July 2005
Volume 46, Issue 7
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Lens  |   July 2005
Effect of Oxidized βB3-Crystallin Peptide on Lens βL-Crystallin: Interaction with βB2-Crystallin
Author Affiliations
  • E. G. Padmanabha Udupa
    From the Departments of Ophthalmology and
  • K. Krishna Sharma
    From the Departments of Ophthalmology and
    Biochemistry, University of Missouri, Columbia, Missouri.
Investigative Ophthalmology & Visual Science July 2005, Vol.46, 2514-2521. doi:10.1167/iovs.05-0031
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      E. G. Padmanabha Udupa, K. Krishna Sharma; Effect of Oxidized βB3-Crystallin Peptide on Lens βL-Crystallin: Interaction with βB2-Crystallin. Invest. Ophthalmol. Vis. Sci. 2005;46(7):2514-2521. doi: 10.1167/iovs.05-0031.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the interaction of oxidized βB3-crystallin peptide (residues 152-166) with βL-crystallin and to identify peptide-interaction sites.

methods. Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine βL-crystallin were conducted at 55°C and 37°C, respectively. Assays were performed in the presence of oxidized and nonoxidized βB3-crystallin peptides and in the presence of α-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4′-dianilino-1,1′ binaphthyl-5,5′ disulfonic acid) binding study. Oxidized βB3-peptide binding sites were identified by sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) labeling and mass spectrometric analysis.

results. Aggregation and relative light-scattering of βL-crystallin was higher in the presence of oxidized βB3-crystallin peptide than with βL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of α-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized βB3-peptide at 37°C. Bis-ANS binding to βL-crystallin treated with oxidized βB3-peptide was two to three times higher than in the controls at 37°C. The oxidized βB3-peptide preferentially interacted with βB2-crystallin. The data were confirmed by mass spectrometric analysis.

conclusions. Oxidized βB3-peptide interacts with βB2-crystallin and enhances its aggregation and precipitation. Peptide-induced aggregation and increased hydrophobicity of the lens crystallin at 37°C are relevant to crystallin aggregation in the aging lenses.

The stability of crystallins and their ability to associate in appropriate intermolecular interactions are critical for transparency and refraction of the eye lens. With aging, crystallins show aggregation, cross-linking, and insolubilization. Compared with normal lenses, cataract lenses exhibit more fragmentation of lens proteins due to the action of proteolytic enzymes. 1 2 3 Low-molecular-weight peptides originating from α-, β- and γ-crystallins have been isolated and characterized from the lens. 4 5 6 7 Accumulation of these crystallin fragments may be a cause of age-related lens opacity. It has been hypothesized that the interaction of crystallin fragments (short peptides) with lens proteins may increase the formation of high-molecular-weight aggregates and scattering of light, 8 but this interaction has not been studied in detail. 
β-Crystallins are major protein constituents of the mammalian lens, where their stability and association in higher-order complexes are necessary for lens clarity and refraction. They constitute approximately half of the soluble crystallins in aged lenses. 9 β-Crystallins associate into dimers, tetramers, and higher-order aggregates and are critical for maintaining lens transparency. 10 11 All β-crystallins have N-terminal extensions; the “basic” β-crystallins also have C-terminal extensions, which the “acidic” β-crystallins lack. 12 The sequence extensions of crystallins have been suggested to play an important role in the oligomerization of the lens proteins. 13 14 15 Among the β-crystallins, βB2-crystallin is the most stable. 16 βB2-crystallin is the predominant subunit, present in all size classes of β-crystallins and versatile in its interaction, being able to self-associate into dimers. It can also interact with other acidic or basic subunits of β-crystallins to form dimer and larger aggregates. 12 The high solubility of βB2-crystallin and its propensity to form noncovalent associations with less-soluble β-crystallins may contribute to the solubility of β-crystallins. 17 18 Further, it has been hypothesized that in aged lenses, where most of the α-crystallin becomes water insoluble, βB2-crystallin may play a dominant role in keeping the remaining crystallins in soluble form. 9 19  
Analysis of the water-insoluble lens proteins has shown that several aggregated species increase in concentration and become water insoluble with aging and cataractogenesis. 19 Further, the presence of covalent multimers (>90 kDa) composed of crystallin fragments has been reported in human lenses. 20 Crystallin fragments have also been found in the water-soluble, high-molecular-weight fractions and in water-insoluble protein fractions of age-matched human cataractous and normal lenses. 21 We have shown that the peptides derived from oxidized βL-crystallins modulate and enhance the aggregation of denaturing βL-crystallin and other proteins. 8 Moreover, we have shown that oxidized βB3-peptide (residues 152-166) can interact and bind to denaturing γ-crystallins and modulate their aggregation. 22 The present study further explores the interaction between oxidized βB3-crystallin peptide (residues 152-166) and βL-crystallin to determine the possible role of crystallin fragments in the development of insolubility of lens crystallins. Using a photoactive cross-linker 23 and mass spectrometry, we have identified the oxidized βB3-peptide interacting sites in βB2-crystallin. 
Materials and Methods
Synthetic peptides were procured from Invitrogen (Carlsbad, CA); bis-ANS (4,4′-dianilino-1,1′ binaphthyl-5,5′ disulfonic acid) from Molecular Probes (Eugene, OR); sulfo-SBED (sulfo-succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) and monomeric avidin gel (ImmunoPure) from Pierce (Rockford, IL); sequencing-grade modified trypsin from Promega (Madison, WI); and trypsin inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride), from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade. 
Purification of Crystallin
Fresh bovine lenses (<2 years old) were purchased from Pel-Freez Biologicals (Rogers, AR). Lenses were stored at −70°C until use. The thawed lenses were decapsulated and homogenized by stirring in 50 mM phosphate buffer (pH 7.4) containing 0.1 M NaCl (buffer A) at 4°C. The βL-crystallin was isolated from bovine lens extracts after chromatography (Sephadex G-200; Roche Diagnostics, Indianapolis, IN), as described previously. 24 α-Crystallin was isolated from young bovine lens cortical extracts by gel filtration on a purification column (Sephadex G-200; Roche Diagnostics) and ion-exchange chromatography on a TMAE column (Fractogel; Merck, Darmstadt, Germany), as described previously. 25 Protein and peptide concentrations were measured by the bicinchoninic acid method. 26  
Oxidation of βB3-Crystallin Peptide (152AINGTWVGYEFPGYR166) and Control Peptides
Before oxidation, all synthetic peptides were purified by reversed-phase high-performance liquid chromatography (HPLC; C-18 column; Grace Vydac, Hesperia, CA). Purified peptide (1–2 mg of each) was dissolved in 25 to 50 μL of dimethyl formamide (DMF), diluted to 1 mL in buffer A, and dialyzed with a 0.5-kDa membrane against buffer A. A known concentration of each of the peptides was oxidized by 100 μM H2O2 in the presence of 100 μM CuSO4 27 for 16 to 18 hours at 25°C and separated from the oxidants by reversed-phase HPLC on a C-18 column. Oxidation of the peptides was confirmed by nanospray quadrupole time of flight mass spectrometry (Qq-ToF-MS) analysis. 22  
Thermal Denaturation and Light-Scattering Assay
Thermal aggregation studies of the βL-crystallin in the presence and absence of α-crystallin were performed as described previously. 24 In brief, a known amount of substrate protein βL-crystallin (3.8 μM) was heat denatured in 1 mL of buffer A at 55°C for 65 minutes. The assay was also performed in the presence and absence of α-crystallin (0.015 μM) and with different concentrations of oxidized βB3-crystallin peptide (23-58 μM), nonoxidized βB3-crystallin peptide (58 μM), and control oxidized peptide (DRRIFWWSLRSAPG; 69 μM, a nonlenticular, synthetic peptide, or 42TSLSPFYLRPPSFLRAPSWF61; 63 μM, a human αB peptide). βL-Crystallin aggregation was measured by recording the relative light-scattering at 360 nm as a function of time in a spectrophotometer (Shimadzu, Columbia, MD) equipped with a temperature-controlled multicell transporter. A similar experiment was set up at 37°C for 10 hours with βL-crystallins and oxidized and nonoxidized βB3-crystallin peptides or control oxidized peptide to study the effect of oxidized βB3-peptides on βL-crystallin aggregation at a physiological temperature. 
Oxidized peptide-induced exposure of hydrophobic sites in the target protein was demonstrated by performing the bis-ANS binding studies. Bis-ANS is a fluorescence probe used to study the hydrophobic sites in proteins. 28 βL-Crystallin (3.8 μM) was treated with 34 μM of oxidized and nonoxidized βB3-crystallin peptide separately and incubated at 37°C. βL-Crystallin by itself was the control. At timed intervals, a 166-μL sample from each tube was treated with 20 μM of bis-ANS and excited at 390 nm, and the relative fluorescence emission was measured at 490 nm in a spectrofluorometer (Jasco, Easton, MD). To determine whether the increased light-scattering effect of oxidized βB3-peptide on βL-crystallin was due to increased crystallin precipitation, we processed the heat-denatured βL-crystallin precipitate, with and without oxidized peptide. The precipitate was collected by centrifugation at 12,000g for 15 minutes, washed with deionized water, and centrifuged. The procedure was repeated three times, to ensure that the precipitate was free of soluble peptides and proteins. The precipitate was then dissolved in 200 μL of freshly prepared 6 M urea solution and subjected to reversed-phase HPLC on a C-18 column. The bound proteins were eluted with a 0% to 60% linear gradient of acetonitrile containing 0.1% trifluoroacetic acid (TFA) for 75 minutes, with a flow rate of 1 mL/min. Absorbance was monitored at 220 nm as well as 280 nm. 
Identification of Oxidized Peptide Binding Sites in βL-Crystallin
Oxidized βB3-crystallin peptide was first derivatized with a trifunctional cross-linker, sulfo-SBED, as described previously. 22 Sulfo-SBED–labeled oxidized peptide (58 μM) was mixed with βL-crystallin (3.8 μM) and incubated at 55°C for 65 minutes. The precipitate was collected by centrifugation at 12,000g. It was then washed and again centrifuged at 12,000g three times to eliminate uninteracted labeled peptides and soluble βL-crystallin. The precipitate was collected again (all steps were performed in the dark), resuspended in buffer and photolyzed, as explained previously. 22 Similarly, sulfo-SBED–derivatized nonoxidized βB3-peptide and control oxidized peptides were treated with βL-crystallin, and the precipitate was photolyzed. The photolyzed complex of βL-crystallin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the photo-incorporation of sulfo-SBED cross-linker to the βL-crystallin was confirmed by Western blot with avidin horseradish peroxidase (HRP). The photolyzed complex of βL-crystallin and oxidized βB3-peptide was then processed, as described previously. 22 Biotinylated βL-crystallin peptides were enriched by monomeric avidin gel (ImmunoPure; Pierce), as described previously. 22 Sulfo-SBED–labeled βL-crystallin peptides were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS; Voyager DE Pro; Applied Biosystems, Foster City, CA), and the peptide sequences were determined by nanospray QqToF MS analysis. Based on the identification of the precursor ions of sulfo-SBED in each of the labeled peptides, 22 the amino acid sequences were assigned by using automated analysis of collision-induced dissociation (CID) spectra. 29  
Results
The extent of H2O2- and Cu2+-induced oxidation of the βB3-crystallin peptide and control peptides was monitored and analyzed, as explained previously. 22 Oxidation resulted in a mixed population of peptides containing oxidized tyrosine and tryptophan residues (MS/MS data not shown). Likewise, oxidation of tyrosine and tryptophan residues was observed in control peptides. These changes can be attributed to the free radicals generated by the Cu2+ and H2O2 mixture during the initial oxidation step. 22  
Effect of Oxidized βB3-Crystallin Peptide on Aggregation of βL-Crystallin
Thermal denaturation of βL-crystallin resulted in aggregation and light-scattering. The addition of the oxidized βB3-crystallin peptide (23 μM) enhanced the light-scattering of denaturing βL-crystallin, compared with light-scattering with nonoxidized βB3-peptide. Furthermore, the increased light-scattering by oxidized βB3-peptide was concentration dependent (Fig. 1) . Use of an increased amount of oxidized βB3-peptide (58 μM) resulted in >80% increased light-scattering. In contrast, the addition of nonoxidized βB3-peptide (23–58 μM) did not result in a similar increase. At higher concentrations of oxidized βB3-peptide, the aggregated crystallin started precipitating (Fig. 1) . However, enhanced light-scattering was not due to the added βB3-peptides, as peptides themselves did not show any light-scattering under the experimental conditions (Fig. 1) . Our data indicate that the oxidation of βB3-peptide was necessary to exhibit the enhanced light-scattering, as the nonoxidized βB3-crystallin peptide had little effect (<5%) on aggregation or light-scattering. The control oxidized peptides used in the assay did not show a significant increase in aggregation (<5%). A light-scattering and aggregation experiment between oxidized βB3-peptide and βL-crystallin was conducted at 37°C for an extended time to determine the effect of peptides on βL-crystallins at a physiological temperature. The degree of light-scattering by βL-crystallin itself or in the presence of nonoxidized βB3-peptide at 37°C was significantly lower than that at 55°C (Fig. 2) . However, at 37°C the aggregation and relative light-scattering by βL-crystallin in the presence of oxidized βB3-peptide was considerably higher than with βL-crystallin alone (Fig. 2) , indicating a physiologically significant peptide interaction with lens crystallins, causing light-scattering. 
After demonstrating the ability of the oxidized βB3-crystallin peptide to enhance the relative light-scattering of denaturing βL-crystallin, we investigated the aggregation-influencing property of the oxidized βB3-peptide in the presence of α-crystallin at 55°C. As expected, α-crystallin suppressed the aggregation of denatured βL-crystallin due to its chaperoning property (data is not shown). The data were similar to that published earlier (see Fig. 6in Ref. 8 ). Further, light-scattering was not observed with α-crystallin alone or when α-crystallin and oxidized peptides were used together in the assay. The data show the ability of the oxidized βB3-peptide to interact with target protein despite a chaperone protein in the system. The increased light-scattering suggests that oxidized βB3-peptide behaves like an anti-chaperone protein, meaning the peptide may prevent the binding of chaperone protein to the denaturing protein by binding itself to the substrate protein. Alternately, oxidized βB3-peptide may bind to α-crystallin and disturb its chaperone property. Further studies are needed to determine the mechanism. 
The oxidized-peptide–induced exposure of hydrophobic sites in βL-crystallin at 37°C was demonstrated by increased bis-ANS binding (Fig. 3) . In a separate set of experiments, βL-crystallin was treated with oxidized or nonoxidized βB3-peptide at 37°C, and the aliquots were withdrawn at timed intervals and treated with bis-ANS. Whereas there was a gradual increase in bis-ANS binding to βL-crystallin, as well as to peptide-treated βL-crystallin, the oxidized βB3-peptide–treated βL-crystallin showed a two- to threefold higher degree of bis-ANS binding (Fig. 3) . In addition, a rapid increase in bis-ANS binding occurred in the initial phase of the oxidized peptide interaction, compared with bis-ANS binding in the βL-crystallin control and the nonoxidized peptide–treated βL-crystallin (Fig. 3) . After the initial increase in bis-ANS fluorescence for approximately 200 minutes of incubation, there was no additional increase in bis-ANS fluorescence, as aggregated crystallins started precipitating. The observation of higher bis-ANS binding in the initial phase of the interaction of βL-crystallin with oxidized peptide in our experiment implies that peptide-induced conformational change in the target protein enhances bis-ANS binding. The relatively constant or slightly decreased bis-ANS binding in the oxidized peptide-treated βL-crystallin in the later phase of the interaction indicates aggregation reaching the saturation level as well as precipitation of aggregated crystallins. The experiment with a control peptide showed a reduction in bis-ANS binding, similar to the results for nonoxidized βB3-peptide and βL-crystallin (Fig. 3)
The increased aggregation of βL-crystallins in the presence of oxidized βB3-peptide was accompanied by increased precipitation of the denatured protein. This increase was confirmed by analyzing the precipitated crystallins, with and without oxidized peptide after thermal aggregation at 55°C. The aggregated precipitates were dissolved in 6 M urea and analyzed by reversed-phase HPLC on a C-18 column (Fig. 4) . Precipitation of βL-crystallin increased when the oxidized βB3-peptide interacted with it, compared with βL-crystallin alone or when nonoxidized βB3-peptide was included in the assay with βL-crystallin. Relatively higher absorbance was observed in a protein peak eluting at 25.6 minutes (Fig. 4 , trace b), when oxidized βB3-peptide and βL-crystallin were used compared with βL-crystallin alone or βL-crystallin treated with nonoxidized βB3-peptide. The protein peak eluting at 25.6 minutes in Figure 4corresponded to βB2-crystallin (on the basis of MS analysis). Therefore, it appears that oxidized βB3-peptide preferentially interacts with βB2-crystallin and enhances its aggregation and precipitation. 
Oxidized βB3-Peptide Binding Sites in βL-Crystallin
The oxidized βB3-peptide interacting sites in βL-crystallin were identified by the sulfo-SBED–labeling method. 22 23 The photoinsertion of sulfo-SBED to the βL-crystallin was confirmed by SDS-PAGE and Western blot analysis (Fig. 5) . The Western blot data showed the interaction of oxidized βB3-peptide with β-crystallin fraction and βB2-crystallin and the transfer of the label from the peptide to the protein (Fig. 4B , lane 4). Further, there was no label transfer from sulfo-SBED–labeled nonoxidized peptide (Fig. 4B , lane 5) or control peptide (Fig. 4B , lane 6) to the βL-crystallins. 
To identify the peptide interaction sites in βL-crystallin, the trypsin digest of βL-crystallin containing biotin-labeled peptides were enriched by monomeric avidin gel and reversed-phase HPLC on a C-18 column. 22 The MALDI-ToF MS spectrum of sulfo-SBED–labeled peptides obtained during reversed-phase HPLC are shown in Figure 6 . Photoinsertion of a molecule of sulfo-SBED (alkylated by iodoacetamide) will add a mass of 605.23 Da (or 621.24 Da in an oxidized form) to the peptide to which it is attached. Hence, sulfo-SBED–derivatized peptides will have a mass of 605.23 or 621.24 Da+peptide mass. Before assigning the amino acid sequence for each sulfo-SBED–labeled peptide, the presence of biotin label in each peptide was confirmed by the detection of the fragment ions generating specifically from sulfo-SBED during MS/MS analysis. 22 The peptide peaks labeled in Figure 6were analyzed further by nanospray QqToF MS by selecting singly, doubly, or triply charged ions. The peptide sequences that contained the label, presumed to be the interaction regions, are listed in Table 1 . Figure 7shows the MS/MS spectrum of a triply charged ion m/z 827.71(+3) of the peptide m/z 2480.2 (+1) from Figure 6 , originating from the N terminus of βB2-crystallin, residues 1ASDHQTQAGKPQPLNPK17, acetylated at its N-terminal end. The sulfo-SBED label was attached to H-4 of this peptide, with a net increase in mass of 621.24 Da. The MS/MS spectra of the remaining sulfo-SBED–labeled peptides are not shown. 
The sulfo-SBED–labeled peptides identified (Table 1)suggest that the oxidized βB3-peptide preferentially interacted with βB2-crystallin. The oxidized βB3-peptide interacted with amino acid residues in N- and C-terminal extensions of the βB2-crysallin (Fig. 8) . Apart from two terminal extensions, the oxidized peptide also interacted with amino acid residues in other regions of βB2-crystallin, which included the following: (1) residues 40-47 at the N-terminal domain; (2) residues 89-144, the region comprising the last segment of the N-terminal domain (residues 89-97), the connecting peptide region (residues 98-106), and the initial segment of the C-terminal domain (residues 107-144); and (3) residues 160-167 in the C-terminal domain. The N- and C-terminal ends of βB2-crystallin are solvent-exposed and flexible. 30 31 32 Of the oxidized βB3-crystallin peptide–interacting regions in βB2-crystallin, amino acid residues 89, 97, 9-104, 106, 127, 129, 130, 132, 133, 137, 160-162, 164, 166, and 167 were found buried in either the PQ interface of the βB2-crystallin dimer or the QR interface of the βB2-crystallin dimer-dimer in an x-ray crystal structure analysis study. 30 Further, based on 1H-NMR studies, it has been suggested that, among the trimer and heterodimer of β-crystallins, the tertiary structure of βB2-crystallin contains more flexible regions. 32 It is also less compact than other subunits. 32 Furthermore, as shown in Table 1 , for few peptides, we were able to assign the site of sulfo-SBED insertion (to histidine), suggesting that the peptide interaction occurred through ionic interactions as well. 
Discussion
β-Crystallins are major protein constituents of the mammalian lens, where their stability and arrangement into higher-order complexes are critical for the maintenance of lens clarity and refraction. The most complex eye lens proteins, β-crystallins made up of several related basic and acidic subunits that combine to form different-sized oligomers, each displaying extensive polydispersity. 11 15 17 33 Lens is known to contain degraded crystallin fragments, the concentration of which increases with advancing age. 6 7 34 35 36 An association between crystallin peptides derived from α-, β-, or γ-crystallins and high-molecular-weight clusters of lens crystallins has been reported. 20 21 In addition, in the cataract lens insoluble high-molecular-weight protein aggregates have been shown to be composed of low-molecular-crystallin fragments and modified/cross-linked crystallins. 37 Although accumulation of crystallin fragments has been implicated in cataract formation, how crystallin fragments contribute to cataractogenesis is unknown. We have reported the modulation of βL-crystallin aggregation by crystallin fragments derived from oxidized βL-crystallin. 8 Because β-crystallins are vital to lens transparency and refraction and make up nearly one half of the soluble crystallins in the aged lenses, the present study was intended to explore further the interaction between oxidized βB3-peptide and the βL-crystallin. 
Proteolysis is a contributing factor in the generation of low-molecular-weight peptides from partially denatured or oxidatively modified crystallins. 35 38 39 Furthermore, peptidase activity is lower in the inner cortex and nucleus than in the outer cortical region of the lens. 35 Decreased peptidase activity may contribute to the increased accumulation of degraded polypeptides in the aging lens. 40 The rate of crystallin damage and accumulation of crystallin fragments may increase with age, due to the diminished activity of many “housekeeping” enzymes. In a prior study, our data revealed the interaction between oxidized βB3-peptide and γ- and α-crystallin. 22 Our current data show the interaction of oxidized βB3-peptide with lens β-crystallins, suggesting that the presence of or accumulation of certain peptides in the lens, which have the ability to interact with lens crystallins, may cause the insolubilization of soluble crystallins with aging. 
It has been suggested that cataract belongs to the group of conformational diseases, such as Alzheimer’s disease, 41 and develops in response to altered surface charges that cause conformational change in crystallins. 42 Peptide-induced conformational change in the target proteins 43 44 and the exposure of the hydrophobic sites has been reported. 43 The results of this study suggest that the binding of oxidized βB3-peptide to the βL-crystallin causes conformational change that leads to increased exposure of hydrophobic regions (Fig. 3) , resulting in an enhanced interaction between the exposed hydrophobic sites. This interaction increases the aggregation of βL-crystallin, even at a physiological temperature (Fig. 2) . That a peptide-induced conformational change occurs is supported by the observation of two- to threefold higher bis-ANS binding in the initial phase of the interaction of oxidized βB3-peptide and βL-crystallin at 37°C (Fig. 3) . Moreover, bis-ANS fluorescence increased much earlier (maximum at 200 minutes, Fig. 3 ) than did the increase in the light-scattering at 37°C (maximum at 400 minutes, Fig. 2 ) suggesting the occurrence of peptide-induced hydrophobic changes before crystallin aggregation. Because hydrophobic sites are believed to play a major role in protein–protein interaction, we hypothesize that oxidized βB3-crystallin peptide binds to the crystallin protein and induces conformational change, leading to additional hydrophobic interaction between the exposed hydrophobic sites. 
Lens β-crystallins are structural proteins with conserved two-domain structure with variable N- and C-terminal extensions. These extensions are known to be involved in quaternary interactions within β-crystallin oligomers and with other lens proteins. 45 βB2-crystallin is a stable lens protein that helps to keep other lens crystallins soluble, a significant attribute in the aging lenses, where most of the α-crystallin becomes water insoluble. 9 19 In addition, βB2-crystallin subunit not only self-associates to a homodimer but also readily forms a heterodimer with βB3- and βA4-crystallins and a larger aggregate with βA3-crystallin. 12 Our study revealed the preferential interaction between oxidized βB3-peptide and βB2-crystallin, which suggests the susceptibility of βB2-crystallin toward oxidized βB3-peptides. MS/MS analysis of oxidized βB3-crystallin peptide–interacted sites in βB2-crystallin (Table 1 , Fig. 8 ) indicates that oxidized peptide readily interacts with amino acid residues in both N- and C-terminal ends of βB2-crystallin. The N- and C-terminal ends of βB2-crystallin are solvent-exposed and flexible in the βB2-dimer. 30 31 The N-terminal end of βB2-crystallin is flexible, even in the tetramer and higher-order aggregation state, whereas the C-terminal end of βB2-crystallin may be involved in tetramer and higher-order aggregation of β-crystallin. 46 The association of crystallins into higher-order complexes is thought to be of critical importance for maintaining lens transparency. 47 Apart from interacting with the two terminal extensions, the oxidized βB3-peptide also interacted with amino acid residues on several regions of βB2-crystallin, as indicated in Table 1and Figure 8 , suggesting extensive interaction. 
It has been proposed that even at high concentrations, crystallin complexes undergo significant levels of monomer exchange instead of existing as static structures. 11 Our data showing the interaction of oxidized peptide with βB2-crystallin at a physiological temperature suggest that binding of peptides and a change in the conformation of βB2-crystallin could interfere with association or dissociation and subunit exchange within the β-crystallins. However, further studies are needed to validate the implications of conformational change in crystallins due to peptide binding. The presence of low-molecular-weight polypeptides (4–8 kDa) in the opaque region, but not in the clear regions, of human brunescent cataracts lens was demonstrated by Horwitz et al. 5 They also reported that brunescent lens nuclei, which are cataractous, possess significant quantities of oxidized protein and also contain crystallin fragments. Because it has been demonstrated that lens fibers also contain a fully functional ubiquitin–proteasome pathway capable of degrading oxidized proteins and peptides in the lens, 48 the interaction between oxidized peptides and lens crystallins in vivo may be possible only when there is reduced ubiquitin–proteasome activity or increased production of oxidized peptides that exceed the capacity of the ubiquitin–proteasome system to degrade it. We have identified several low-molecular-weight crystallin fragments in aged and cataract human lenses that cause enhanced in vitro crystallin aggregation and exhibit antichaperone-like properties (Sharma KK, et al. IOVS 2004;45:ARVO E-Abstract 3378). Based on our previous study 22 and the present data, we hypothesize that accumulation of oxidized lens crystallin fragments due to incomplete hydrolysis by peptidases and the ubiquitin system may result in their interaction with other lens crystallins. This process may contribute to protein aggregation and light-scattering in vivo. Studies are in progress to establish the role of low-molecular-weight peptides on the aggregation of α-, β-, and γ-crystallins in aged and cataractous human lenses. 
 
Figure 1.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 55°C. βL-Crystallin alone (•); βL-crystallin+23 μM oxidized βB3-peptide (▴); βL-crystallin+34 μM oxidized βB3-peptide (□); βL-crystallin+58 μM oxidized βB3-peptide (▪); βL-crystallin+58 μM nonoxidized βB3-peptide (▵); βL-crystallin+63 μM oxidized human αB-peptide 42TSLSPFYLRPPSFLRAPSWF61 (♦); βL-crystallin+69 μM oxidized non-lenticular peptide DRRIFWWSLRSAPG (○); and 58 μM oxidized βB3-peptide alone (⋄).
Figure 1.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 55°C. βL-Crystallin alone (•); βL-crystallin+23 μM oxidized βB3-peptide (▴); βL-crystallin+34 μM oxidized βB3-peptide (□); βL-crystallin+58 μM oxidized βB3-peptide (▪); βL-crystallin+58 μM nonoxidized βB3-peptide (▵); βL-crystallin+63 μM oxidized human αB-peptide 42TSLSPFYLRPPSFLRAPSWF61 (♦); βL-crystallin+69 μM oxidized non-lenticular peptide DRRIFWWSLRSAPG (○); and 58 μM oxidized βB3-peptide alone (⋄).
Figure 2.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 37°C. Symbols are as described in Figure 1 .
Figure 2.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 37°C. Symbols are as described in Figure 1 .
Figure 3.
 
Bis-ANS fluorescence of βL-crystallin incubated at 37°C and treated with oxidized and nonoxidized βB3-peptide and control peptide at 37°C. In each assay 3.8 μM of βL-crystallin was used, and 0.66 μM was withdrawn at timed intervals and treated with bis-ANS (20 μM). βL-Crystallin alone (•); βL-crystallin+34 μM oxidized βB3-peptide (▪); βL-crystallin+34 μM nonoxidized βB3-peptide (♦); and βL-crystallin+69 μM control oxidized peptide, DRRIFWWSLRSAPG (▵). Oxidized peptide and control peptide showed low bis-ANS binding (<1 unit).
Figure 3.
 
Bis-ANS fluorescence of βL-crystallin incubated at 37°C and treated with oxidized and nonoxidized βB3-peptide and control peptide at 37°C. In each assay 3.8 μM of βL-crystallin was used, and 0.66 μM was withdrawn at timed intervals and treated with bis-ANS (20 μM). βL-Crystallin alone (•); βL-crystallin+34 μM oxidized βB3-peptide (▪); βL-crystallin+34 μM nonoxidized βB3-peptide (♦); and βL-crystallin+69 μM control oxidized peptide, DRRIFWWSLRSAPG (▵). Oxidized peptide and control peptide showed low bis-ANS binding (<1 unit).
Figure 4.
 
Reversed-phase HPLC chromatogram of an aggregation precipitate of βL-crystallin (3.8 μM) dissolved in 6 M urea. βL-Crystallin alone (trace a); βL-crystallin treated with 34 μM oxidized βB3-crystallin peptide (trace b); and βL-crystallin treated with 34 μM nonoxidized βB3-crystallin peptide (trace c).
Figure 4.
 
Reversed-phase HPLC chromatogram of an aggregation precipitate of βL-crystallin (3.8 μM) dissolved in 6 M urea. βL-Crystallin alone (trace a); βL-crystallin treated with 34 μM oxidized βB3-crystallin peptide (trace b); and βL-crystallin treated with 34 μM nonoxidized βB3-crystallin peptide (trace c).
Figure 5.
 
SDS-PAGE (A) and Western blot (B) analyses of an aggregation precipitate of βL-crystallin treated with sulfo-SBED–labeled peptides. Lanes 1 and 6: βL-crystallin treated with the control oxidized peptide DRRIFWWSLRSAPG; lanes 2 and 5: βL-crystallin treated with nonoxidized βB3-peptide; lanes 3 and 4: βL-crystallin treated with oxidized βB3-peptide; and lane 7: prestained molecular weight marker.
Figure 5.
 
SDS-PAGE (A) and Western blot (B) analyses of an aggregation precipitate of βL-crystallin treated with sulfo-SBED–labeled peptides. Lanes 1 and 6: βL-crystallin treated with the control oxidized peptide DRRIFWWSLRSAPG; lanes 2 and 5: βL-crystallin treated with nonoxidized βB3-peptide; lanes 3 and 4: βL-crystallin treated with oxidized βB3-peptide; and lane 7: prestained molecular weight marker.
Figure 6.
 
MALDI-ToF MS spectrum of sulfo-SBED–labeled βL-crystallin peptides purified by reversed-phase HPLC on a C-18 column after monomeric avidin gel chromatography.
Figure 6.
 
MALDI-ToF MS spectrum of sulfo-SBED–labeled βL-crystallin peptides purified by reversed-phase HPLC on a C-18 column after monomeric avidin gel chromatography.
Table 1.
 
Peptide Sequences of Sulfo-SBED-Labeled βB2-Crystallin Peptides Seen in MALDI-ToF MS Spectrum
Table 1.
 
Peptide Sequences of Sulfo-SBED-Labeled βB2-Crystallin Peptides Seen in MALDI-ToF MS Spectrum
Peptide Mass (Da), m/z Peptide Sequence
1001.44 (+2) 108ITLYENPNFTGK119
1323.54 (+1) 198GAFH * PSS204
1524.80 (+1) 40LKETGVEK47
1568.80 (+1) 160GLQYLLEK167
1773.82 (+1) 135GYQEKVSSVR144
2480.20 (+1) 1ASDH * QTQAGKPQPLNPK17
2661.40 (+1) 90TDSLSSLRPIKVDSQEH * K107
2816.40 (+1) 89RTDSLSSLRPIKVDSQEH * K107
2966.20 (+1) 120KMEVIDDDVPSFHAH * GYQEK139
Figure 7.
 
Nanospray QqToF MS/MS spectrum of sulfo-SBED–labeled βB2-crystallin peptide (residues 1-17) with m/z, 2480.20 (+1). The triply charged peptide ion at m/z, 827.71 (+3) was selected for MS/MS analysis. Inset: the identified fragment ions generated from the peptide sequence 1ASDHQTQAGKPQPLNPK17 (acetylated at its N terminus) and the sulfo-SBED label was attached to H-4.
Figure 7.
 
Nanospray QqToF MS/MS spectrum of sulfo-SBED–labeled βB2-crystallin peptide (residues 1-17) with m/z, 2480.20 (+1). The triply charged peptide ion at m/z, 827.71 (+3) was selected for MS/MS analysis. Inset: the identified fragment ions generated from the peptide sequence 1ASDHQTQAGKPQPLNPK17 (acetylated at its N terminus) and the sulfo-SBED label was attached to H-4.
Figure 8.
 
Amino acid sequence of bovine βB2-crystallin (1-204). Bold sequences: oxidized βB3-crystallin peptide interaction regions.
Figure 8.
 
Amino acid sequence of bovine βB2-crystallin (1-204). Bold sequences: oxidized βB3-crystallin peptide interaction regions.
The authors thank Beverly DaGue (Proteomics Center, University of Missouri, Columbia, MO) for performing the mass spectrometry analysis. 
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Figure 1.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 55°C. βL-Crystallin alone (•); βL-crystallin+23 μM oxidized βB3-peptide (▴); βL-crystallin+34 μM oxidized βB3-peptide (□); βL-crystallin+58 μM oxidized βB3-peptide (▪); βL-crystallin+58 μM nonoxidized βB3-peptide (▵); βL-crystallin+63 μM oxidized human αB-peptide 42TSLSPFYLRPPSFLRAPSWF61 (♦); βL-crystallin+69 μM oxidized non-lenticular peptide DRRIFWWSLRSAPG (○); and 58 μM oxidized βB3-peptide alone (⋄).
Figure 1.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 55°C. βL-Crystallin alone (•); βL-crystallin+23 μM oxidized βB3-peptide (▴); βL-crystallin+34 μM oxidized βB3-peptide (□); βL-crystallin+58 μM oxidized βB3-peptide (▪); βL-crystallin+58 μM nonoxidized βB3-peptide (▵); βL-crystallin+63 μM oxidized human αB-peptide 42TSLSPFYLRPPSFLRAPSWF61 (♦); βL-crystallin+69 μM oxidized non-lenticular peptide DRRIFWWSLRSAPG (○); and 58 μM oxidized βB3-peptide alone (⋄).
Figure 2.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 37°C. Symbols are as described in Figure 1 .
Figure 2.
 
Facilitated aggregation of βL-crystallin (3.8 μM) in the presence of oxidized and nonoxidized βB3-peptide and control peptides at 37°C. Symbols are as described in Figure 1 .
Figure 3.
 
Bis-ANS fluorescence of βL-crystallin incubated at 37°C and treated with oxidized and nonoxidized βB3-peptide and control peptide at 37°C. In each assay 3.8 μM of βL-crystallin was used, and 0.66 μM was withdrawn at timed intervals and treated with bis-ANS (20 μM). βL-Crystallin alone (•); βL-crystallin+34 μM oxidized βB3-peptide (▪); βL-crystallin+34 μM nonoxidized βB3-peptide (♦); and βL-crystallin+69 μM control oxidized peptide, DRRIFWWSLRSAPG (▵). Oxidized peptide and control peptide showed low bis-ANS binding (<1 unit).
Figure 3.
 
Bis-ANS fluorescence of βL-crystallin incubated at 37°C and treated with oxidized and nonoxidized βB3-peptide and control peptide at 37°C. In each assay 3.8 μM of βL-crystallin was used, and 0.66 μM was withdrawn at timed intervals and treated with bis-ANS (20 μM). βL-Crystallin alone (•); βL-crystallin+34 μM oxidized βB3-peptide (▪); βL-crystallin+34 μM nonoxidized βB3-peptide (♦); and βL-crystallin+69 μM control oxidized peptide, DRRIFWWSLRSAPG (▵). Oxidized peptide and control peptide showed low bis-ANS binding (<1 unit).
Figure 4.
 
Reversed-phase HPLC chromatogram of an aggregation precipitate of βL-crystallin (3.8 μM) dissolved in 6 M urea. βL-Crystallin alone (trace a); βL-crystallin treated with 34 μM oxidized βB3-crystallin peptide (trace b); and βL-crystallin treated with 34 μM nonoxidized βB3-crystallin peptide (trace c).
Figure 4.
 
Reversed-phase HPLC chromatogram of an aggregation precipitate of βL-crystallin (3.8 μM) dissolved in 6 M urea. βL-Crystallin alone (trace a); βL-crystallin treated with 34 μM oxidized βB3-crystallin peptide (trace b); and βL-crystallin treated with 34 μM nonoxidized βB3-crystallin peptide (trace c).
Figure 5.
 
SDS-PAGE (A) and Western blot (B) analyses of an aggregation precipitate of βL-crystallin treated with sulfo-SBED–labeled peptides. Lanes 1 and 6: βL-crystallin treated with the control oxidized peptide DRRIFWWSLRSAPG; lanes 2 and 5: βL-crystallin treated with nonoxidized βB3-peptide; lanes 3 and 4: βL-crystallin treated with oxidized βB3-peptide; and lane 7: prestained molecular weight marker.
Figure 5.
 
SDS-PAGE (A) and Western blot (B) analyses of an aggregation precipitate of βL-crystallin treated with sulfo-SBED–labeled peptides. Lanes 1 and 6: βL-crystallin treated with the control oxidized peptide DRRIFWWSLRSAPG; lanes 2 and 5: βL-crystallin treated with nonoxidized βB3-peptide; lanes 3 and 4: βL-crystallin treated with oxidized βB3-peptide; and lane 7: prestained molecular weight marker.
Figure 6.
 
MALDI-ToF MS spectrum of sulfo-SBED–labeled βL-crystallin peptides purified by reversed-phase HPLC on a C-18 column after monomeric avidin gel chromatography.
Figure 6.
 
MALDI-ToF MS spectrum of sulfo-SBED–labeled βL-crystallin peptides purified by reversed-phase HPLC on a C-18 column after monomeric avidin gel chromatography.
Figure 7.
 
Nanospray QqToF MS/MS spectrum of sulfo-SBED–labeled βB2-crystallin peptide (residues 1-17) with m/z, 2480.20 (+1). The triply charged peptide ion at m/z, 827.71 (+3) was selected for MS/MS analysis. Inset: the identified fragment ions generated from the peptide sequence 1ASDHQTQAGKPQPLNPK17 (acetylated at its N terminus) and the sulfo-SBED label was attached to H-4.
Figure 7.
 
Nanospray QqToF MS/MS spectrum of sulfo-SBED–labeled βB2-crystallin peptide (residues 1-17) with m/z, 2480.20 (+1). The triply charged peptide ion at m/z, 827.71 (+3) was selected for MS/MS analysis. Inset: the identified fragment ions generated from the peptide sequence 1ASDHQTQAGKPQPLNPK17 (acetylated at its N terminus) and the sulfo-SBED label was attached to H-4.
Figure 8.
 
Amino acid sequence of bovine βB2-crystallin (1-204). Bold sequences: oxidized βB3-crystallin peptide interaction regions.
Figure 8.
 
Amino acid sequence of bovine βB2-crystallin (1-204). Bold sequences: oxidized βB3-crystallin peptide interaction regions.
Table 1.
 
Peptide Sequences of Sulfo-SBED-Labeled βB2-Crystallin Peptides Seen in MALDI-ToF MS Spectrum
Table 1.
 
Peptide Sequences of Sulfo-SBED-Labeled βB2-Crystallin Peptides Seen in MALDI-ToF MS Spectrum
Peptide Mass (Da), m/z Peptide Sequence
1001.44 (+2) 108ITLYENPNFTGK119
1323.54 (+1) 198GAFH * PSS204
1524.80 (+1) 40LKETGVEK47
1568.80 (+1) 160GLQYLLEK167
1773.82 (+1) 135GYQEKVSSVR144
2480.20 (+1) 1ASDH * QTQAGKPQPLNPK17
2661.40 (+1) 90TDSLSSLRPIKVDSQEH * K107
2816.40 (+1) 89RTDSLSSLRPIKVDSQEH * K107
2966.20 (+1) 120KMEVIDDDVPSFHAH * GYQEK139
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