From the § Mason Eye Institute, The hydrophobic binding sites in Recently, the ability of native We have shown earlier that both A- and B-subunits in 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, Preparation of Photoincorporation of Bis-ANS into
Department of Biochemistry, University
of Missouri, Columbia, Missouri 65212.
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-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-
-crystallin complex resulted in incorporation of the probe to both
A- and
B-subunits. Prior binding of denatured alcohol dehydrogenase to
-crystallin significantly decreased the photoincorporation of
bis-ANS to
-crystallin. Localization of bis-ANS incorporated into
A-crystallin resulted in the identification of residues QSLFR and
HFSPEDLTVK as the fluorophore binding regions. In
B-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of
A-crystallin and DRFSVNLNVK and VLGDVIEVHGK of
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
-crystallin. Localization of 1,5-AZNS binding site in
B-crystallin lead to the identification of HFSPEEK sequence as the
interacting site in this subunit of
-crystallin. Glycated
-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
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-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).
-Crystallin exists as a polydisperse aggregate
with an average molecular mass of 800 kDa (2). The two types of
subunits, designated
A and
B, each of which has a molecular mass
of 20 kDa, arrange themselves in yet undefined ways to form the
aggregate (2). During aging,
-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
-crystallin have high homology to small heat shock
proteins (3, 4) and are highly conserved between species.
-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
-crystallin or its subunits has remained
an enigma and challenge for researchers.
-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
-crystallin aggregate are involved in the binding of
-crystallin to target proteins during the display of chaperone-like activity (13, 23). A direct correlation between the extent of
-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.
-crystallin
interact with bis-ANS1 in 1:1
stoichiometry at 37 °C (26). The number of bis-ANS molecules binding
to
-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
B-crystallin (25) or
-crystallin (26) diminishes the
chaperone-like activity of the protein. In the present study we have
determined the bis-ANS binding sequences in
-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
-crystallin.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
385 = 16,790 cm
1 M
1
(28). Lysyl endopeptidase was purchased from Wako Bioproducts. Sequence
grade trypsin was obtained from Sigma.
L-crystallin (29) was
isolated from bovine lenses (15). All other chemicals were of the
highest grade commercially available.
-Crystallin--
-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
-crystallin thus
obtained was >99% pure as judged by SDS-PAGE, and this preparation
was used in this study.
-Crystallin--
Photoincorporation of bis-ANS to
-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
-crystallin during 15-min photolysis was
determined by quantitative densitometry function of Image-1 system
(Universal Imaging Corp.).
-crystallin prevents bis-ANS binding and photoincorporation,
-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 A- and
B-crystallin--
The
photolyzed
-crystallin-bis-ANS complex was treated with 5 mM dithiothreitol for 2 h and filtered. The
A- and
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
A- and
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 A- and
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 -Crystallin and
Identification of Binding Site in
B-crystallin--
The
photoincorporation of 1,5-AZNS to
-crystallin was accomplished using
a procedure described by Dockter and Koseki (33). 1 mM
1,5-AZNS and 1.25 µM
-crystallin were used in this
study. Following photoincorporation, A- and B- subunits of
-crystallin were separated by HPLC as above using a C18 column.
Although the fluorophore was incorporated to both the subunits, only
B-subunit was further analyzed to determine the 1,5-AZNS
incorporation site. Labeled
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 -Crystallin, Chaperone Assay, and ANS
Binding--
Glycation of
-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
L-crystallin for chaperone-like
activity (15). The interaction of glycated
-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.
-Crystallin incubated
without ascorbic acid and processed similarly was used as the
control.
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RESULTS |
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Interaction of Bis-ANS with -Crystallin--
The binding of
bis-ANS, the environment-sensitive probe, to
-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
-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
-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
-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
-crystallin-bis-ANS was dialyzed to remove the free bis-ANS prior to
photolysis, it is unlikely that nonspecific incorporation of the
fluorophore to
-crystallin occurred during our experiments with
bis-ANS and
-crystallin.
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Effect of Denaturing Protein Bound to -Crystallin on the
Photoincorporation of Bis-ANS--
Earlier we showed that prior
binding of bis-ANS to
-crystallin diminishes the chaperone-like
activity of the protein (26). To determine whether the binding of
denaturing proteins to
-crystallin at the chaperone site can affect
subsequent bis-ANS photoincorporation,
-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
-crystallin when ADH was heat-denatured and allowed to
bind to
-crystallin prior to the addition of the fluorophore
(compare lanes 1 and 2 in Fig. 1).
Photoincorporation of Bis-ANS to -Crystallin Subunits and
Localization of Incorporated Bis-ANS--
To determine the bis-ANS
binding sites in
-crystallin, the fluorophore was initially allowed
to bind to purified
-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
-crystallin-bis-ANS complex by UV-A
light (366 nm) resulted in covalent incorporation of the fluorophore to
both
A- and
B-subunits as we reported earlier (26).
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Interaction of 1,5-AZNS with -Crystallin--
1,5-AZNS is
another, less specific, photoreactive agent used to study the
hydrophobic sites in proteins (33, 35). When bovine
-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
-crystallin (800 kDa), suggesting that on average there exists one
1,5-AZNS binding site per subunit of
-crystallin at 25 °C.
Further analysis of 1,5-AZNS-
-crystallin by HPLC revealed that both
A- and
B-crystallins were labeled with 1,5-AZNS during the
experiment (data not shown), as with bis-ANS (26).
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Effect of Glycation of -Crystallin on Its Chaperone-like
Function and Interaction with ANS--
Earlier studies have shown that
glycation of
-crystallin reduces its chaperone-like activity (36).
To determine whether this was due to an alteration of the hydrophobic
chaperone site in
-crystallin, we measured the interaction of
glycated
-crystallin with hydrophobic probe ANS. ANS, like bis-ANS
is a polarity-sensitive reagent.
-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
-crystallin was tested with
L-crystallin in a heat denaturation
assay (15), a marked decrease in chaperone-like activity was observed
(Fig. 6).
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DISCUSSION |
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The presence of surface hydrophobic sites on -crystallin has
been known for a number of years (2). Since the demonstration of
chaperone-like activity with
-crystallin (9), considerable interest
has been shown in the hydrophobic sites within
-crystallins as these
sites have been implicated in the chaperone-like function of the
protein (13, 23-27). The hydrophobic sites in
-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
-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
B-crystallin (25). Prior binding of denatured proteins to
-crystallin resulted in diminished photoincorporation of the
fluorophore bis-ANS (Fig. 1, lane 2) compared with
-crystallin alone. Earlier we showed that prior binding of bis-ANS
to
-crystallin partially suppresses its chaperone-like activity
(26). Taken together these data suggest that both ADH and bis-ANS share
common binding sites in
-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 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
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
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
A-crystallin were labeled with bis-ANS. Since a loss in
chaperone activity of
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 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
B-crystallin, where
the authors observed incorporation of bis-ANS to the
NH2-terminal domain of the protein. Since prior exposure of
-crystallin to urea can affect the bis-ANS binding (26), it is yet
to be determined whether the bis-ANS binding to rat
B-crystallin was
influenced by urea used during the isolation of the recombinant
protein. Of the two bis-ANS binding sequences identified in
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
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
B-crystallin sequence
83-90. The 1,5-AZNS-labeled peptide is the sequence between the two
bis-ANS binding sequences in
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
B-crystallin. Nevertheless, both bind at the highly conserved region
of the protein (3, 4).
The two bis-ANS binding sequences in B-crystallin are separated by
10 amino acids, which have a major in vitro glycation site
(43). Glycation of
-crystallin decreases ANS binding. Glycation also
reduces chaperone-like activity of
-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
B-crystallin that may be
part of the hydrophobic/chaperone site.
The most hydrophobic region of 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
B-crystallin identified during the present
study belong to a region of high homology between HSP18.1 and
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
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 - 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
-crystallin subunits
and their interaction with bis-ANS may result in labeling of more than
one peptide sequence as a binding site.
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ACKNOWLEDGEMENT |
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We thank Dr. B. J. Ortwerth for helpful discussions on this project and critically reading the manuscript.
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FOOTNOTES |
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* 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.
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REFERENCES |
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