Identification of 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic Acid Binding Sequences in alpha -Crystallin*

K. Krishna SharmaDagger §, G. Suresh Kumar§, A. Scott Murphy§, and Kathryn Kester§

From the § Mason Eye Institute, Department of Ophthalmology and Dagger  Department of Biochemistry, University of Missouri, Columbia, Missouri 65212.

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The hydrophobic binding sites in alpha -crystallin were evaluated using fluorescent probes 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS), 8-anilino-1-naphthalene sulfonate (ANS), and 1-azidonaphthalene 5-sulfonate (1,5-AZNS). The photolysis of bis-ANS-alpha -crystallin complex resulted in incorporation of the probe to both alpha A- and alpha B-subunits. Prior binding of denatured alcohol dehydrogenase to alpha -crystallin significantly decreased the photoincorporation of bis-ANS to alpha -crystallin. Localization of bis-ANS incorporated into alpha A-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In alpha B-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of alpha A-crystallin and DRFSVNLNVK and VLGDVIEVHGK of alpha B-crystallin were earlier identified as part of the sequences involved in their interaction with target proteins during the molecular chaperone-like action. The hydrophobic probe, 1,5-AZNS, also interacted with both subunits of alpha -crystallin. Localization of 1,5-AZNS binding site in alpha B-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of alpha -crystallin. Glycated alpha -crystallin displayed decreased ANS fluorescence and loss of chaperone-like function, suggesting the involvement of glycation site as well as ANS binding site in chaperone-like activity display.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

alpha -Crystallin is one of the most predominant eye lens proteins. Its concentration in lens fiber cells is about 40% of the total protein in lens (1). alpha -Crystallin exists as a polydisperse aggregate with an average molecular mass of 800 kDa (2). The two types of subunits, designated alpha A and alpha B, each of which has a molecular mass of 20 kDa, arrange themselves in yet undefined ways to form the aggregate (2). During aging, alpha -crystallin undergoes extensive modifications culminating in the formation of super aggregates and highly cross-linked light-scattering molecules (2). The sequences of the subunits of alpha -crystallin have high homology to small heat shock proteins (3, 4) and are highly conserved between species. alpha -Crystallin subunits, once thought to be lens-specific, are now widely known to be present in other tissues as well (5-8). Despite extensive studies carried out in the past, the quarternary structure or the structure-function of alpha -crystallin or its subunits has remained an enigma and challenge for researchers.

Recently, the ability of native alpha -crystallin to suppress the aggregation of heat-denatured (9-20), UV-irradiated (20, 21), as well as chemically denatured (22) proteins and enzymes has been demonstrated. It has been proposed that surface hydrophobic sites in the alpha -crystallin aggregate are involved in the binding of alpha -crystallin to target proteins during the display of chaperone-like activity (13, 23). A direct correlation between the extent of alpha -crystallin hydrophobicity and chaperone-like activity has been demonstrated by several studies (13, 23-27). However, the amino acid sequence that contributes to the site responsible for the binding of denatured proteins and hydrophobic site specific probes is not fully understood.

We have shown earlier that both A- and B-subunits in alpha -crystallin interact with bis-ANS1 in 1:1 stoichiometry at 37 °C (26). The number of bis-ANS molecules binding to alpha -crystallin increases if the protein is exposed to higher temperatures or denaturing agents prior to the addition of the fluorophore (26, 27). Furthermore, it has been shown that binding of bis-ANS to alpha B-crystallin (25) or alpha -crystallin (26) diminishes the chaperone-like activity of the protein. In the present study we have determined the bis-ANS binding sequences in alpha -crystallin by photocross-linking, peptide mapping and sequencing. The data presented here also show that the bis-ANS binding sequences are also the chaperone sites in alpha -crystallin.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- bis-ANS, ANS, and 1,5-AZNS were obtained from Molecular Probes, Inc. (Junction City, OR). The stock solutions of bis-ANS were prepared in 95% alcohol, and the concentration was determined by absorbance at 385 nm using an extinction coefficient, epsilon  385 = 16,790 cm-1 M-1 (28). Lysyl endopeptidase was purchased from Wako Bioproducts. Sequence grade trypsin was obtained from Sigma. beta L-crystallin (29) was isolated from bovine lenses (15). All other chemicals were of the highest grade commercially available.

Preparation of alpha -Crystallin-- alpha -Crystallin was isolated from young bovine lens cortex by gel filtration on Sephadex G-200 and ion-exchange chromatography on trimethylaminoethyl-fractogel column (EM-Separations) as described earlier (26, 30). The alpha -crystallin thus obtained was >99% pure as judged by SDS-PAGE, and this preparation was used in this study.

Photoincorporation of Bis-ANS into alpha -Crystallin-- Photoincorporation of bis-ANS to alpha -crystallin was carried out as described earlier (26), with slight modification of the original procedure described by Seale et al. (31). Following photolysis, the sample was analyzed by HPLC and SDS-PAGE, and the fluorescence associated with protein bands was documented by photography using TMAX 100 film (Eastman Kodak Co.) under UV light (360 nm). The gel was later stained with Coomassie Blue. The efficiency of bis-ANS incorporation to alpha -crystallin during 15-min photolysis was determined by quantitative densitometry function of Image-1 system (Universal Imaging Corp.).

To investigate whether prior binding of denatured proteins to alpha -crystallin prevents bis-ANS binding and photoincorporation, alpha -crystallin and alcohol dehydrogenase (ADH) (1:6 ratio) were incubated at 48 °C for 1 h. Following incubation, the reaction mixture was cooled to 25 °C, and bis-ANS was added. The final bis-ANS concentration was 12.5 µM. Photolysis of the sample was carried out as above for 15 min, and the aliquots were subjected to SDS-PAGE under reducing conditions. The fluorescent bands were photographed as above and the gel was stained with Coomassie Blue.

Separation of Bis-ANS-labeled alpha A- and alpha B-crystallin-- The photolyzed alpha -crystallin-bis-ANS complex was treated with 5 mM dithiothreitol for 2 h and filtered. The alpha A- and alpha B-subunits were separated from one another by HPLC using a C18 column (218TP1010 from The Separation Group, Hesparia, CA) and linear gradient (0-60% over a period of 1 h) formed between 0.065% trifluoroacetic acid in water and 0.065% trifluoroacetic acid in acetonitrile. The flow rate was 1 ml/min. The elution was monitored by absorbance (280 nm) and fluorescence (390 nm excitation and 490 nm emission). bis-ANS-labeled alpha A- and alpha B-crystallins were further purified by SDS-PAGE and recovered by electroelution, and the SDS was removed by ether precipitation (32).

Identification of Bis-ANS-labeled Peptides-- The bis-ANS-labeled alpha A- and alpha B-crystallins were digested with lysyl endopeptidase (1:30, enzyme/protein) for 4 h at 37 °C. The peptides were separated by reverse phase HPLC on a Vydac C18 column (218TP54) equilibrated with 20 mM sodium phosphate buffer, pH 6.5 + 5% acetonitrile (solvent A). The elution of bound peptides was carried out with a linear gradient (0-60%) formed by solvent A and solvent B (20 mM phosphate buffer, pH 6.5, in 95% acetonitrile). A flow rate of 1 ml/min over 120 min was maintained, and 1-ml fractions were collected. The elution was monitored at 220 nm for absorption and fluorescence (390 nm excitation and 490 nm emission). The amino-terminal sequences of bis-ANS-labeled peptides were determined by Edman degradation on an Applied Biosystems PROCISE CLC protein sequencing system.

Photoincorporation of 1,5-AZNS to alpha -Crystallin and Identification of Binding Site in alpha B-crystallin-- The photoincorporation of 1,5-AZNS to alpha -crystallin was accomplished using a procedure described by Dockter and Koseki (33). 1 mM 1,5-AZNS and 1.25 µM alpha -crystallin were used in this study. Following photoincorporation, A- and B- subunits of alpha -crystallin were separated by HPLC as above using a C18 column. Although the fluorophore was incorporated to both the subunits, only alpha B-subunit was further analyzed to determine the 1,5-AZNS incorporation site. Labeled alpha B-crystallin was digested with sequencing grade trypsin (1:50 ratio), and the resulting peptides were separated as described earlier (30). The elution profile was monitored at 220 nm. All fractions were tested for fluorescence in a Perkin-Elmer Spectrophotometer model 650-40 (excitation and emission maxima of 334 and 440 nm, respectively). The major fluorescent peptide eluting at 45 min from the HPLC column was subjected to amino acid sequencing in an Applied Biosystems 470A sequencer.

Glycation of alpha -Crystallin, Chaperone Assay, and ANS Binding-- Glycation of alpha -crystallin was carried out in 0.1 M phosphate buffer, pH 7.0, using 10 mg/ml protein and 20 mM L-ascorbic acid (34). After incubation at 37 °C for 4 weeks, the reaction mixture was dialyzed, and the glycated protein was tested with beta L-crystallin for chaperone-like activity (15). The interaction of glycated alpha -crystallin (0.25 µM) with ANS was examined by fluorescence measurement in a Perkin-Elmer Spectrofluorimeter model 650-40. The samples with ANS were excited at 390 nm, and the emission was measured at 490 nm in a cuvette with 1-cm path length and slit width of 5 nm. The ratio of protein to probe was approximately 1:50. alpha -Crystallin incubated without ascorbic acid and processed similarly was used as the control.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Interaction of Bis-ANS with alpha -Crystallin-- The binding of bis-ANS, the environment-sensitive probe, to alpha -crystallin results in severalfold increases in fluorescence intensity of the probe, and the emission maxima is blue shifted to ~490 nm from its emission maximum of 533 nm in aqueous medium (27). We have shown recently that bis-ANS interacts strongly with alpha -crystallin, and the bound fluorophore cannot be removed by dialysis (26). Covalent bonds are formed between protein-bound bis-ANS and the amino acids forming the binding pocket when the complexes are exposed to long UV light (26, 31). The photoincorporation of bis-ANS to alpha -crystallin is directly proportional to the duration of photolysis. By image analysis we estimated that about 15% of the bound bis-ANS covalently cross-links to alpha -crystallin in the 15 min of photolysis used in our study (Fig. 1, lane 1). Although photolysis for longer durations results in higher amount of photoincorporation, there is increased subunit cross-linking and generation of high molecular weight species. Therefore the studies described here were limited to 15 min of photolysis. As alpha -crystallin-bis-ANS was dialyzed to remove the free bis-ANS prior to photolysis, it is unlikely that nonspecific incorporation of the fluorophore to alpha -crystallin occurred during our experiments with bis-ANS and alpha -crystallin.


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Fig. 1.   Bis-ANS labeling of alpha -crystallin in the presence and absence of alcohol dehydrogenase. The experimental details are under "Experimental Procedures." Lanes 1 and 3, bis-ANS + alpha -crystallin after photolysis; lanes 2 and 4, alpha -crystallin + ADH + bis-ANS after photolysis. Bis-ANS was added to sample in lanes 2 and 4 after 1-h incubation of the proteins at 48 °C. The 20- and 39-kDa bands in lanes 1 and 2 are bis-ANS-labeled alpha -crystallin and ADH, respectively. Left panel, fluorescence; right panel, Coomassie Blue R-250 stain.

Effect of Denaturing Protein Bound to alpha -Crystallin on the Photoincorporation of Bis-ANS-- Earlier we showed that prior binding of bis-ANS to alpha -crystallin diminishes the chaperone-like activity of the protein (26). To determine whether the binding of denaturing proteins to alpha -crystallin at the chaperone site can affect subsequent bis-ANS photoincorporation, alpha -crystallin and ADH (1: 6 ratio) were incubated at 48 °C for 1 h prior to the addition of bis-ANS and photolysis. SDS-PAGE of such an experiment is shown in Fig. 1. The result shows a significant decrease in photoincorporation of bis-ANS to alpha -crystallin when ADH was heat-denatured and allowed to bind to alpha -crystallin prior to the addition of the fluorophore (compare lanes 1 and 2 in Fig. 1).

Photoincorporation of Bis-ANS to alpha -Crystallin Subunits and Localization of Incorporated Bis-ANS-- To determine the bis-ANS binding sites in alpha -crystallin, the fluorophore was initially allowed to bind to purified alpha -crystallin by the addition of saturating amounts of the probe and removal of the excess by dialysis to minimize nonspecific photoincorporation of free bis-ANS activated during photolysis. Photolysis of the alpha -crystallin-bis-ANS complex by UV-A light (366 nm) resulted in covalent incorporation of the fluorophore to both alpha A- and alpha B-subunits as we reported earlier (26).

In order to identify the sites of bis-ANS incorporation into alpha A- and alpha B-crystallins, alpha -crystallin was modified with bis-ANS, and the subunits were separated by HPLC and purified by SDS-PAGE. The purified bis-ANS-modified proteins were later digested with lysyl endopeptidase. The resulting fluorescent peptides were separated from other peptides by reverse phase HPLC. The HPLC profile of alpha A-crystallin peptides and the fluorescence is shown in Fig. 2. Edman degradation of fluorescent peptides eluting at 37 and 40 min revealed the same NH2-terminal sequence HFSPE. These peptides probably have the sequence HFSPEDLTVK and HFSPEDLTVKVQEDFVEIHGK, corresponding to residues 79-88 and 79-99 of alpha A-crystallin (Fig. 3), since we used lysyl endopeptidase to digest alpha A-crystallin. Incomplete digestion at Lys-88 due to the bis-ANS incorporation at/or near Lys-88 may have generated the latter peptide. Furthermore, the peptide eluting at 40 min yielded low levels of Phe (10 pmol) in the second cycle compared with 20 pmol of Ser in the third sequencing cycle, indicating that Phe-80 was modified by bis-ANS during photoincorporation. We could not determine the location of bis-ANS insertion site in peptide eluting at 37 min by examining the results of five sequence cycles used to determine the identity of the peptide.


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Fig. 2.   HPLC separation of bis-ANS-labeled alpha A-crystallin peptides. alpha A-crystallin was isolated from the bis-ANS-labeled alpha -crystallin and digested with lysyl endopeptidase as described under "Experimental Procedures." The peptides were separated by reverse phase HPLC, and the relative fluorescence is shown by a graph. Solid line, fluorescence (390 excitation/490 emission); broken line, A220. Inset, HPLC profile of alpha A-peptide eluting at 89 min in Fig. 2 digested with trypsin.


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Fig. 3.   alpha A- and alpha B-crystallin sequence showing the bis-ANS binding region. The bis-ANS binding sequences are shown in bold. The alcohol dehydrogenase binding sequences in alpha B-crystallin and mellitin binding sequence in alpha A-crystallin are taken from Refs. 30 and 40, respectively, and underlined. The mellitin binding region in alpha B-crystallin marked with a double underline is taken from Ref. 40. The 1,5-AZNS binding sequence in alpha B-crystallin is shown in italics.

The fluorescent peptide eluting at 89 min showed the NH2-terminal sequence RTLGPF. The peptide showed a mobility equivalent to 6.5 kDa on SDS-PAGE (data not shown). Therefore, the peptide eluting at 89 min is likely to represent the residues 12-70 of alpha A-crystallin. Analysis of the fluorescent material eluting at 72 min failed to show any amino acid during sequencing cycles. Since the same peak also did not contain appreciable 220 nm absorption, it is likely that the fluorescence at this region may be due to the peptide-bound unstable bis-ANS or its derivative. To localize the bis-ANS-bound amino acid in peptide eluting at 89 min (Fig. 2), the peptide was subjected to trypsin digestion and HPLC analysis. Fig. 2, inset, shows the HPLC profile of the trypsin digest. The single fluorescent peptide eluting at 66 min (Fig. 2, inset) was sequenced as described earlier. The observed sequence for the 66-min peptide, QSLFR, corresponds to residues 50-54 of alpha A-crystallin (Fig. 3). During the sequencing of this peptide the yield of Phe was low (82 pmol) compared with other amino acids (which were in the range of 110-160 pmol), suggesting a possible modification of Phe-53 in alpha A-crystallin by bis-ANS.

The two fluorescent peptides of alpha B-crystallin, generated by lysyl endopeptidase digestion, eluted from the HPLC column at 38 min as a doublet (Fig. 4). The same peaks also showed maximal fluorescence emission at 490 nm when excited at 390 nm and revealed NH2-terminal sequence DRFSV and VLGDV. These two alpha B-peptides can only be from the sequence DRFSVNLDVK and VLGDVIEVHGK (Fig. 3), since we have used lysyl endopeptidase for digestion. We were unable to conclude which of the amino acid in alpha B-crystallin formed a cross-link with bis-ANS by examining the results from five cycles of sequencing reaction used to determine the identity of the fluorescence peptide. Although the fluorescent material eluting at 72 min was the largest fluorescent peak, it gave no amino acids during sequencing cycles. Since the same peak also did not contain measurable 220 nm absorption, it is likely that the fluorescence at this region may be due to the peptide-bound unstable bis-ANS or its derivative. Since the chemistry of the reaction is not known at the present time, further studies are required to see whether the 72-in fluorescent peak was due to the bis-ANS that was originally bound to the two peptides we have identified or to a different peptide.


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Fig. 4.   HPLC separation of bis-ANS-labeled alpha B-crystallin peptides. Bis-ANS-alpha B-crystallin was prepared and digested and subjected to HPLC separation as described under "Experimental Procedures." Solid line, fluorescence (390 excitation/490 emission); broken line, A220.

Interaction of 1,5-AZNS with alpha -Crystallin-- 1,5-AZNS is another, less specific, photoreactive agent used to study the hydrophobic sites in proteins (33, 35). When bovine alpha -crystallin was treated with 1 mM 1,5-AZNS at 25 °C and photolyzed, about ~40 nmol of 1,5-AZNS was incorporated to each mole of alpha -crystallin (800 kDa), suggesting that on average there exists one 1,5-AZNS binding site per subunit of alpha -crystallin at 25 °C. Further analysis of 1,5-AZNS-alpha -crystallin by HPLC revealed that both alpha A- and alpha B-crystallins were labeled with 1,5-AZNS during the experiment (data not shown), as with bis-ANS (26).

The 1,5-AZNS binding site in alpha B-crystallin was determined by peptide mapping and sequencing of the fluorophore-containing peptide. A peptide eluting from C18 column at 45 min (Fig. 5) with a sequence HFSPEELK was found to have incorporated 1,5-AZNS. This sequence corresponds to residues 83-90 in bovine alpha B-crystallin (Fig. 3). Examination of the chromatographic profiles obtained during the sequencing of the fluorescent peptide showed that Asp in the peptide was modified by 1,5-AZNS during photolysis. Although alpha A-crystallin was also labeled with 1,5-AZNS, we did not analyze the sample further.


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Fig. 5.   HPLC separation of 1,5 AZNS-labeled alpha B-crystallin peptides. 1,5-AZNS-alpha B-crystallin was digested with trypsin and subjected to HPLC separation as described under "Experimental Procedures." Solid line, A220; broken line, fluorescence. The arrow shows the elution region of the fluorescent peptide sequenced.

Effect of Glycation of alpha -Crystallin on Its Chaperone-like Function and Interaction with ANS-- Earlier studies have shown that glycation of alpha -crystallin reduces its chaperone-like activity (36). To determine whether this was due to an alteration of the hydrophobic chaperone site in alpha -crystallin, we measured the interaction of glycated alpha -crystallin with hydrophobic probe ANS. ANS, like bis-ANS is a polarity-sensitive reagent. alpha -Crystallin glycated with ascorbate for 4 weeks showed a 25% decrease in its ability to increase ANS fluorescence compared with the controls. When the same glycated alpha -crystallin was tested with beta L-crystallin in a heat denaturation assay (15), a marked decrease in chaperone-like activity was observed (Fig. 6).


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Fig. 6.   Effect of glycation on chaperone-like activity of alpha -crystallin. The assays were done as described under "Experimental Procedures" using beta L-crystallin and glycated alpha -crystallin. Squares, beta L-crystallin; circles, beta L-crystallin + alpha -crystallin; triangles, beta L-crystallin + glycated alpha -crystallin. 200 µg of beta L-crystallin and 30 µg of alpha -crystallin or glycated alpha -crystallin were used in this study.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The presence of surface hydrophobic sites on alpha -crystallin has been known for a number of years (2). Since the demonstration of chaperone-like activity with alpha -crystallin (9), considerable interest has been shown in the hydrophobic sites within alpha -crystallins as these sites have been implicated in the chaperone-like function of the protein (13, 23-27). The hydrophobic sites in alpha -crystallin and its subunits have been studied during recent years using probes such as ANS (37), bis-ANS (26, 27), and pyrene (13). We have shown recently that UV photolysis of the alpha -crystallin-bound bis-ANS leads to photoincorporation of the fluorophore to the protein subunits similar to that seen with chaperone GroEL (31) and HSP18.1 (38) and alpha B-crystallin (25). Prior binding of denatured proteins to alpha -crystallin resulted in diminished photoincorporation of the fluorophore bis-ANS (Fig. 1, lane 2) compared with alpha -crystallin alone. Earlier we showed that prior binding of bis-ANS to alpha -crystallin partially suppresses its chaperone-like activity (26). Taken together these data suggest that both ADH and bis-ANS share common binding sites in alpha -crystallin. The sequence analysis of binding sites discussed below confirms this view. Similar sharing of bis-ANS binding site and denaturing protein binding site in heat shock protein 18.1 has been reported recently (38).

The role of hydrophobic sites and the amino acid residues that contribute to their makeup within the multimeric chaperone GroEL has been confirmed (31). The available data show that the bis-ANS binding sequences are part of the chaperone sites in GroEL (31, 39). The bis-ANS binding sites in small heat shock proteins have also been identified (38), but the chaperone sites in those proteins are yet to be determined. The two bis-ANS binding sequences in alpha A-crystallin, HFSPEDLTVK and HFSPEDLTVKVQEDFVEIHGK (Fig. 3), in part represent the chaperone site we identified earlier (40). On the basis of deuterium exchange studies Smith et al. (41) have also proposed that residues 72-75, in alpha A-crystallin, are a potential chaperone site. It should be noted that under the experimental conditions described here to determine bis-ANS binding sequences, other hydrophobic sequences in alpha A-crystallin, namely the residues 3-10, 27-37, or 130-145, did not label with bis-ANS. It is possible that those sequences may be buried inside the protein molecule. The only other sequence that was labeled with bis-ANS was QSLFR peptide. Although it is not a hydrophobic sequence by itself, the sequence can be interpreted as part of an extended hydrophobic region between residues 44 and 57 (Fig. 3). It should also be noted that none of the peptides arising from the COOH terminus of alpha A-crystallin were labeled with bis-ANS. Since a loss in chaperone activity of alpha A-crystallin has been correlated with COOH-terminal truncation (20, 42), and no bis-ANS binding site has been identified in that region, further studies are needed to determine the role of this region in chaperoning.

The two regions in alpha B-crystallin identified as bis-ANS binding sequences are at the COOH-terminal domain (Fig. 3). This is in contrast with the recent report published by Smulders and de Jong (25) on the photoincorporation of bis-ANS to recombinant rat alpha B-crystallin, where the authors observed incorporation of bis-ANS to the NH2-terminal domain of the protein. Since prior exposure of alpha -crystallin to urea can affect the bis-ANS binding (26), it is yet to be determined whether the bis-ANS binding to rat alpha B-crystallin was influenced by urea used during the isolation of the recombinant protein. Of the two bis-ANS binding sequences identified in alpha B-crystallin during the present study (Fig. 3), the FSVNLDVK portion of the DRFSVNLDVK sequence is the same as the mellitin binding sequence we determined by cross-linking studies,2 and the VLGDVIEVHGK sequence is one of the alcohol dehydrogenase binding sites determined earlier (30). The DRFSVNLDVK sequence follows the alcohol dehydrogenase interacting site in alpha B-crystallin reported by us earlier (30). In a separate experiment we have determined that another hydrophobic site-specific probe, 1,5-AZNS, binds to alpha B-crystallin sequence 83-90. The 1,5-AZNS-labeled peptide is the sequence between the two bis-ANS binding sequences in alpha B-crystallin. The structural differences between bis-ANS and 1,5-AZNS may have contributed to the difference in the site of incorporation of these two probes to alpha B-crystallin. Nevertheless, both bind at the highly conserved region of the protein (3, 4).

The two bis-ANS binding sequences in alpha B-crystallin are separated by 10 amino acids, which have a major in vitro glycation site (43). Glycation of alpha -crystallin decreases ANS binding. Glycation also reduces chaperone-like activity of alpha -crystallin (Fig. 6). The data from this study suggest that the glycation-induced loss of chaperone-like activity reported earlier (36) may be due to the modification of Lys residues 90 and 92 of alpha B-crystallin that may be part of the hydrophobic/chaperone site.

The most hydrophobic region of alpha B-crystallin, residues 28-34, proposed as a potential chaperone site by deuterium exchange studies (39) was not labeled by bis-ANS during our study. The two bis-ANS binding sequences in alpha B-crystallin identified during the present study belong to a region of high homology between HSP18.1 and alpha B-crystallin (38). Earlier studies have shown that this region in HSP18.1 is the primary bis-ANS binding region (38). On the basis of these data it can be stated that the entire third exon sequence of alpha B-crystallin, which has a high degree of homology to other heat shock proteins (3), is responsible for its chaperone-like function.

Although we estimated the binding of one bis-ANS molecule per subunit of alpha - crystallin (26), in this study we see two sequences in each subunit as bis-ANS binding sites. The occurrence of two UV-sensitive anilinonaphthalene centers in bis-ANS and the activation of either one of the centers and insertion to the adjacent amino acid in the binding pocket may be the cause for the observation of two peptides as binding sites. Alternately, multiple conformation of alpha -crystallin subunits and their interaction with bis-ANS may result in labeling of more than one peptide sequence as a binding site.

    ACKNOWLEDGEMENT

We thank Dr. B. J. Ortwerth for helpful discussions on this project and critically reading the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant EY 11981 and a grant-in aid from Research to Prevent Blindness, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 573-884-4100; E-mail: opthks{at}showme.missouri.edu.

1 The abbreviations used are: bis-ANS, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid; HPLC, high pressure liquid chromatography; 1,5-AZNS, 1-azidonaphthalene 5-sulfonate; ANS, 8-anilino-1-naphthalenesulfonate; HSP, heat shock protein; PAGE, polyacrylamide gel electrophoresis; ADH, alcohol dehydrogenase.

2 K. Krishna Sharma and G. S. Kumar, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Bloemendal, H. (1981) in Molecular and Cellular Biology of the Eye Lens (Bloemendal, H., ed), pp. 1-47, John Wiley & Sons, New York
  2. Groenen, P. J. T. A., Merck, K. B., de Jong, W. W., and Bloemendal, H. (1994) Eur. J. Biochem. 225, 1-19[Abstract]
  3. Ingolia, T. D., and Craig, E. A. (1982) Proc. Natl. Acad. Sci., U. S. A. 79, 2360-2364[Abstract]
  4. Sax, C. M., and Piatigorsky, J. (1994) Adv. Enzymol. Relat. Areas Mol. Biol. 69, 155-201[Medline] [Order article via Infotrieve]
  5. Bhat, S. P., and Nagineni, C. N. (1989) Biochem. Biophys. Res. Commun. 158, 319-325[Medline] [Order article via Infotrieve]
  6. Iwaki, T., Kume-Iwaki, T., Liem, R. K. H., and Goldman, J. E. (1989) Cell 57, 71-78[Medline] [Order article via Infotrieve]
  7. Kato, K., Shinohara, H., Kurobe, N., Goto, S., Inaguma, Y., and Ohshima, K. (1991) Biochim. Biophys. Acta 1080, 173-180[Medline] [Order article via Infotrieve]
  8. Srinivasan, A. N., Nagineni, C. N., and Bhat, S. P. (1992) J. Biol. Chem. 267, 23337-23341[Abstract/Free Full Text]
  9. Horwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10449-10453[Abstract]
  10. Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520[Abstract/Free Full Text]
  11. Rao, P. V., Horwitz, J., and Zigler, J. S., Jr. (1993) Biochem. Biophys. Res. Commun. 190, 786-793[CrossRef][Medline] [Order article via Infotrieve]
  12. Merk, K. B., Groenen, P. J. T. A., Voorter, C. E. M., de Haard-Hoekman, W. A., Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. 268, 1046-1052[Abstract/Free Full Text]
  13. Raman, B., and Rao, Ch. M. (1994) J. Biol. Chem. 269, 27264-27268[Abstract/Free Full Text]
  14. Wang, K., and Spector, A. (1994) J. Biol. chem. 269, 13601-13608[Abstract/Free Full Text]
  15. Sharma, K. K., and Ortwerth, B. J. (1995) Exp. Eye Res. 61, 413-421[Medline] [Order article via Infotrieve]
  16. Smulders, R. H. P. H., Merh, K. B., Aendekerk, J., Horwitz, J., Takemoto, L., Slingsby, C., Bloemendal, H., and de Jong, W. W. (1995) Eur. J. Biochem. 232, 834-838[Abstract]
  17. Das, K. P., Petrash, J. M., and Surewicz, W. K. (1996) J. Biol. Chem. 271, 10449-10452[Abstract/Free Full Text]
  18. Plater, M. L., Goode, D., and Crabbe, J. M. (1996) J. Biol. Chem. 271, 28558-28566[Abstract/Free Full Text]
  19. Clark, J. I., and Haung, Q. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15185-15189[Abstract/Free Full Text]
  20. Andley, U. P., Mathur, S., Griest, T. A., and Petrash, J. M. (1996) J. Biol. Chem. 271, 31973-31980[Abstract/Free Full Text]
  21. Borkman, R. F., Knight, G., and Obi, B. (1996) Exp. Eye Res. 62, 141-148[CrossRef][Medline] [Order article via Infotrieve]
  22. Farahbakhsh, Z. T., Huang, Q.-L., Ding, L.-L., Altenbach, C., Steinhoff, H.-J., Horwitz, J., and Hubbell, W. L. (1995) Biochemistry 34, 509-517[Medline] [Order article via Infotrieve]
  23. Raman, B., and Rao, Ch. M. (1997) J. Biol. Chem. 272, 23559-23564[Abstract/Free Full Text]
  24. Das, B. K., and Liang, J. N. (1997) Biochem. Biophys. Res. Commun. 236, 370-374[CrossRef][Medline] [Order article via Infotrieve]
  25. Smulders, R. H. P. H., and de Jong, W. W. (1997) FEBS Lett. 409, 101-104[CrossRef][Medline] [Order article via Infotrieve]
  26. Sharma, K. K., Kaur, H., Kumar, S. G., and Kester, K. (1998) J. Biol. Chem. 273, 8965-8970[Abstract/Free Full Text]
  27. Das, K. P., and Surewicz, W. K. (1995) FEBS Lett. 369, 321-325[CrossRef][Medline] [Order article via Infotrieve]
  28. Sudhakar, K., and Fay, P. J. (1996) J. Biol. Chem. 271, 23015-23021[Abstract/Free Full Text]
  29. Bloemendal, M., and Bloemendal, H. (1995) Exp. Eye Res. 61, 757-761[Medline] [Order article via Infotrieve]
  30. Sharma, K. K., Kaur, H., and Kester, K. (1997) Biochem. Biophys. Res. Commun. 239, 217-222[CrossRef][Medline] [Order article via Infotrieve]
  31. Seale, J. W., Martinez, J. L., and Horowitz, P. M. (1995) Biochemistry 34, 7443-7449[Medline] [Order article via Infotrieve]
  32. Hager, D. A., and Burgess, R. R. (1980) Anal. Biochem. 109, 76-86[Medline] [Order article via Infotrieve]
  33. Dockter, M. E., and Koseki, T. (1983) Biochemistry 22, 3954-3961[Medline] [Order article via Infotrieve]
  34. Prabhakaram, M., and Ortwerth, B. J. (1992) Exp. Eye Res. 55, 451-459[Medline] [Order article via Infotrieve]
  35. Stevens, D. J., and Gennis, R. B. (1980) J. Biol. Chem. 255, 379-383[Abstract/Free Full Text]
  36. Cherian, M., and Abraham, E. (1995) Biochem. Biophys. Res. Commun. 208, 675-679[CrossRef][Medline] [Order article via Infotrieve]
  37. Sun, T.-X., Das, B. P., and Liang, J. N. (1997) J. Biol. Chem. 272, 6220-6225[Abstract/Free Full Text]
  38. Lee, G. J., Roseman, A. M., Saibil, H. R., and Vierling, E. (1997) EMBO J. 16, 659-671[Abstract/Free Full Text]
  39. Fenton, W. A., Kashi, Y., Furtak, K., and Horwich, A. L. (1994) Nature 371, 614-619[CrossRef][Medline] [Order article via Infotrieve]
  40. Sharma, K. K., and Kumar, S. G. (1997) FASEB J. 11, A908 (Abstr. 9)
  41. Smith, J. B., Liu, Y., and Smith, D. L. (1996) Exp. Eye Res. 63, 125-128[CrossRef][Medline] [Order article via Infotrieve]
  42. Takemoto, L., Emmons, T., and Horwitz, J. (1993) Biochem. J. 294, 435-438[Medline] [Order article via Infotrieve]
  43. Ortwerth, B. J., Slight, S. H., Prabhakaram, M., Sun, Y., and Smith, J. B. (1992) Biochim. Biophys. Acta 1117, 207-215[Medline] [Order article via Infotrieve]


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