IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2005;46:2514-2521.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0031
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Udupa, E. G. P.
Right arrow Articles by Sharma, K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Udupa, E. G. P.
Right arrow Articles by Sharma, K. K.

Effect of Oxidized ßB3-Crystallin Peptide on Lens ßL-Crystallin: Interaction with ßB2-Crystallin

E. G. Padmanabha Udupa1 and K. Krishna Sharma1,2

1From the Departments of Ophthalmology and 2Biochemistry, University of Missouri, Columbia, Missouri.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the interaction of oxidized ßB3-crystallin peptide (residues 152-166) with ßL-crystallin and to identify peptide-interaction sites.

METHODS. Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine ßL-crystallin were conducted at 55°C and 37°C, respectively. Assays were performed in the presence of oxidized and nonoxidized ßB3-crystallin peptides and in the presence of {alpha}-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4'-dianilino-1,1' binaphthyl-5,5' disulfonic acid) binding study. Oxidized ßB3-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 ßL-crystallin was higher in the presence of oxidized ßB3-crystallin peptide than with ßL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of {alpha}-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized ßB3-peptide at 37°C. Bis-ANS binding to ßL-crystallin treated with oxidized ßB3-peptide was two to three times higher than in the controls at 37°C. The oxidized ßB3-peptide preferentially interacted with ßB2-crystallin. The data were confirmed by mass spectrometric analysis.

CONCLUSIONS. Oxidized ßB3-peptide interacts with ßB2-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 {alpha}-, ß- and {gamma}-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.

ß-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 ß-Crystallins associate into dimers, tetramers, and higher-order aggregates and are critical for maintaining lens transparency.10 11 All ß-crystallins have N-terminal extensions; the "basic" ß-crystallins also have C-terminal extensions, which the "acidic" ß-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 ß-crystallins, ßB2-crystallin is the most stable.16 ßB2-crystallin is the predominant subunit, present in all size classes of ß-crystallins and versatile in its interaction, being able to self-associate into dimers. It can also interact with other acidic or basic subunits of ß-crystallins to form dimer and larger aggregates.12 The high solubility of ßB2-crystallin and its propensity to form noncovalent associations with less-soluble ß-crystallins may contribute to the solubility of ß-crystallins.17 18 Further, it has been hypothesized that in aged lenses, where most of the {alpha}-crystallin becomes water insoluble, ßB2-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 ßL-crystallins modulate and enhance the aggregation of denaturing ßL-crystallin and other proteins.8 Moreover, we have shown that oxidized ßB3-peptide (residues 152-166) can interact and bind to denaturing {gamma}-crystallins and modulate their aggregation.22 The present study further explores the interaction between oxidized ßB3-crystallin peptide (residues 152-166) and ßL-crystallin to determine the possible role of crystallin fragments in the development of insolubility of lens crystallins. Using a photoactive cross-linker23 and mass spectrometry, we have identified the oxidized ßB3-peptide interacting sites in ßB2-crystallin.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
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 ßL-crystallin was isolated from bovine lens extracts after chromatography (Sephadex G-200; Roche Diagnostics, Indianapolis, IN), as described previously.24 {alpha}-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 ßB3-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 CuSO427 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 ßL-crystallin in the presence and absence of {alpha}-crystallin were performed as described previously.24 In brief, a known amount of substrate protein ßL-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 {alpha}-crystallin (0.015 µM) and with different concentrations of oxidized ßB3-crystallin peptide (23-58 µM), nonoxidized ßB3-crystallin peptide (58 µM), and control oxidized peptide (DRRIFWWSLRSAPG; 69 µM, a nonlenticular, synthetic peptide, or 42TSLSPFYLRPPSFLRAPSWF61; 63 µM, a human {alpha}B peptide). ßL-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 ßL-crystallins and oxidized and nonoxidized ßB3-crystallin peptides or control oxidized peptide to study the effect of oxidized ßB3-peptides on ßL-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 ßL-Crystallin (3.8 µM) was treated with 34 µM of oxidized and nonoxidized ßB3-crystallin peptide separately and incubated at 37°C. ßL-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 ßB3-peptide on ßL-crystallin was due to increased crystallin precipitation, we processed the heat-denatured ßL-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 ßL-Crystallin
Oxidized ßB3-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 ßL-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 ßL-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 ßB3-peptide and control oxidized peptides were treated with ßL-crystallin, and the precipitate was photolyzed. The photolyzed complex of ßL-crystallin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the photo-incorporation of sulfo-SBED cross-linker to the ßL-crystallin was confirmed by Western blot with avidin horseradish peroxidase (HRP). The photolyzed complex of ßL-crystallin and oxidized ßB3-peptide was then processed, as described previously.22 Biotinylated ßL-crystallin peptides were enriched by monomeric avidin gel (ImmunoPure; Pierce), as described previously.22 Sulfo-SBED–labeled ßL-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
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The extent of H2O2- and Cu2+-induced oxidation of the ßB3-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 ßB3-Crystallin Peptide on Aggregation of ßL-Crystallin
Thermal denaturation of ßL-crystallin resulted in aggregation and light-scattering. The addition of the oxidized ßB3-crystallin peptide (23 µM) enhanced the light-scattering of denaturing ßL-crystallin, compared with light-scattering with nonoxidized ßB3-peptide. Furthermore, the increased light-scattering by oxidized ßB3-peptide was concentration dependent (Fig. 1) . Use of an increased amount of oxidized ßB3-peptide (58 µM) resulted in >80% increased light-scattering. In contrast, the addition of nonoxidized ßB3-peptide (23–58 µM) did not result in a similar increase. At higher concentrations of oxidized ßB3-peptide, the aggregated crystallin started precipitating (Fig. 1) . However, enhanced light-scattering was not due to the added ßB3-peptides, as peptides themselves did not show any light-scattering under the experimental conditions (Fig. 1) . Our data indicate that the oxidation of ßB3-peptide was necessary to exhibit the enhanced light-scattering, as the nonoxidized ßB3-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 ßB3-peptide and ßL-crystallin was conducted at 37°C for an extended time to determine the effect of peptides on ßL-crystallins at a physiological temperature. The degree of light-scattering by ßL-crystallin itself or in the presence of nonoxidized ßB3-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 ßL-crystallin in the presence of oxidized ßB3-peptide was considerably higher than with ßL-crystallin alone (Fig. 2) , indicating a physiologically significant peptide interaction with lens crystallins, causing light-scattering.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Facilitated aggregation of ßL-crystallin (3.8 µM) in the presence of oxidized and nonoxidized ßB3-peptide and control peptides at 55°C. ßL-Crystallin alone (•); ßL-crystallin+23 µM oxidized ßB3-peptide ({blacktriangleup}); ßL-crystallin+34 µM oxidized ßB3-peptide ({square}); ßL-crystallin+58 µM oxidized ßB3-peptide ({blacksquare}); ßL-crystallin+58 µM nonoxidized ßB3-peptide ({triangleup}); ßL-crystallin+63 µM oxidized human {alpha}B-peptide 42TSLSPFYLRPPSFLRAPSWF61 ({diamondsuit}); ßL-crystallin+69 µM oxidized non-lenticular peptide DRRIFWWSLRSAPG ({circ}); and 58 µM oxidized ßB3-peptide alone ({diamond}).

 


View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. Facilitated aggregation of ßL-crystallin (3.8 µM) in the presence of oxidized and nonoxidized ßB3-peptide and control peptides at 37°C. Symbols are as described in Figure 1 .

 
After demonstrating the ability of the oxidized ßB3-crystallin peptide to enhance the relative light-scattering of denaturing ßL-crystallin, we investigated the aggregation-influencing property of the oxidized ßB3-peptide in the presence of {alpha}-crystallin at 55°C. As expected, {alpha}-crystallin suppressed the aggregation of denatured ßL-crystallin due to its chaperoning property (data is not shown). The data were similar to that published earlier (see Fig. 6 in Ref. 8 ). Further, light-scattering was not observed with {alpha}-crystallin alone or when {alpha}-crystallin and oxidized peptides were used together in the assay. The data show the ability of the oxidized ßB3-peptide to interact with target protein despite a chaperone protein in the system. The increased light-scattering suggests that oxidized ßB3-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 ßB3-peptide may bind to {alpha}-crystallin and disturb its chaperone property. Further studies are needed to determine the mechanism.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 6. MALDI-ToF MS spectrum of sulfo-SBED–labeled ßL-crystallin peptides purified by reversed-phase HPLC on a C-18 column after monomeric avidin gel chromatography.

 
The oxidized-peptide–induced exposure of hydrophobic sites in ßL-crystallin at 37°C was demonstrated by increased bis-ANS binding (Fig. 3) . In a separate set of experiments, ßL-crystallin was treated with oxidized or nonoxidized ßB3-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 ßL-crystallin, as well as to peptide-treated ßL-crystallin, the oxidized ßB3-peptide–treated ßL-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 ßL-crystallin control and the nonoxidized peptide–treated ßL-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 ßL-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 ßL-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 ßB3-peptide and ßL-crystallin (Fig. 3) .



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Bis-ANS fluorescence of ßL-crystallin incubated at 37°C and treated with oxidized and nonoxidized ßB3-peptide and control peptide at 37°C. In each assay 3.8 µM of ßL-crystallin was used, and 0.66 µM was withdrawn at timed intervals and treated with bis-ANS (20 µM). ßL-Crystallin alone (•); ßL-crystallin+34 µM oxidized ßB3-peptide ({blacksquare}); ßL-crystallin+34 µM nonoxidized ßB3-peptide ({diamondsuit}); and ßL-crystallin+69 µM control oxidized peptide, DRRIFWWSLRSAPG ({triangleup}). Oxidized peptide and control peptide showed low bis-ANS binding (<1 unit).

 
The increased aggregation of ßL-crystallins in the presence of oxidized ßB3-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 ßL-crystallin increased when the oxidized ßB3-peptide interacted with it, compared with ßL-crystallin alone or when nonoxidized ßB3-peptide was included in the assay with ßL-crystallin. Relatively higher absorbance was observed in a protein peak eluting at 25.6 minutes (Fig. 4 , trace b), when oxidized ßB3-peptide and ßL-crystallin were used compared with ßL-crystallin alone or ßL-crystallin treated with nonoxidized ßB3-peptide. The protein peak eluting at 25.6 minutes in Figure 4 corresponded to ßB2-crystallin (on the basis of MS analysis). Therefore, it appears that oxidized ßB3-peptide preferentially interacts with ßB2-crystallin and enhances its aggregation and precipitation.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4. Reversed-phase HPLC chromatogram of an aggregation precipitate of ßL-crystallin (3.8 µM) dissolved in 6 M urea. ßL-Crystallin alone (trace a); ßL-crystallin treated with 34 µM oxidized ßB3-crystallin peptide (trace b); and ßL-crystallin treated with 34 µM nonoxidized ßB3-crystallin peptide (trace c).

 
Oxidized ßB3-Peptide Binding Sites in ßL-Crystallin
The oxidized ßB3-peptide interacting sites in ßL-crystallin were identified by the sulfo-SBED–labeling method.22 23 The photoinsertion of sulfo-SBED to the ßL-crystallin was confirmed by SDS-PAGE and Western blot analysis (Fig. 5) . The Western blot data showed the interaction of oxidized ßB3-peptide with ß-crystallin fraction and ßB2-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 ßL-crystallins.



View larger version (64K):
[in this window]
[in a new window]
  		FIGURE 5. SDS-PAGE (A) and Western blot (B) analyses of an aggregation precipitate of ßL-crystallin treated with sulfo-SBED–labeled peptides. Lanes 1 and 6: ßL-crystallin treated with the control oxidized peptide DRRIFWWSLRSAPG; lanes 2 and 5: ßL-crystallin treated with nonoxidized ßB3-peptide; lanes 3 and 4: ßL-crystallin treated with oxidized ßB3-peptide; and lane 7: prestained molecular weight marker.

 
To identify the peptide interaction sites in ßL-crystallin, the trypsin digest of ßL-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 6 were 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 7 shows 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 ßB2-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.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Peptide Sequences of Sulfo-SBED-Labeled ßB2-Crystallin Peptides Seen in MALDI-ToF MS Spectrum

 


View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 7. Nanospray QqToF MS/MS spectrum of sulfo-SBED–labeled ßB2-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.

 
The sulfo-SBED–labeled peptides identified (Table 1) suggest that the oxidized ßB3-peptide preferentially interacted with ßB2-crystallin. The oxidized ßB3-peptide interacted with amino acid residues in N- and C-terminal extensions of the ßB2-crysallin (Fig. 8) . Apart from two terminal extensions, the oxidized peptide also interacted with amino acid residues in other regions of ßB2-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 ßB2-crystallin are solvent-exposed and flexible.30 31 32 Of the oxidized ßB3-crystallin peptide–interacting regions in ßB2-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 ßB2-crystallin dimer or the QR interface of the ßB2-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 ß-crystallins, the tertiary structure of ßB2-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.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 8. Amino acid sequence of bovine ßB2-crystallin (1-204). Bold sequences: oxidized ßB3-crystallin peptide interaction regions.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
ß-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, ß-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 {alpha}-, ß-, or {gamma}-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 ßL-crystallin aggregation by crystallin fragments derived from oxidized ßL-crystallin.8 Because ß-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 ßB3-peptide and the ßL-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 ßB3-peptide and {gamma}- and {alpha}-crystallin.22 Our current data show the interaction of oxidized ßB3-peptide with lens ß-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 proteins43 44 and the exposure of the hydrophobic sites has been reported.43 The results of this study suggest that the binding of oxidized ßB3-peptide to the ßL-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 ßL-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 ßB3-peptide and ßL-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 ßB3-crystallin peptide binds to the crystallin protein and induces conformational change, leading to additional hydrophobic interaction between the exposed hydrophobic sites.

Lens ß-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 ß-crystallin oligomers and with other lens proteins.45 ßB2-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 {alpha}-crystallin becomes water insoluble.9 19 In addition, ßB2-crystallin subunit not only self-associates to a homodimer but also readily forms a heterodimer with ßB3- and ßA4-crystallins and a larger aggregate with ßA3-crystallin.12 Our study revealed the preferential interaction between oxidized ßB3-peptide and ßB2-crystallin, which suggests the susceptibility of ßB2-crystallin toward oxidized ßB3-peptides. MS/MS analysis of oxidized ßB3-crystallin peptide–interacted sites in ßB2-crystallin (Table 1 , Fig. 8 ) indicates that oxidized peptide readily interacts with amino acid residues in both N- and C-terminal ends of ßB2-crystallin. The N- and C-terminal ends of ßB2-crystallin are solvent-exposed and flexible in the ßB2-dimer.30 31 The N-terminal end of ßB2-crystallin is flexible, even in the tetramer and higher-order aggregation state, whereas the C-terminal end of ßB2-crystallin may be involved in tetramer and higher-order aggregation of ß-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 ßB3-peptide also interacted with amino acid residues on several regions of ßB2-crystallin, as indicated in Table 1 and 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 ßB2-crystallin at a physiological temperature suggest that binding of peptides and a change in the conformation of ßB2-crystallin could interfere with association or dissociation and subunit exchange within the ß-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 study22 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 {alpha}-, ß-, and {gamma}-crystallins in aged and cataractous human lenses.


   Acknowledgements
 
The authors thank Beverly DaGue (Proteomics Center, University of Missouri, Columbia, MO) for performing the mass spectrometry analysis.


   Footnotes
 
Supported by National Eye Institute Grants EY09855 and EY014795 and by a departmental grant from Research to Prevent Blindness, Inc.

Submitted for publication January 10, 2005; revised March 18, 2005; accepted March 31, 2005.

Disclosure: E.G.P. Udupa, None; K.K. Sharma, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: K. Krishna Sharpa, Department of Ophthalmology, University of Missouri, Columbia, MO 65212; sharmak{at}health.missouri.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. David LL, Shearer TR, Shih M. Sequence analysis of lens ß-crystallins suggests involvement of calpain in cataract formation. J Biol Chem. 1993;268:1937–1940.[Abstract/Free Full Text]
  2. Zhan H, Yamamoto Y, Shumiya S, et al. Peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses derived from Shumiya cataract rats. Histochem J. 2001;33:511–521.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Baruch A, Greenbaum D, Levy ET, et al. Defining a link between gap junction communication, proteolysis, and cataract formation. J Biol Chem. 2001;276:28999–29006.[Abstract/Free Full Text]
  4. Roy D, Garner MH, Spector A, Carper D, Russell P. Investigation of Nakano lens proteins. Exp Eye Res. 1982;34:909–920.[CrossRef][ISI][Medline][Order article via Infotrieve]
  5. Horwitz J, Hansen JS, Cheung CC, et al. Presence of low molecular weight polypeptides in human brunescent cataracts. Biochem Biophys Res Commun. 1983;113:65–71.[CrossRef][ISI][Medline][Order article via Infotrieve]
  6. Srivastava OP. Age-related increase in concentration and aggregation of degraded polypeptides in human lenses. Exp Eye Res. 1988;47:525–543.[CrossRef][ISI][Medline][Order article via Infotrieve]
  7. Srivastava OP, Srivastava K, Silney C. Levels of crystallin fragments and identification of their origin in water soluble high molecular weight (HMW) proteins of human lenses. Curr Eye Res. 1996;15:511–520.[ISI][Medline][Order article via Infotrieve]
  8. Senthilkumar R, Chaerkady R, Sharma KK. Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens ßL-crystallins. J Biol Chem. 2002;277:39136–39143.[Abstract/Free Full Text]
  9. Ma Z, Hanson SR, Lampi KJ, David LL, Smith DL, Smith JB. Age-related changes in human lens crystallins identified by HPLC and mass spectrometry. Exp Eye Res. 1998;67:21–30.[CrossRef][ISI][Medline][Order article via Infotrieve]
  10. Hejtmancik JF, Wingfield PT, Chambers C, et al. Association properties of ßB2- and ßA3-crystallin: ability to form dimers. Protein Eng. 1997;10:1347–1352.[Abstract/Free Full Text]
  11. Hejtmancik JF, Wingfield PT, Sergeev YV. ß-Crystallin association. Exp Eye Res. 2004;79:377–383.
  12. Slingsby C, Bateman OA. Quaternary interactions in eye lens ß-crystallins: basic and acidic subunits of ß-crystallins favor heterologous association. Biochemistry. 1990;29:6592–6599.[CrossRef][Medline][Order article via Infotrieve]
  13. Werten PJ, Lindner RA, Carver JA, de Jong WW. Formation of ßA3/ßB2-crystallin mixed complexes: involvement of N- and C-terminal extensions. Biochim Biophys Acta. 1999;1432:286–292.[CrossRef][Medline][Order article via Infotrieve]
  14. Norledge BV, Mayr EM, Glockshuber R, et al. The X-ray structures of two mutant crystallin domains shed light on the evolution of multi-domain proteins. Nat Struct Biol. 1996;3:267–274.[CrossRef][ISI][Medline][Order article via Infotrieve]
  15. Nalini V, Bax B, Driessen H, Moss DS, Lindley PF, Slingsby C. Close packing of an oligomeric eye lens ß-crystallin induces loss of symmetry and ordering of sequence extensions. J Mol Biol. 1994;236:1250–1258.[CrossRef][ISI][Medline][Order article via Infotrieve]
  16. Zhang Z, David LL, Smith DL, Smith JB. Resistance of human ßB2-crystallin to in vivo modification. Exp Eye Res. 2001;73:203–211.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Bateman OA, Slingsby C. Structural studies on ßH-crystallin from bovine eye lens. Exp Eye Res. 1992;55:127–133.[ISI][Medline][Order article via Infotrieve]
  18. Zhang Z, Smith DL, Smith JB. Human ß-crystallins modified by backbone cleavage, deamidation and oxidation are prone to associate. Exp Eye Res. 2003;77:259–272.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Hanson SR, Hasan A, Smith DL, Smith JB. The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage. Exp Eye Res. 2000;71:195–207.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Srivastava OP, Kirk MC, Srivastava K. Characterization of covalent multimers of crystallins in aging human lenses. J Biol Chem. 2004;279:10901–10909.[Abstract/Free Full Text]
  21. Harrington V, McCall S, Huynh S, Srivastava K, Srivastava OP. Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses. Mol Vis. 2004;10:476–489.[ISI][Medline][Order article via Infotrieve]
  22. Udupa E G P, Sharma KK. Effect of oxidized ßB3-crystallin peptide (152-166) on thermal aggregation of bovine lens {gamma}-crystallin: identification of peptide interacting sites. Exp Eye Res. 2005;80:185–196.[CrossRef][ISI][Medline][Order article via Infotrieve]
  23. Santhoshkumar P, Sharma KK. Identification of a region in alcohol dehydrogenase that binds to {alpha}-crystallin during chaperone action. Biochim Biophys Acta. 2002;1598:115–121.[Medline][Order article via Infotrieve]
  24. Sharma KK, Ortwerth BJ. Effect of cross-linking on the chaperone-like function of {alpha}-crystallin. Exp Eye Res. 1995;61:413–421.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Das KP, Petrash JM, Surewicz WK. Conformational properties of substrate proteins bound to a molecular chaperone {alpha}-crystallin. J Biol Chem. 1996;271:10449–10452.[Abstract/Free Full Text]
  26. Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76–85.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Kato Y, Uchida K, Kawakishi S. Oxidative fragmentation of collagen and prolyl peptide by Cu (II)/H2O2: conversion of proline residue to 2-pyrrolidone. J Biol Chem. 1992;267:23646–23651.[Abstract/Free Full Text]
  28. Chamberlain AK, Marqusee S. Comparison of equilibrium and kinetic approaches for determining protein folding mechanisms. Adv Prot Chem. 2000;33:283–327.
  29. Yates JR, III, Carmack E, Hays L, Link AJ, Eng JK. Automated protein identification using micro column liquid chromatography tandem mass spectrometry. Methods Mol Biol. 1999;112:553–569.[Medline][Order article via Infotrieve]
  30. Bax B, Lapatto R, Nalini V, et al. X-ray analysis of ßB2-crystallin and evolution of oligomeric lens proteins. Nature. 1990;347:776–780.[CrossRef][Medline][Order article via Infotrieve]
  31. Carver JA, Cooper PG, Truscott RJ. 1H-NMR spectroscopy of ßB2-crystallin from bovine eye lens: conformation of the N- and C-terminal extensions. Eur J Biochem. 1993;213:313–320.[ISI][Medline][Order article via Infotrieve]
  32. Cooper PG, Carver JA, Truscott RJ. 1H-NMR spectroscopy of bovine lens ß-crystallin: the role of the ßB2-crystallin C-terminal extension in aggregation. Eur J Biochem. 1993;213:321–328.[ISI][Medline][Order article via Infotrieve]
  33. Chiou SH, Azari P, Himmel ME, Lin HK, Chang WP. Physicochemical characterization of ß-crystallins from bovine lenses: hydrodynamic and aggregation properties. J Protein Chem. 1989;8:19–32.[CrossRef][ISI][Medline][Order article via Infotrieve]
  34. Shearer TR, David LL, Anderson RS, Azuma M. Review of selenite cataract. Curr Eye Res. 1992;11:357–369.[ISI][Medline][Order article via Infotrieve]
  35. Sharma KK, Kester K. Peptide hydrolysis in lens: role of leucine aminopeptidase, aminopeptidase III, prolyloligopeptidase and acylpeptidehydrolase. Curr Eye Res. 1996;15:363–369.[ISI][Medline][Order article via Infotrieve]
  36. Chongcharoen K, Sharma KK. Characterization of trypsin-modified bovine lens acylpeptide hydrolase. Biochem Biophys Res Commun. 1998;247:136–141.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Kamei A. Characterization of water-insoluble proteins in normal and cataractous human lens. Jpn J Ophthalmol. 1990;34:216–224.[Medline][Order article via Infotrieve]
  38. Srivastava OP, Ortwerth BJ. Age-related and distributional changes in the trypsin inhibitor activity of bovine lens. Exp Eye Res. 1983;36:695–709.[CrossRef][ISI][Medline][Order article via Infotrieve]
  39. David LL, Shearer TR. Purification of calpain II from rat lens and determination of endogenous substrates. Exp Eye Res. 1986;42:227–238.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Sharma KK, Ortwerth BJ. Aminopeptidase III activity in normal and cataractous lenses. Cur Eye Res. 1986;5:373–380.
  41. Harding JJ. Conformational changes in human lens proteins in cataract. Biochem J. 1972;129:97–100.[ISI][Medline][Order article via Infotrieve]
  42. Lapko VN, Purkiss AG, Smith DL, Smith JB. Deamidation in human {gamma}S-crystallin from cataractous lenses is influenced by surface exposure. Biochemistry. 2002;41:8638–8648.[CrossRef][Medline][Order article via Infotrieve]
  43. Randall LL. Peptide binding by chaperone SecB: implications for recognition of nonnative structure. Science. 1992;257:241–245.[Abstract/Free Full Text]
  44. Sato AK, Zarutskie JA, Rushe MM, et al. Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1. J Biol Chem. 2000;275:2165–2173.[Abstract/Free Full Text]
  45. Werten PJ, Carver JA, Jaenicke R, de Jong WW. The elusive role of the N-terminal extension of ßA3- and ßA1-crystallin. Protein Eng. 1996;9:1021–1028.[Abstract/Free Full Text]
  46. Le Breton ER, Carver JA. Solution conformation of bovine lens {alpha}- and ßB2-crystallin terminal extensions. Int J Peptide Protein Res. 1996;47:9–19.[ISI][Medline][Order article via Infotrieve]
  47. Hejtmancik JF, Piatigorsky J. Lens Proteins and their molecular biology. Albert DM Jakobiec FA Azar DT Gragoudas ES eds. Principles and Practice of Ophthalmology. 2000;1409–1428. WB Saunders Philadelphia.
  48. Pereira P, Shang F, Hobbs M, Girao H, Taylor A. Lens fibers have a fully functional ubiquitin-proteasome pathway. Exp Eye Res. 2003;76:623–631.[CrossRef][ISI][Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
P. Santhoshkumar, P. Udupa, R. Murugesan, and K. K. Sharma
Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation
J. Biol. Chem., March 28, 2008; 283(13): 8477 - 8485.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Udupa, E. G. P.
Right arrow Articles by Sharma, K. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Udupa, E. G. P.
Right arrow Articles by Sharma, K. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS