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Phe71 Is Essential for Chaperone-like Function in A-crystallin*

Received for publication, August 13, 2001, and in revised form, September 21, 2001Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M107737200

Puttur Santhoshkumar and K. Krishna Sharma

From the Departments ofOphthalmology and Biochemistry, University of Missouri, Columbia, Missouri 65212

Experiments with mini- A-crystallin (KFVIFLD- VKHFSPEDLTVK) showed that Phe71 in A-crystallincould be essential for the chaperone-like action of theprotein (Sharma, K. K., Kumar, R. S., Kumar, G. S., andQuinn, P. T. (2000) J. Biol. Chem. 275, 37673771). In thepresent study we replaced Phe71 in rat A-crystallinwith Gly by site-directed mutagenesis and then com-pared the structural and functional properties of themutant protein with the wild-type protein. There wereno differences in molecular size or intrinsic tryptophanfluorescence between the proteins. However, 1,1-bi(4-anilino)naphthalene-5,5-disulfonic acid interaction in-dicated a higher hydrophobicity for the mutant protein.

Both wild-type and mutant proteins displayed similarsecondary structure during far UV CD experiments.Near UV CD signal showed a slight difference in thetertiary structure around the 285295 region for the twoproteins. The mutant protein was totally inactive in sup-pressing the aggregation of reduced insulin, heat-dena-tured citrate synthase, and alcohol dehydrogenase.However, a marginal suppression ofL-crystallin aggre-gation was observed when mutant A-crystallin was in-cluded. These results suggest that Phe71 contributes tothe chaperone-like action ofA-crystallin. Therefore weconclude that the 7088-region in A-crystallin, identi-fied by us earlier, is the functional chaperone site inA-crystallin.

-Crystallins are major refractive proteins in the vertebrate

eye lens. When isolated from the lens they exist as polydisperse

aggregates having an average molecular mass of800 kDa (1,

2). -Crystallin is composed of two subunits, A and B, which

have considerable sequence homology between them and with

other heat shock proteins (3, 4). Recently -crystallin subunits

were also reported to be present in nonlenticular tissues like

heart, brain, and kidney (58). The significance of their pres-

ence in nonlenticular tissues is not clear. However, the in-

creased expression of B-crystallin observed in a variety of

neurological disorders has drawn significant medical attention

(911). Like other small heat shock proteins, -crystallin can

sequester certain unfolding proteins in vitro, by preventing

their aggregation and insolubilization (1215). Both subunitsof-crystallin show chaperone-like activity to different extents

(1618). Complex formation with L- and -crystallin and de-

creased chaperone-like function during aging has indicated the

importance of-crystallin in maintaining the transparency of

the lens (2, 1921). During chaperone-like action, hydrophobic

surfaces in -crystallin interact with specific sites in non-na-

tive target proteins (2224). Earlier we were able to map the

site in A- and B-crystallin responsible for chaperone-like

action using photoactive cross-linkers and hydrophobic probes

(2527). Our studies with bis-ANS1 and the hydrophobic pro-

tein mellitin have shown that there is an overlapping of chap-

erone site and hydrophobic site in A-crystallin. Further, using

a synthetic peptide (mini- A-crystallin), we were able to dem-

onstrate the importance of sequence 7088 in the chaperone-

like action of A-crystallin (28). The experiments with trun-

cated forms of mini-A-crystallin had suggested that Phe71 in

A-crystallin might be critical for chaperone-like function. Inthe present study, we did a site-directed mutagenesis of Phe71

to Gly in A-crystallin and compared the structural and func-

tional properties of this mutant protein with the wild-type

protein.

Recombinant A- and B-crystallins show similar structural

and functional properties to crystallins isolated from lens tis-

sues and are widely used in the characterization of the protein.

Site-directed mutations of recombinant protein provide an ex-

cellent means of studying the role of constituent amino acids in

the functional properties of the protein. Earlier, several site-

directed mutations were conducted on -crystallin either to

identify the region responsible for chaperone-like function or to

explain the role of -crystallin in hereditary cataracts and

certain other diseases (2938). The majority of these studies

report either no change in chaperone-like function or a partial

loss of this function. We report here, for the first time, a

complete loss in the functional property of a mutant A-crys-

tallin at and slightly above physiological temperatures. The

results also indicate the presence of additional sites in -crys-

tallin that become available at elevated temperatures. We con-

clude here that the region identified by us earlier as chaperone

site contributes to the chaperone-like activity ofA-crystallin.

EXPERIMENTAL PROCEDURES

Preparation of the Mutant CloneRat A-crystallin cDNA cloned inpET21b was kindly donated by Dr. Suraj Bhat (UCLA). AF71G mu-

tant was constructed using a QuikChange site-directed mutagenesis kit(Stratagene). The following set of primers were used: 5-CTGACCGG-GACAAGGGTGTCATCTTCTTGG-3 and 5-CCAAGAAGATGACAC-

CCTTGTCCCGGTCAG-3. The mutation was confirmed by automatedDNA sequencing.

Expression and Purification of Wild-type and Mutant A-crystallin

The proteins were expressed in Escherichia coli BL21(DE3) cells (No-vagen) as described by Horwitz et al. (39). The proteins were isolatedfrom the cell pellet using Bugbuster protein extract reagent (Novagen).In brief, 1 g of cells was suspended in 5 ml of reagent at room temper-ature and vortexed gently. Protease inhibitor mixture set III (Novagen)

was then added. The cell suspension was treated with 1 l (25 units) ofbenzonase/ml of Bugbuster reagent and incubated at room temperatureon a shaking platform for 30 min. The extract was centrifuged at

* This work is supported in part by National Institutes of HealthGrant EY11981 and a grant-in-aid from Research to Prevent Blindness.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact. To whom correspondence should be addressed: Mason Eye Inst.,

Dept. of Ophthalmology, 1 Hospital Dr., University of Missouri, Colum-bia, MO 65212. E-mail: [email protected].

1 The abbreviations used are: bis-ANS, 1,1-bi(4-anilino)naphthalene-5,5-disulfonic acid; CS, citrate synthase; ADH, alcohol dehydrogenase;HPLC, high pressure liquid chromatography.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 50, Issue of December 14, pp. 4709447099, 2001 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org47094

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17,000 g for 2 h, and the supernatant was filtered through a 0.2-mfilter. The filtrate was loaded onto a Bio-Rad High Q anion exchangecolumn and eluted using a linear gradient of NaCl (0 1 M) in 20 mMTris-HCl (pH 8) at a flow rate of 2 ml/min. The fractions containing therecombinant crystallin (determined by SDS-PAGE) were pooled andconcentrated. The concentrated protein was purified further on a C18

reverse phase HPLC using a water-acetonitrile gradient containing0.1% trifluoroacetic acid. The peaks corresponding to wild-type andmutant A-crystallin were pooled, dried on a Speedvac, resuspended in6 M urea, and dialyzed extensively against 0.05 M phosphate (PO4)

buffer containing 0.15 M NaCl, with several changes, for a period of 2days. The purity of the proteins was checked by SDS-PAGE, and themass was determined by mass spectrometry.

Molecular Size DeterminationSize determination was carried outusing a Amersham Biosciences Hiload 16/60 Superdex 200 gel filtrationcolumn equilibrated with 0.05 M PO

4buffer containing 0.15 M NaCl (pH

7.4). The mass was calculated from the calibration curve generated by

using Sigma molecular weight marker standards.Tryptophan Fluorescence MeasurementsThe intrinsic fluorescence

spectra of the wild-type and mutant A-crystallin were recorded usinga Jasco spectrofluorometer FP-750. Protein samples of 200 g/ml in0.05 M PO4 buffer containing 0.15 M NaCl were used. The excitation wasset to 295 nm, and the emission was recorded between 300 and 400 nm.

bis-ANS Fluorescence MeasurementTo 100 g of wild-type andmutant protein taken in 0.05 M PO

4containing 0.15 M NaCl (pH 7.4)

was added 20 l of 10 mM bis-ANS dissolved in ethanol. The sample wasexcited at 365 nm, and the fluorescence spectrum was measured be-

tween 400 and 600 nm using a Jasco spectrofluorometer.Circular Dichroism StudiesProtein secondary and tertiary struc-

tural changes were investigated by far and near UV CD measurementsusing an AVIV circular dichroism spectrometer. The concentration ofthe proteins used was 1.5 and 0.35 mg/ml for near and far UV CD,respectively. The reported CD spectra are the averages of four scans.

Chaperone-like ActivityThe ability of the wild-type and mutantproteins to prevent protein aggregation was determined using severalsubstrates. The extent of aggregation was measured by monitoring thelight scattering at 360 nm in a Shimadzu spectrophotometer.

Insulin Aggregation AssayThe aggregation of insulin (0.4 mg/ml)(Sigma) in 0.05 M PO4 buffer containing 0.15 M NaCl (pH 7.2) was

initialized by the addition of 25 l of 1 M dithiothreitol in the presenceof wild-type and mutant proteins. The aggregation was monitored atroom temperature.

CS Aggregation AssayCS (75 g) (Roche Molecular Biochemicals)in 1 ml of 40 mM HEPES-NaOH buffer (pH 7.4) was heated at 43 C for

1 h in the presence of various amounts of mutant and wild-type pro-teins. The light scattering was measured as described above.

ADH Aggregation AssayADH (250 g) (Sigma) was heated at 45 Cin the presence of various amounts of mutant and wild-type A-crys-tallin in 0.05 M PO

4buffer containing 0.15 M NaCl (pH 7.4). The

scattering of light was measured up to 100 min.

L

-Crystallin Aggregation AssayPurified bovine L-crystallin (250g) in 0.05 M PO4 buffer containing 0.15 M NaCl (pH 7.4) was heated at55 C in the presence of different amounts of recombinant proteins. Theaggregation was monitored up to 1 h.

RESULTS

Characterization of Recombinant A-crystallinIn the pres-

ent study, the recombinant proteins were purified by a combi-

nation of ion exchange and reverse phase HPLC. The purified

proteins were dissolved in urea and refolded by extensive dial-

ysis. The proteins thus obtained were highly pure (Fig. 1). TheES mass spectrometry analysis revealed molecular masses of

19,799 and 19,709 daltons, which would be expected for the

wild-type and mutant A-crystallin respectively. Like the

-crystallin subunits isolated from eye lens, recombinant pro-

teins exist in oligomeric form. For analysis of the molecular

mass of the homoaggregates, the purified recombinant proteins

were chromatographed on a Superdex-200 column. Both wild-

type and AF71G mutant proteins showed similar elution pro-

files, corresponding to an oligomeric mass of 7.1 105 daltons

(Fig. 2). This is slightly higher than the earlier published

values for rat A-crystallin (37). The discrepancy can be attrib-

uted to the different buffer conditions used in analysis, because

it is known that the mass of the purified protein varies depend-

ing on the buffer condition (40, 41). During these studies we

also observed a similar mass for reconstituted homopolymers of

bovine lens A-crystallin.

The structural differences between the wild-type and mutant

proteins were analyzed by spectroscopic methods. Tryptophans

of the protein have a fixed solvent accessibility, and any change

in their environment leads to an altered fluorescence emissionpattern and intensity. Our results show no change in the tryp-

tophan region of the recombinant proteins, as evidenced by the

similar fluorescence emission maximum (340 nm) and intensity

(Fig. 3). Unfolding of proteins increases the exposure of hydro-

phobic surfaces that can be probed with bis-ANS fluorescence

(26). We see an increase in bis-ANS binding to the AF71G

mutant (Fig. 4), indicating an increased hydrophobicity com-

pared with wild-type protein.

The secondary and tertiary structures of wild-type and mu-

tant A-crystallin were determined by far and near UV CD

spectral analysis. The far UV profile showed a characteristic

-sheet conformation with a slight increase in the negative

intensity of the mutant protein (Fig. 5). Both proteins showed

similar amounts of-helix, -sheet, and random coil (42). Near

FIG. 1. SDS-PAGE analysis of recombinant protein purifica-tion. Lane 1, crude cell extract; lane 2, recombinant protein after ionexchange; lane 3, recombinant protein after reverse phase; lane 4,molecular mass markers.

FIG. 2. Gel permeation profiles of refolded wild-type and mu-tant protein on a Superdex 200 column.

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UV CD spectra showed a slight increase in the negative inten-

sity of the mutant protein (Fig. 6). Although significant por-

tions of the near UV spectra for the two proteins were similar,

only minor changes were seen in the 285295 nm region of the

spectra, suggesting some differences in the tyrosine and/or

tryptophan microenvironments of the mutant protein com-

pared with the wild-type A-crystallin. Surprisingly enough,

there was no alteration in the signal caused by phenylalanine

in the 250 270-nm region. In summary, the data in Fig. 6 do

not suggest a significant difference in the tertiary structure

between wild-type and mutant A-crystallin.

The Chaperone-like Activity of F71G A-crystallinThe con-

sequence of mutation on recombinant crystallin chaperone-like

activity was determined under different conditions. Reduction

of insulin results in the separation of the subunits and precip-itation of B chain that can be followed by measurement of light

scattering. The presence of -crystallin subunits in the assay

prevents the aggregation of insulin B chain, and the solution

remains clear. Fig. 7 shows the dithiothreitol-induced aggrega-

tion kinetics of insulin in the presence of both wild-type and

mutant A-crystallins. The wild-type protein showed suppres-

sion of insulin B chain aggregation that increased with the

concentration of the protein in the assay tube. However, the

mutant A-crystallin completely failed to prevent the forma-

tion of light-scattering aggregates. In fact, a marginal increase

in light scattering was observed in some assays. Higher con-

centrations of mutant protein had no effect on the aggregation

of polypeptide. The chaperone-like activity of the recombinant

proteins was also investigated at different temperatures. Fig. 8

FIG. 3 Intrinsic fluorescence intensity of purified wild-typeand mutant A-crystallin. Protein samples (200 g) in phosphatebuffer were excited at 295 nm.

FIG. 4. Interaction of bis-ANS with mutant and wild-typeA-crystallin.

FIG. 5 Far UV CD spectra of recombinant proteins.

FIG. 6. Near UV CD spectra of recombinant proteins.

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shows the thermal aggregation of CS in the presence of wild-

type and mutant proteins. Although the wild-type protein (50

g) completely suppressed the aggregation of CS (75 g), the

mutant protein, as with insulin, failed to prevent the aggrega-

tion of denaturing CS. We also analyzed the ability of recom-

binant proteins to suppress the aggregation of ADH at 45 C.

The wild-type A-crystallin showed increased suppression of

denaturing protein aggregation with increasing concentration

(Fig. 9A). Although the mutant protein appeared to suppress

the aggregation of ADH at initial time points, the aggregation

at 80 min was comparable with ADH by itself (Fig. 9B). In-creasing the concentration of mutant protein had no effect on

the aggregation of ADH. We also compared the abilities of

mutant and wild-type A-crystallin to prevent the heat-in-

duced aggregation of L-crystallin at 55 C (Fig. 10). Unlike

other substrates, mutant A-crystallin showed a significant

protection ofL-crystallin with increasing concentration. How-

ever, compared with the wild-type A-crystallin, the mutant

A-crystallin was 610-fold less effective in suppressing

L-aggregation.

DISCUSSION

A-crystallin subunit has been categorized into three do-

mains: an N-terminal domain containing residues 1 66, a C-

terminal or -crystallin domain (central core) comprising resi-

dues 64 105, and an extended C-terminal including residues106173 (43 45). Most of the mutational studies on A-crys-

tallin were conducted either on the N-terminal domain or the

C-terminal extension. Derham and Harding (46) have reviewed

the mutations conducted by different laboratories on -crystal-

lin and have recently reanalyzed the chaperone-like activity of

several mutants (35). In the present study, we produced an

A-crystallin mutant by substituting Phe71 in the core region

with a neutral amino acid Gly. This residue is highly conserved

in A-crystallin and is located in the region identified as the

chaperone site of A-crystallin (28). Biophysical characteriza-

tion of the recombinant protein revealed no change in the

oligomer size or tryptophan fluorescence. Because the -crys-

tallin molecule has only one tryptophan at position 9, the

intrinsic tryptophan fluorescence data may be of limited value

FIG. 7 Reduction of insulin (0.4 mg) by dithiothreitol in thepresence of wild-type and mutant A-crystallin. A, insulin alone;

BD, insulin with 0.1, 0.2, and 0.3 mg of mutant A-crystallin, respec-tively; E and F, insulin with 0.05 and 0.1 mg of wild-type A-crystallin,respectively.

FIG. 8. Aggregation of CS at 43 C in the presence of wild-typeand mutant A-crystallin. A, CS (75 g); B, CS mutant (50 g); C,CS mutant (100 g); D, CS wild type (25 g); E, CS wild type (50g).

FIG. 9. Thermal aggregation of ADH (250g) in the presenceofvarious amounts of wild-type and mutantA-crystallin at 45 C.

A, in the presence of wild type; B, in the presence of mutantA-crystallin.


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to describe the structural changes in the central core or C-

terminal domain as a consequence of the mutation. However, it

will be a valuable tool for analyzing the stability of the N-

terminal domain of the mutant protein. When the mutant

protein was heated up to 60 C, we did not observe any aggre-

gation or shift in tryptophan fluorescence emission wavelength

or intensity (data not shown), suggesting that the heat stability

of the protein was not affected by the mutation.

It has been hypothesized that hydrophobic sites in -crystal-

lin are responsible for chaperone-like activity (22, 23). How-

ever, this is not free of controversy (29, 47). In the present

study we see a complete loss in the chaperone-like function of

mutant A-crystallin at and slightly above physiological tem-

peratures despite an increase in hydrophobicity. Smulders et

al. (29) observed an increase in the chaperone-like activity of an

AF74N mutant with a slight decrease in ANS binding. They

concluded that there is no correlation between surface hydro-

phobicity and chaperone-like activity. Experiments with super-

A-crystallin have indicated that the disappearance of chaper-

one-like activity may be independent of hydrophobicity (47).

Further, Reddy et al. (18) have shown recently that hydropho-

bicity is not the sole determinant of chaperone-like activity in

-crystallin. Recently, the studies with mini-A-crystallin

showed that both hydrophobicity and -sheet conformation of

the functional element are essential for chaperone-like activity(48). Although we see increased exposure of hydrophobic sur-

faces in the mutant, it is quite unlikely that all exposed hydro-

phobic patches would be involved in suppressing the substrate

protein aggregation. We, as well as others, have observed bis-

ANS binding to residues other than those necessary for chap-

erone activity (27, 49). Taking these observations together, one

can conclude that although hydrophobicity is important, the

extent of hydrophobicity does not reflect the chaperone-like

activity of the protein.

The AF71G mutant has similar secondary structure to that

of the wild type. However, the tertiary structure shows some

minor changes around the 285295-nm region. The signal in

this region is produced by tyrosine or tryptophan residues.

Because we did not observe any change in the tryptophan

fluorescence intensity, it is possible that the alteration is in the

tyrosine region. Interestingly, two tyrosine residues in A-

crystallin are found near the bis-ANS-binding region 5054

(27). This may explain the increased bis-ANS binding of the

mutant protein. However, it is unlikely that such a minor

difference in the near UV CD signal would completely abolish

the chaperone-like activity of the molecule. The AR116C mu-

tant, with structural alterations at many levels, showed only a

25% decrease in chaperone-like activity (37, 38). Further, it has

been shown that -crystallin could preserve its chaperone func-

tion despite some irreversible structural changes (50).

We have measured the chaperone-like function of the

AF71G mutant under different conditions and observed a

complete loss in the activity of the mutant up to 45 C. How-

ever, at elevated temperatures the mutant showed some sup-

pression of L-crystallin aggregation. It has been shown that

-crystallin undergoes a structural transition around 55 C,

resulting in the exposure of more hydrophobic patches (23, 24,

26). Our study on the stabilization of restriction enzyme (51) as

well as studies conducted by others (35, 52) indicates the pres-

ence of multiple sites in -crystallin for chaperone function.

Based on the experiments with mini-A-crystallin (28) and the

complete loss of chaperone-like function of the mutant protein

at physiological temperatures in this study, we conclude that

the region identified by us earlier (residues 71 88) contributes

to the chaperone-like function.

Plater et al. (30) reported that the F27R mutation in the

N-terminal domain of B-crystallin completely abolishes its

chaperone-like activity at higher temperatures, which led them

to conclude that this conserved residue is vital for chaperone

function. Their report is controversial because later studies

have shown that the mutant F27R is fully active (34, 35).

Earlier, work showed that proteins resulting from mutation of

V72N and F74N in the core region ofA-crystallin had normal

activity (29). However, their conclusion was based on a single

assay conducted at 58 C. We also found some activity of the

mutant with L-crystallin around this temperature. Also, un-

like Phe71, the Val72 and Phe74 residues show variations in

different vertebrate lens species. The conserved Phe71 residue

appears to be important for suppressing the aggregation of

proteins. Other factors like charge, hydrophobicity, and struc-

tural integrity may influence the functional property to differ-

ent extents. Recently, Kumar and Rao (53) produced a chimeric

-crystallin by swapping the domains ofA- and B-crystallin

and tested its effect on the chaperone-like activity. Interest-

ingly, the ANBC chimeric protein which contained residues

179 ofA-crystallin, including a part of the functional site in

A-crystallin, was completely inactive in suppressing the ag-

gregation of insulin. However, the BNAC chimeric protein

containing the complete ADH binding sequence (25) and a part

of the functional site in A-crystallin had enhanced chaperone-

like activity. This suggests that other residues in the chaperonesite of A-crystallin are also important in suppressing the

aggregation of proteins. Therefore it would be interesting to

study the role of other conserved residues on chaperone-like

action.

AcknowledgmentsWe are grateful to Dr. P. D. Prasad for the helpin site-directed mutagenesis and Jelena Kocergin in the expression andpurification of recombinant proteins.

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FIG. 10.Aggregation ofL

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