SEC-S 4000 gel filtration column (7.8 mm x 300 mm, Phenomenex, Torracance, CA). Fractions corresponding to the HMW and LMW forms of α-A crystallin were collected, followed by reverse phase chromatography of each fraction to purify α-A crystallin [3]. The βL-crystallin fraction from human lenses was prepared using a Sephacryl S 300 HR gel-filtration column (26 mm x 600 mm, Pharmacia, Tokyo, Japan), which was equilibrated with 0.1 M Na₂SO₄, 0.1 M KH₂PO₄ phosphate buffer (pH 7.5). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, CA) with bovine serum albumin as a standard.

Chaperone activity assay for heat-induced aggregation of βL-crystallin

A chaperone activity assay was used to measure the ability of α-crystallin to protect βL-crystallin against heat-induced aggregation. βL-Crystallin (200 μg) in 0.1 M KH₂PO₄ buffer (pH 7.5) with or without 20 or 40 μg of αA-crystallin was incubated at 60 °C. The final volume of each reaction mixture was 0.7 ml [βL-crystallin/αA-crystallin=10/1 or 5/1 (w/w)]. Aggregation of βL-crystallin was monitored by absorption due to light scattering at a wavelength of 360 nm. The chaperone activities of α-A crystallin from HMW and LMW fractions from old donors (50 year old) and the LMW fraction from a young donor (2 year old) were compared.

Fluorescence measurement

Fluorescence was measured with a F-4500 Hitachi fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Bis-ANS (4,4'dianilino-1,1'-binaphthalene-5,5'disulfonic acid, Molecular Probes, Junction City, OR) was used to probe the change in hydrophobicity, with an excitation wavelength at 395 nm and emission scanning between 420 and 550 nm [25]. Aliquots (20 μl) of bis-ANS (4.5 x 10^-4 M) were added to 3 ml of protein sample (0.1 mg/ml) and allowed to incubate at room temperature for 15 min before measurement.

Preparation of antibodies of anti-1-154 degraded αA-crystallin

Polyclonal antibodies against the native forms of αA- and αB-crystallins were produced as previously described [26]. An antibody against the truncated form of bovine αA-crystallin (1-154) was produced in rabbits using a synthetic antigen peptide corresponding to residues 149-154 (GVDAGH) with a cysteine residue added to the N-terminus as a site for conjugation to carrier protein. Affinity purification of antibodies was carried out using antigenic peptides immobilized on epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech, Buckinghamshire, England).

SDS-PAGE and western blot analysis

The HMW and LMW αA-crystallin fractions from 50 year old and LMW αA-crystallin from 2 year old samples were electrophoresed by the method of Laemmli [27] using 15% SDS-polyacrylamide gels. The proteins in the gels were stained with Coomassie brilliant blue and/or transferred onto PVDF membranes and the blotted proteins were incubated with the first antibody (anti-1-154 degraded αA-crystallin antibody) followed by incubation with peroxidase-conjugated anti-rabbit IgG as the second antibody. The antigen polypeptides were visualized using diaminobenzidine as substrate.

Enzymatic digestion and isolation of peptides

All preparations of α-crystallins were digested with trypsin (Wako, Osaka, Japan) for 20 h at 37 °C in buffer containing 0.1 M Tris-HCl buffer, 20 mM CaCl₂, pH 7.6 at an enzyme-to-substrate ratio of 1/50 (mol/mol). The resulting tryptic peptides (T) were separated by RP-HPLC (LC-10A, Shimadzu, Kyoto, Japan) using a C18 column (TSK gel-ODS -80 TM, 4.6 x 250 mm, Tosoh, Tokyo, Japan) with a linear gradient of 0-40% acetonitrile plus 0.1% trifluroacetic acid at a flow rate of 0.8 ml/min. Absorbance was monitored at 215 nm, and the peptides were collected using a fraction collector (FRC10 Shimadzu, Kyoto, Japan).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)

All spectra were obtained using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS, Kompact MALDI IV, Shimadzu Kyoto, Japan). The MALDI-TOFMS spectrometer was operated with a 337-nm nitrogen laser and an ion acceleration voltage of 20 kV. Data were collected in reflection mode as signals of positive ions. For the matrix, α-cyano-4-hydroxycinnamic acid (CHCA, 10 mg) was dissolved in 1 ml of solution containing two parts of 0.1% (v/v) trifluroacetic acid in water to one part of acetonitrile. The sample peptide (0.5 ml) was added to an equal volume (0.5 ml) of the matrix solution on the plate, and then dried. Each sample was present at a level of a few picomoles per spot.

Determination of D/L ratio of amino acids

Peptide samples were lyophilized in tubes and hydrolyzed with gas-phase 6 N HCl for 7 h at 108 °C. After hydrolysis, the samples were derivatized with o-phthalaldehyde (OPA) and n-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) to form diastereoisomers. The D/L ratio of amino acids was determined by RP-HPLC with a C18 column (Nova-Pak ODS, 3.9 x 300 mm, Waters, Japan) using fluorescence detection (wavelengths: excitation, 344 nm; emission, 433 nm). The samples were eluted at 30 °C with a linear gradient of 5-47% acetonitrile plus 3% tetrahydrofuran in 0.1 M acetate buffer (pH 6.0) over 120 min at a flow rate of 0.8 ml/min.

Amino acid sequence analysis

Amino acid sequences were determined by Edman degradation on a pulsed-liquid protein sequencer, equipped with an on-line phenylthiohydantoin (PTH) amino acid analyzer (Applied Biosystems 477A/120A, Foster City, CA).

Results

Chaperone activity

Since chaperone activity has been proposed to be a major function of α-crystallin, we measured α-A crystallin's chaperone activity through its ability to protect another protein from heat-induced aggregation. As shown in Figure 1, aggregation of βL-crystallin increased at 60 °C in the absence of α-A crystallin, but this aggregation was completely suppressed by the presence of α-A crystallin from the LMW fraction of lenses from 2 year old sources. However, protection from aggregation decreased markedly using αA-crystallin from the LMW fraction of older lenses (50 year old sources), with a further decrease in activity using αA-crystallin from the HMW fraction of the same lens [βL/αA=10/1 (w/w)]. The chaperone activity of HMW αA-crystallin decreased dramatically in the case of βL/αA=5/1 (w/w). Since HMW αA-crystallin did not aggregate at the same concentration without βL-crystallin, the aggregation is due to the mixture of βL-crystallin and HMW αA-crystallin.

Fluorescence

Bis-ANS is an anionic hydrophobic fluorescent probe that binds to the polar interface, and has been used in the study of protein hydrophobicity [28]. Figure 2 shows that the fluorescence intensity of the HMW form of αA -crystallin is higher than that of the LMW form, indicating that the HMW aggregated form of αA-crystallin was more hydrophobic than the LMW form of αA-crystallin, which could possibly result from partial unfolding of the molecule.

SDS-PAGE analysis

Figure 3 shows SDS-PAGE of HMW and LMW αA-crystallin from the 50 year old sample and the 2 year old sample of human lenses. The monomeric αA-crystallin (molecular weight of 20 kDa) band from HMW the 50 year old sample was not distinct, which may be caused by an increase in crosslinked products and other post-translational modifications.

Characterization of antibody against the truncated form of αA-crystallin

To evaluate the specificity of the antibody against the truncated form of 1-154 αA-crystallin, the α-crystallin fraction prepared from bovine lenses was analyzed by western blotting (Figure 4). The antibody against the native form of αA-crystallin reacts with both the intact approximately 20-kDa and truncated forms (Figure 4). In contrast, the anti-truncated form of αA-crystallin antibody recognizes only the truncated form and does not react with its intact 20 kDa form (Figure 5). Immunostaining with the antibody in the presence of antigenic peptide results in disappearance of the immunoreactive band (Figure 4, lane C). These results indicate that the antibody possesses strict specificity towards the C-terminal sequence of the truncated form of αA-crystallin (1-154). There are several possible reasons why the anti-truncated αA-crystallin (1-154) antibody does not cross-react at all with the antigenic peptide sequence (GVDAGH) present in the native αA-crystallin molecule. First, the corresponding portion in native form may not be exposed to the outside of the molecule, therefore it does not fit the antigen binding domain of the antibody; or the newly formed amino group on the C-terminal histidine of truncated αA-crystallin is an essential part of the epitope for the antibody.

Degradation of αA-crystallin peptide from HMW and LMW fractions

Figure 5 shows western blotting of HMW and LMW αA-crystallin using anti-1-154 degraded αA-crystallin antibody. Since the antibody specifically cross reacted with degraded αA-crystallin 1-154 but not with full-length αA-crystallin 1-173, the result indicated an increased degradation of the HMW αA-crystallin compared with that of the LMW αA-crystallin.

Isomerization and racemization of the Asp-151 and Asp-58 residue in HMW and LMW αA-crystallin

We have reported previously that Asp-151 [7-12] and Asp-58 [13] in αA-crystallin is stereochemically labile to allow the conversion of L-Asp to D-Asp or isomerization of α-Asp to β-Asp during aging. Here, we measured the ratio of Asp-151 and Asp-58 isomerization and racemization in HMW αA-crystallin to that in LMW αA-crystallin from 50 year old donor lenses. First, we identified the Asp-151-containing peptides, namely, T18 peptides (IQTGLD151ATHAER) of HMW and LMW αA-crystallin by sequence and mass analysis.

The T18 peptide was separated into two peaks by RP-HPLC owing to the difference between α- and β-linkages of the Asp-151 residue (Figure 6). We designated the β-Asp-151-containing T18 peptide T18-β, and the α-Asp-151 containing peptide T18-α. Although the molecular masses of T18-β and T18-α are both m/z 1,311.6, consistent with the theoretical mass of the peptide, T18-β can be distinguished from T18-α by protein sequencing. Sequence analysis of T18-α gave the expected sequence from Ile-146 to Arg-157, whereas T18-beta gave a shortened sequence from Ile-146 to Leu-150 because β-linkages are resistant to Edman degradation. The β/α ratio of Asp-151 of HMW αA-crystallins was markedly higher than that of LMW αA-crystallins. Subsequently, we also identified the Asp-58-containing peptides, namely, T6 peptides (TVLD58SGISGVR) of HMW and LMW αA-crystallin and analyzed the linkage of Asp-58 by a similar methodology. Figure 7 shows the ratio of isomerization of Asp-151 and Asp-58 in αA-crystallin from HMW and LMW fractions. The β/α ratio of Asp-151 of HMW αA-crystallin was extremely high, reaching a value of about 3.0, and the ratio of β/α of Asp 58 of αA-crystallin from HMW is also higher than that of LMW αA-crystallin. The Mann-Whitney test indicated that the average β/α ratio of Asp-151 and Asp-58 residues in HMW αA-crystallin was significantly higher than those of LMW αA-crystallin (for Asp-151: p<0.05; for Asp-58: p<0.05). We analyzed the D/L ratios of the β- and α-Asp 151 residues from HMW and LMW αA-crystallins in the 50 year old donors, respectively (Figure 8). Statistical analysis showed that the average D/L ratio of the β-Asp-151 residue in LMW αA-crystallin was significantly higher than that of HMW αA-crystallin (p<0.05). In LMW αA-crystallin, the D/L ratio of β-Asp-151 residue was higher than 1.0, which was consistent with our previous results using normal lenses from elderly human donors [8-12]. Since racemization is defined as a reversible first order reaction, when the D/L ratio reaches 1.0, racemization is in equilibrium. Therefore, the D/L ratios which were higher than 1.0 would not be defined as racemization, but as the stereoinversion of L-Asp to its D-isomer. On the other hand the D/L ratios of α-Asp-151 residues of LMW and HMW αA-crystallin are low (approximately 0.2). There were no significant differences in the D/L ratios for either LMW or HMW αA-crystallin (p=0.39). In contrast, as shown in Figure 9, the D/L ratio of β-Asp-58 was higher than 1.0 either in HMW or LMW αA-crystallin, which is also consistent with our previous result [13]. The statistical analysis showed that there were no differences in the D/L ratios of the Asp-58 residue between HMW and LMW αA-crystallin (p=0.48).

In contrast, the D/L ratios of α-Asp-58 of LMW αA-crystallin was significantly higher than that of HMW αA-crystallin (p<0.05), although the values were low (approximately 0.05-0.3).

Discussion

Formation of very high molecular weight aggregates of lens proteins is known to occur during aging, and may be relevant to cataract formation in human eye lenses. Although the mechanism of HMW aggregate formation has not yet been established, aggregated proteins may be precursors of components that eventually become insoluble. Therefore in the present study we have characterized the HMW form of αA-crystallin from normal lenses and compared our results with those obtained from previous studies of αA-crystallin obtained from total proteins of human cataractous lenses. As shown in Figure 1, the chaperone activity of αA-crystallin from young (2 year old) lens completely prevented the aggregation of βL-crystallin, while αA-crystallin from the LMW fraction of older (50 year old) lens was reduced in activity, with further reductions in chaperone activity using αA-crystallin from the HMW fraction. To form the HMW fraction, we therefore hypothesize that there might occur partial conformational changes, resulting at least in part from increased amounts of degraded polypeptides and/or various post-translational modifications. Since the process of aggregation to form HMW components most likely results in insolublization, the HMW form of αA-crystallin should be more hydrophobic than native α-crystallin. Figure 2 shows that the hydrophobicity of αA-crystallin in the HMW aggregates was indeed greater than αA-crystallin in the LMW fraction. The results strongly suggest that HMW aggregates of αA-crystallin result from exposure of buried beta-pleated sheets and increased hydrophobic interactions, which is consistent with an earlier report [29].

We have also found a new cleavage site which is a post-translational modification between residues His-154 and Ala-155 in αA-crystallin from old human lens. Since this cleavage was not found in αA-crystallin from young lens, the cleavage probably results from aging [9]. Therefore, we prepared a specific antibody against 1-154 αA-crystallin and examined the amounts of the degraded αA-crystallin in either HMW or LMW fractions by western blotting. There was a significant difference in the amounts of degraded products between HMW and LMW fractions, that is, the amounts of cleaved αA-crystallin increased markedly in HMW protein (Figure 5). The results indicate that the HMW fraction contains a large amount of degraded αA-crystallin, which may alter the tertiary structure of the protein.

In addition, we have analyzed the racemization and isomerization of Asp-151 and Asp-58 residues in HMW and LMW fractions of αA-crystallin. In previous studies we have investigated D-β-Asp formation of Asp-151 and Asp-58 in αA-crystallin obtained from total proteins of aged and cataractous human lenses [8-13,24]. As shown in Figure 10, the D-Asp formation might occur via a succinimide intermediate. L-Succinimide is readily formed by intramolecular cyclization of L-α-Asp. L-Succinimide can be converted to D-succinimide by racemization, and both succinimides can be hydrolyzed to D-β-Asp, L-β-Asp, D-α- Asp and L-α-Asp residues, respectively. This reaction depends on the rate of succinimide formation, which is expected to depend on the neighboring residue of the Asp residue. When the neighboring amino acid of the Asp residue has a small side chain, succinimide occurs easily because there is no steric hindrance. Since Asp-151 and Asp-58 residues in αA-crystallin are followed by alanine and serine residues, succinimides are easily formed. On the other hand, a previous study indicated that the formation of D-Asp needed the native higher order structure of αA-crystallin itself, because when the native structure of 80 year old human αA-crystallin which has a high D/L ratio of Asp-151 (5.0) was unfolded by heating in 6 M urea, the D/L ratio of Asp-151 decreased dramatically to 1.0 [10]. The result suggested that the area surrounding the Asp-151 or Asp-58 residues might have a chiral environment that allows for D-Asp formation in αA-crystallin.

The native structure of aged αA-crystallin may be distorted by various post-translational modifications, and the function of D-Asp may be to avoid the unfolding of the structure of αA-crystallin which is caused by this distortion. In the present study, the D/L ratio of Asp-151 of LMW αA-crystallin was higher than 1.0, while the value of the HMW form dramatically decreased. The tendency towards a lower D/L ratio was observed in both cataractous lenses[12,13] and γ-ray irradiated lenses, in a dose dependent manner [24].

The function of D-Asp therefore might be to prevent unfolding of αA-crystallin, which seems to occur in the formation of cataracts or following irradiation. Taking these results together with our findings here, it seems likely that the area surrounding Asp-151 differs in HMW αA-crystallin from the area in LMW αA-crystallin. In contrast, there are no differences in the D/L ratio of β-Asp-58 between LMW and HMW. The higher order structure of surrounding Asp-58 may remain in the native state compared with the region surrounding Asp-151. We also determined the β/α ratios of Asp-151 and Asp-58 in either HMW or LMW αA-crystallin. The results are consistent with previous results [12,13,24]. As shown in Figure 10, both L- and D-succinimide are easily hydrolyzed. The probability of succinimide opening to β-Asp or α-Asp is not known, although it favors β-Asp over α-Asp in small peptides and in human αA-crystallin. The increase of β-Asp formation in protein should alter the structure and possible function because the peptide backbone goes through the beta-carboxyl group.

In summary, our study suggests that modifications such as an increase in degradation, hydrophobicity, isomerization, and a decrease in racemization may destroy the higher-order structure, including the hydrophobic protein binding site of α-crystallin and thus reduce its chaperone activity. Further studies are therefore needed to elucidate in full the effects of post-translational modifications on chaperone activity and protein structure.

Acknowledgements

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the National Institutes of Health.

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