FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002602401> ?p ?o ?g. }

• W2002602401 endingPage "15478" @default.
• W2002602401 startingPage "15474" @default.
• W2002602401 abstract "The hydrophobic binding sites in α-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-α-crystallin complex resulted in incorporation of the probe to both αA- and αB-subunits. Prior binding of denatured alcohol dehydrogenase to α-crystallin significantly decreased the photoincorporation of bis-ANS to α-crystallin. Localization of bis-ANS incorporated into αA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In αB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of αA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of αB-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 α-crystallin. Localization of 1,5-AZNS binding site in αB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of α-crystallin. Glycated α-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. The hydrophobic binding sites in α-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-α-crystallin complex resulted in incorporation of the probe to both αA- and αB-subunits. Prior binding of denatured alcohol dehydrogenase to α-crystallin significantly decreased the photoincorporation of bis-ANS to α-crystallin. Localization of bis-ANS incorporated into αA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In αB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of αA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of αB-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 α-crystallin. Localization of 1,5-AZNS binding site in αB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of α-crystallin. Glycated α-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. α-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 (1Bloemendal H. Bloemendal H. Molecular and Cellular Biology of the Eye Lens. John Wiley & Sons, New York1981: 1-47Google Scholar). α-Crystallin exists as a polydisperse aggregate with an average molecular mass of 800 kDa (2Groenen P.J.T.A. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar). The two types of subunits, designated αA and αB, each of which has a molecular mass of 20 kDa, arrange themselves in yet undefined ways to form the aggregate (2Groenen P.J.T.A. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar). During aging, α-crystallin undergoes extensive modifications culminating in the formation of super aggregates and highly cross-linked light-scattering molecules (2Groenen P.J.T.A. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar). The sequences of the subunits of α-crystallin have high homology to small heat shock proteins (3Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci., U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (677) Google Scholar, 4Sax C.M. Piatigorsky J. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 155-201PubMed Google Scholar) and are highly conserved between species. α-Crystallin subunits, once thought to be lens-specific, are now widely known to be present in other tissues as well (5Bhat S.P. Nagineni C.N. Biochem. Biophys. Res. Commun. 1989; 158: 319-325Crossref PubMed Scopus (495) Google Scholar, 6Iwaki T. Kume-Iwaki T. Liem R.K.H. Goldman J.E. Cell. 1989; 57: 71-78Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 7Kato K. Shinohara H. Kurobe N. Goto S. Inaguma Y. Ohshima K. Biochim. Biophys. Acta. 1991; 1080: 173-180Crossref PubMed Scopus (197) Google Scholar, 8Srinivasan A.N. Nagineni C.N. Bhat S.P. J. Biol. Chem. 1992; 267: 23337-23341Abstract Full Text PDF PubMed Google Scholar). Despite extensive studies carried out in the past, the quarternary structure or the structure-function of α-crystallin or its subunits has remained an enigma and challenge for researchers. Recently, the ability of native α-crystallin to suppress the aggregation of heat-denatured (9Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar, 10Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 11Rao P.V. Horwitz J. Zigler Jr., J.S. Biochem. Biophys. Res. Commun. 1993; 190: 786-793Crossref PubMed Scopus (110) Google Scholar, 12Merk K.B. Groenen P.J.T.A. Voorter C.E.M. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W.W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 13Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 14Wang K. Spector A. J. Biol. chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar, 15Sharma K.K. Ortwerth B.J. Exp. Eye Res. 1995; 61: 413-421Crossref PubMed Scopus (44) Google Scholar, 16Smulders R.H.P.H. Merh K.B. Aendekerk J. Horwitz J. Takemoto L. Slingsby C. Bloemendal H. de Jong W.W. Eur. J. Biochem. 1995; 232: 834-838Crossref PubMed Scopus (61) Google Scholar, 17Das K.P. Petrash J.M. Surewicz W.K. J. Biol. Chem. 1996; 271: 10449-10452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Plater M.L. Goode D. Crabbe J.M. J. Biol. Chem. 1996; 271: 28558-28566Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 19Clark J.I. Haung Q. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15185-15189Crossref PubMed Scopus (64) Google Scholar, 20Andley U.P. Mathur S. Griest T.A. Petrash J.M. J. Biol. Chem. 1996; 271: 31973-31980Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), UV-irradiated (20Andley U.P. Mathur S. Griest T.A. Petrash J.M. J. Biol. Chem. 1996; 271: 31973-31980Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 21Borkman R.F. Knight G. Obi B. Exp. Eye Res. 1996; 62: 141-148Crossref PubMed Scopus (63) Google Scholar), as well as chemically denatured (22Farahbakhsh Z.T. Huang Q.-L. Ding L.-L. Altenbach C. Steinhoff H.-J. Horwitz J. Hubbell W.L. Biochemistry. 1995; 34: 509-517Crossref PubMed Scopus (203) Google Scholar) proteins and enzymes has been demonstrated. It has been proposed that surface hydrophobic sites in the α-crystallin aggregate are involved in the binding of α-crystallin to target proteins during the display of chaperone-like activity (13Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 23Raman B. Rao Ch. M. J. Biol. Chem. 1997; 272: 23559-23564Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). A direct correlation between the extent of α-crystallin hydrophobicity and chaperone-like activity has been demonstrated by several studies (13Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 23Raman B. Rao Ch. M. J. Biol. Chem. 1997; 272: 23559-23564Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 24Das B.K. Liang J.N. Biochem. Biophys. Res. Commun. 1997; 236: 370-374Crossref PubMed Scopus (53) Google Scholar, 25Smulders R.H.P.H. de Jong W.W. FEBS Lett. 1997; 409: 101-104Crossref PubMed Scopus (44) Google Scholar, 26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (278) Google Scholar). 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 α-crystallin interact with bis-ANS 1The 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. in 1:1 stoichiometry at 37 °C (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The number of bis-ANS molecules binding to α-crystallin increases if the protein is exposed to higher temperatures or denaturing agents prior to the addition of the fluorophore (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (278) Google Scholar). Furthermore, it has been shown that binding of bis-ANS to αB-crystallin (25Smulders R.H.P.H. de Jong W.W. FEBS Lett. 1997; 409: 101-104Crossref PubMed Scopus (44) Google Scholar) or α-crystallin (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) diminishes the chaperone-like activity of the protein. In the present study we have determined the bis-ANS binding sequences in α-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 α-crystallin. 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, ε385 = 16,790 cm−1m−1(28Sudhakar K. Fay P.J. J. Biol. Chem. 1996; 271: 23015-23021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Lysyl endopeptidase was purchased from Wako Bioproducts. Sequence grade trypsin was obtained from Sigma. βL-crystallin (29Bloemendal M. Bloemendal H. Exp. Eye Res. 1995; 61: 757-761Crossref PubMed Scopus (6) Google Scholar) was isolated from bovine lenses (15Sharma K.K. Ortwerth B.J. Exp. Eye Res. 1995; 61: 413-421Crossref PubMed Scopus (44) Google Scholar). All other chemicals were of the highest grade commercially available. α-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 (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 30Sharma K.K. Kaur H. Kester K. Biochem. Biophys. Res. Commun. 1997; 239: 217-222Crossref PubMed Scopus (114) Google Scholar). The α-crystallin thus obtained was >99% pure as judged by SDS-PAGE, and this preparation was used in this study. Photoincorporation of bis-ANS to α-crystallin was carried out as described earlier (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), with slight modification of the original procedure described by Seale et al. (31Seale J.W. Martinez J.L. Horowitz P.M. Biochemistry. 1995; 34: 7443-7449Crossref PubMed Scopus (37) Google Scholar). 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 α-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 α-crystallin prevents bis-ANS binding and photoincorporation, α-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. The photolyzed α-crystallin-bis-ANS complex was treated with 5 mm dithiothreitol for 2 h and filtered. The αA- and αB-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 αA- and αB-crystallins were further purified by SDS-PAGE and recovered by electroelution, and the SDS was removed by ether precipitation (32Hager D.A. Burgess R.R. Anal. Biochem. 1980; 109: 76-86Crossref PubMed Scopus (813) Google Scholar). The bis-ANS-labeled αA- and αB-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. The photoincorporation of 1,5-AZNS to α-crystallin was accomplished using a procedure described by Dockter and Koseki (33Dockter M.E. Koseki T. Biochemistry. 1983; 22: 3954-3961Crossref PubMed Scopus (9) Google Scholar). 1 mm1,5-AZNS and 1.25 μm α-crystallin were used in this study. Following photoincorporation, A- and B- subunits of α-crystallin were separated by HPLC as above using a C18 column. Although the fluorophore was incorporated to both the subunits, only αB-subunit was further analyzed to determine the 1,5-AZNS incorporation site. Labeled αB-crystallin was digested with sequencing grade trypsin (1:50 ratio), and the resulting peptides were separated as described earlier (30Sharma K.K. Kaur H. Kester K. Biochem. Biophys. Res. Commun. 1997; 239: 217-222Crossref PubMed Scopus (114) Google Scholar). 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 α-crystallin was carried out in 0.1m phosphate buffer, pH 7.0, using 10 mg/ml protein and 20 mml-ascorbic acid (34Prabhakaram M. Ortwerth B.J. Exp. Eye Res. 1992; 55: 451-459Crossref PubMed Scopus (26) Google Scholar). After incubation at 37 °C for 4 weeks, the reaction mixture was dialyzed, and the glycated protein was tested with βL-crystallin for chaperone-like activity (15Sharma K.K. Ortwerth B.J. Exp. Eye Res. 1995; 61: 413-421Crossref PubMed Scopus (44) Google Scholar). The interaction of glycated α-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. α-Crystallin incubated without ascorbic acid and processed similarly was used as the control. The binding of bis-ANS, the environment-sensitive probe, to α-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 (27Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (278) Google Scholar). We have shown recently that bis-ANS interacts strongly with α-crystallin, and the bound fluorophore cannot be removed by dialysis (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). 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 (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 31Seale J.W. Martinez J.L. Horowitz P.M. Biochemistry. 1995; 34: 7443-7449Crossref PubMed Scopus (37) Google Scholar). The photoincorporation of bis-ANS to α-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 α-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 α-crystallin-bis-ANS was dialyzed to remove the free bis-ANS prior to photolysis, it is unlikely that nonspecific incorporation of the fluorophore to α-crystallin occurred during our experiments with bis-ANS and α-crystallin. Earlier we showed that prior binding of bis-ANS to α-crystallin diminishes the chaperone-like activity of the protein (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). To determine whether the binding of denaturing proteins to α-crystallin at the chaperone site can affect subsequent bis-ANS photoincorporation, α-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 α-crystallin when ADH was heat-denatured and allowed to bind to α-crystallin prior to the addition of the fluorophore (compare lanes 1 and 2 in Fig. 1). To determine the bis-ANS binding sites in α-crystallin, the fluorophore was initially allowed to bind to purified α-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 α-crystallin-bis-ANS complex by UV-A light (366 nm) resulted in covalent incorporation of the fluorophore to both αA- and αB-subunits as we reported earlier (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In order to identify the sites of bis-ANS incorporation into αA- and αB-crystallins, α-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 αA-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 αA-crystallin (Fig. 3), since we used lysyl endopeptidase to digest αA-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.Figure 3αA- and αB-crystallin sequence showing the bis-ANS binding region. The bis-ANS binding sequences are shown in bold. The alcohol dehydrogenase binding sequences in αB-crystallin and mellitin binding sequence in αA-crystallin are taken from Refs. 30Sharma K.K. Kaur H. Kester K. Biochem. Biophys. Res. Commun. 1997; 239: 217-222Crossref PubMed Scopus (114) Google Scholar and 40Sharma K.K. Kumar S.G. FASEB J. 1997; 11 (Abstr. 9): A908Google Scholar, respectively, and underlined. The mellitin binding region in αB-crystallin marked with adouble underline is taken from Ref. 40Sharma K.K. Kumar S.G. FASEB J. 1997; 11 (Abstr. 9): A908Google Scholar. The 1,5-AZNS binding sequence in αB-crystallin is shown in italics.View Large Image Figure ViewerDownload (PPT) 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 αA-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 αA-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 αA-crystallin by bis-ANS. The two fluorescent peptides of αB-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 αB-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 αB-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. 1,5-AZNS is another, less specific, photoreactive agent used to study the hydrophobic sites in proteins (33Dockter M.E. Koseki T. Biochemistry. 1983; 22: 3954-3961Crossref PubMed Scopus (9) Google Scholar, 35Stevens D.J. Gennis R.B. J. Biol. Chem. 1980; 255: 379-383Abstract Full Text PDF PubMed Google Scholar). When bovine α-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 α-crystallin (800 kDa), suggesting that on average there exists one 1,5-AZNS binding site per subunit of α-crystallin at 25 °C. Further analysis of 1,5-AZNS-α-crystallin by HPLC revealed that both αA- and αB-crystallins were labeled with 1,5-AZNS during the experiment (data not shown), as with bis-ANS (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The 1,5-AZNS binding site in αB-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 αB-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 αA-crystallin was also labeled with 1,5-AZNS, we did not analyze the sample further. Earlier studies have shown that glycation of α-crystallin reduces its chaperone-like activity (36Cherian M. Abraham E. Biochem. Biophys. Res. Commun. 1995; 208: 675-679Crossref PubMed Scopus (127) Google Scholar). To determine whether this was due to an alteration of the hydrophobic chaperone site in α-crystallin, we measured the interaction of glycated α-crystallin with hydrophobic probe ANS. ANS, like bis-ANS is a polarity-sensitive reagent. α-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 α-crystallin was tested with βL-crystallin in a heat denaturation assay (15Sharma K.K. Ortwerth B.J. Exp. Eye Res. 1995; 61: 413-421Crossref PubMed Scopus (44) Google Scholar), a marked decrease in chaperone-like activity was observed (Fig. 6). The presence of surface hydrophobic sites on α-crystallin has been known for a number of years (2Groenen P.J.T.A. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar). Since the demonstration of chaperone-like activity with α-crystallin (9Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1745) Google Scholar), considerable interest has been shown in the hydrophobic sites within α-crystallins as these sites have been implicated in the chaperone-like function of the protein (13Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 23Raman B. Rao Ch. M. J. Biol. Chem. 1997; 272: 23559-23564Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 24Das B.K. Liang J.N. Biochem. Biophys. Res. Commun. 1997; 236: 370-374Crossref PubMed Scopus (53) Google Scholar, 25Smulders R.H.P.H. de Jong W.W. FEBS Lett. 1997; 409: 101-104Crossref PubMed Scopus (44) Google Scholar, 26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (278) Google Scholar). The hydrophobic sites in α-crystallin and its subunits have been studied during recent years using probes such as ANS (37Sun T.-X. Das B.P. Liang J.N. J. Biol. Chem. 1997; 272: 6220-6225Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), bis-ANS (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (278) Google Scholar), and pyrene (13Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar). We have shown recently that UV photolysis of the α-crystallin-bound bis-ANS leads to photoincorporation of the fluorophore to the protein subunits similar to that seen with chaperone GroEL (31Seale J.W. Martinez J.L. Horowitz P.M. Biochemistry. 1995; 34: 7443-7449Crossref PubMed Scopus (37) Google Scholar) and HSP18.1 (38Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (655) Google Scholar) and αB-crystallin (25Smulders R.H.P.H. de Jong W.W. FEBS Lett. 1997; 409: 101-104Crossref PubMed Scopus (44) Google Scholar). Prior binding of denatured proteins to α-crystallin resulted in diminished photoincorporation of the fluorophore bis-ANS (Fig. 1, lane 2) compared with α-crystallin alone. Earlier we showed that prior binding of bis-ANS to α-crystallin partially suppresses its chaperone-like activity (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Taken together these data suggest that both ADH and bis-ANS share common binding sites in α-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 (38Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (655) Google Scholar). The role of hydrophobic sites and the amino acid residues that contribute to their makeup within the multimeric chaperone GroEL has been confirmed (31Seale J.W. Martinez J.L. Horowitz P.M. Biochemistry. 1995; 34: 7443-7449Crossref PubMed Scopus (37) Google Scholar). The available data show that the bis-ANS binding sequences are part of the chaperone sites in GroEL (31Seale J.W. Martinez J.L. Horowitz P.M. Biochemistry. 1995; 34: 7443-7449Crossref PubMed Scopus (37) Google Scholar, 39Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar). The bis-ANS binding sites in small heat shock proteins have also been identified (38Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (655) Google Scholar), but the chaperone sites in those proteins are yet to be determined. The two bis-ANS binding sequences in αA-crystallin, HFSPEDLTVK and HFSPEDLTVKVQEDFVEIHGK (Fig. 3), in part represent the chaperone site we identified earlier (40Sharma K.K. Kumar S.G. FASEB J. 1997; 11 (Abstr. 9): A908Google Scholar). On the basis of deuterium exchange studies Smith et al. (41Smith J.B. Liu Y. Smith D.L. Exp. Eye Res. 1996; 63: 125-128Crossref PubMed Scopus (63) Google Scholar) have also proposed that residues 72–75, in αA-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 αA-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 αA-crystallin were labeled with bis-ANS. Since a loss in chaperone activity of αA-crystallin has been correlated with COOH-terminal truncation (20Andley U.P. Mathur S. Griest T.A. Petrash J.M. J. Biol. Chem. 1996; 271: 31973-31980Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 42Takemoto L. Emmons T. Horwitz J. Biochem. J. 1993; 294: 435-438Crossref PubMed Scopus (118) Google Scholar), 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 αB-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 (25Smulders R.H.P.H. de Jong W.W. FEBS Lett. 1997; 409: 101-104Crossref PubMed Scopus (44) Google Scholar) on the photoincorporation of bis-ANS to recombinant rat αB-crystallin, where the authors observed incorporation of bis-ANS to the NH2-terminal domain of the protein. Since prior exposure of α-crystallin to urea can affect the bis-ANS binding (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), it is yet to be determined whether the bis-ANS binding to rat αB-crystallin was influenced by urea used during the isolation of the recombinant protein. Of the two bis-ANS binding sequences identified in αB-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, 2K. Krishna Sharma and G. S. Kumar, manuscript in preparation. and the VLGDVIEVHGK sequence is one of the alcohol dehydrogenase binding sites determined earlier (30Sharma K.K. Kaur H. Kester K. Biochem. Biophys. Res. Commun. 1997; 239: 217-222Crossref PubMed Scopus (114) Google Scholar). The DRFSVNLDVK sequence follows the alcohol dehydrogenase interacting site in αB-crystallin reported by us earlier (30Sharma K.K. Kaur H. Kester K. Biochem. Biophys. Res. Commun. 1997; 239: 217-222Crossref PubMed Scopus (114) Google Scholar). In a separate experiment we have determined that another hydrophobic site-specific probe, 1,5-AZNS, binds to αB-crystallin sequence 83–90. The 1,5-AZNS-labeled peptide is the sequence between the two bis-ANS binding sequences in αB-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 αB-crystallin. Nevertheless, both bind at the highly conserved region of the protein (3Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci., U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (677) Google Scholar, 4Sax C.M. Piatigorsky J. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 69: 155-201PubMed Google Scholar). The two bis-ANS binding sequences in αB-crystallin are separated by 10 amino acids, which have a major in vitro glycation site (43Ortwerth B.J. Slight S.H. Prabhakaram M. Sun Y. Smith J.B. Biochim. Biophys. Acta. 1992; 1117: 207-215Crossref PubMed Scopus (36) Google Scholar). Glycation of α-crystallin decreases ANS binding. Glycation also reduces chaperone-like activity of α-crystallin (Fig. 6). The data from this study suggest that the glycation-induced loss of chaperone-like activity reported earlier (36Cherian M. Abraham E. Biochem. Biophys. Res. Commun. 1995; 208: 675-679Crossref PubMed Scopus (127) Google Scholar) may be due to the modification of Lys residues 90 and 92 of αB-crystallin that may be part of the hydrophobic/chaperone site. The most hydrophobic region of αB-crystallin, residues 28–34, proposed as a potential chaperone site by deuterium exchange studies (39Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar) was not labeled by bis-ANS during our study. The two bis-ANS binding sequences in αB-crystallin identified during the present study belong to a region of high homology between HSP18.1 and αB-crystallin (38Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (655) Google Scholar). Earlier studies have shown that this region in HSP18.1 is the primary bis-ANS binding region (38Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (655) Google Scholar). On the basis of these data it can be stated that the entire third exon sequence of αB-crystallin, which has a high degree of homology to other heat shock proteins (3Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci., U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (677) Google Scholar), is responsible for its chaperone-like function. Although we estimated the binding of one bis-ANS molecule per subunit of α- crystallin (26Sharma K.K. Kaur H. Kumar S.G. Kester K. J. Biol. Chem. 1998; 273: 8965-8970Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), 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 α-crystallin subunits and their interaction with bis-ANS may result in labeling of more than one peptide sequence as a binding site. We thank Dr. B. J. Ortwerth for helpful discussions on this project and critically reading the manuscript." @default.
• W2002602401 created "2016-06-24" @default.
• W2002602401 creator A5053005998 @default.
• W2002602401 creator A5053186034 @default.
• W2002602401 creator A5064990790 @default.
• W2002602401 creator A5090821735 @default.
• W2002602401 date "1998-06-01" @default.
• W2002602401 modified "2023-10-16" @default.
• W2002602401 title "Identification of 1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic Acid Binding Sequences in α-Crystallin" @default.
• W2002602401 cites W1537274817 @default.
• W2002602401 cites W1559329092 @default.
• W2002602401 cites W1564205457 @default.
• W2002602401 cites W1574972176 @default.
• W2002602401 cites W1599376419 @default.
• W2002602401 cites W1671787304 @default.
• W2002602401 cites W1827875159 @default.
• W2002602401 cites W1987657904 @default.
• W2002602401 cites W1988230012 @default.
• W2002602401 cites W1989999739 @default.
• W2002602401 cites W1990390559 @default.
• W2002602401 cites W1992194517 @default.
• W2002602401 cites W1996418015 @default.
• W2002602401 cites W1998495384 @default.
• W2002602401 cites W2004688505 @default.
• W2002602401 cites W2005987554 @default.
• W2002602401 cites W2007342308 @default.
• W2002602401 cites W2016410251 @default.
• W2002602401 cites W2019048217 @default.
• W2002602401 cites W2019804051 @default.
• W2002602401 cites W2033123702 @default.
• W2002602401 cites W203357141 @default.
• W2002602401 cites W2034843138 @default.
• W2002602401 cites W2044031095 @default.
• W2002602401 cites W2059310462 @default.
• W2002602401 cites W2064634950 @default.
• W2002602401 cites W2064948261 @default.
• W2002602401 cites W2064980931 @default.
• W2002602401 cites W2068405523 @default.
• W2002602401 cites W2076521338 @default.
• W2002602401 cites W2080545638 @default.
• W2002602401 cites W2083924310 @default.
• W2002602401 cites W2085212730 @default.
• W2002602401 cites W2092457909 @default.
• W2002602401 cites W2093924477 @default.
• W2002602401 cites W2110336102 @default.
• W2002602401 cites W2114886316 @default.
• W2002602401 cites W2127033103 @default.
• W2002602401 cites W2130224280 @default.
• W2002602401 cites W2162735744 @default.
• W2002602401 doi "https://doi.org/10.1074/jbc.273.25.15474" @default.
• W2002602401 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9624133" @default.
• W2002602401 hasPublicationYear "1998" @default.
• W2002602401 type Work @default.
• W2002602401 sameAs 2002602401 @default.
• W2002602401 citedByCount "118" @default.
• W2002602401 countsByYear W20026024012012 @default.
• W2002602401 countsByYear W20026024012013 @default.
• W2002602401 countsByYear W20026024012014 @default.
• W2002602401 countsByYear W20026024012015 @default.
• W2002602401 countsByYear W20026024012016 @default.
• W2002602401 countsByYear W20026024012017 @default.
• W2002602401 countsByYear W20026024012018 @default.
• W2002602401 countsByYear W20026024012019 @default.
• W2002602401 countsByYear W20026024012020 @default.
• W2002602401 countsByYear W20026024012021 @default.
• W2002602401 countsByYear W20026024012022 @default.
• W2002602401 countsByYear W20026024012023 @default.
• W2002602401 crossrefType "journal-article" @default.
• W2002602401 hasAuthorship W2002602401A5053005998 @default.
• W2002602401 hasAuthorship W2002602401A5053186034 @default.
• W2002602401 hasAuthorship W2002602401A5064990790 @default.
• W2002602401 hasAuthorship W2002602401A5090821735 @default.
• W2002602401 hasBestOaLocation W20026024011 @default.
• W2002602401 hasConcept C116834253 @default.
• W2002602401 hasConcept C178790620 @default.
• W2002602401 hasConcept C185592680 @default.
• W2002602401 hasConcept C2779255514 @default.
• W2002602401 hasConcept C55493867 @default.
• W2002602401 hasConcept C59822182 @default.
• W2002602401 hasConcept C7263354 @default.
• W2002602401 hasConcept C86803240 @default.
• W2002602401 hasConceptScore W2002602401C116834253 @default.
• W2002602401 hasConceptScore W2002602401C178790620 @default.
• W2002602401 hasConceptScore W2002602401C185592680 @default.
• W2002602401 hasConceptScore W2002602401C2779255514 @default.
• W2002602401 hasConceptScore W2002602401C55493867 @default.
• W2002602401 hasConceptScore W2002602401C59822182 @default.
• W2002602401 hasConceptScore W2002602401C7263354 @default.
• W2002602401 hasConceptScore W2002602401C86803240 @default.
• W2002602401 hasIssue "25" @default.
• W2002602401 hasLocation W20026024011 @default.
• W2002602401 hasOpenAccess W2002602401 @default.
• W2002602401 hasPrimaryLocation W20026024011 @default.
• W2002602401 hasRelatedWork W1531601525 @default.
• W2002602401 hasRelatedWork W2319480705 @default.
• W2002602401 hasRelatedWork W2384464875 @default.
• W2002602401 hasRelatedWork W2606230654 @default.
• W2002602401 hasRelatedWork W2607424097 @default.

• next