Vol. 46, No.

2, October 1998

~-CRYSTALLIN
ACTIVATION

BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Pages 249-258

DOES
NOT
REQUIRE
TEMPERATURE
F O R ITS C H A P E R O N E - L I K E A C T I V I T Y

Jaya Bhattacharyya and Kali P. Das*

Protein Chemistry Laboratory, Department of Chemistry, Bose Institute,
93/1, A.P.C. Road, Calcutta 700 009, India.
Received March 30, 1998
Received after revision, June 17, 1998

SUMMARY
cx-crystallin acts as a molecular chaperone by preventing the aggregation of
proteins. Although the mechanism for this activity is not understood there is a proposition
that temperature activation at or above 30~ of et-crystallin is an absolute requirement,
thereby suggesting a conformational transition as a trigger for the activity. In an attempt
to unravel the putative temperature-activity relationship, the chaperone-like activity of c~crystallin was studied at a number of temperatures above and below 30~ Chaperone
activity was monitored against aggregation of the insulin-B chain induced by cleavage of
disulfide bond of insulin and also against photo-aggregation of y-crystallin. Contrary to
the above notion, the results indicate that a-crystallin does not require thermal activation
for its chaperone function and that it can efficiently function as a molecular chaperone
even at temperatures below the previously reported transition temperature.
Key words: a-crystallin, molecular chaperone, chaperone-substrate interaction.
INTRODUCTION
(z-crystallin, the major protein of the mammalian eye lens, is believed to play an
important role in the maintenance of the transparency of the lens (1,2). It is composed of
two highly homologous polypeptide chains cxA and etB of 20 KDa each and exists as a
oligomer with an average molecular weight 800 KDa (3-6). Direct information regarding
the structure of this protein is lacking, since neither cx-crystallin oligomer nor its
composite subunits have been crystallized. Moreover, there is disagreement among the
models suggested for its quaternary structure (7-10).

PMSF,
phenylmethanesulphonyl fluoride;
FITC,
fluorescein
isothiocyanate; EDTA, ethylenediaminetetraacetic acid disodium salt; DTT,
dithiothreitol; SDS-PAGE, sodium dodecyl sulphate polyacryalamide gel electrophoresis.

Abbreviations:

*Correspondence author. FAX: 91 33 350 6790; E-mail: kalipada@boseinst.ernet.in

1039-9712/98/140249-10505.00/0
249

Copyright 9 1998 by Academic Press Australia.

All rights ~[ reproduction in any form reserved.

Vol. 46, No. 2, 1998

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and MOLECULAR BIOLOGYINTERNATIONAL

Despite extensive studies on ct-crystallin in the past, no direct evidence as to its

biological activity is known. Recent findings have revealed some knowledge of the origin
and function of this protein (1 1-15). et-crystallin was believed to be lens specific. It is
now known that both ~tA and ctB crystallins are found in many other organs including
heart, lung, spleen etc. (11). The other major finding is that, ot-crystallin bears extensive
sequence homology to a class of plant proteins known as small heat shock proteins (12)
[SHSP]. Finally it is established that c~-crystallin, like other heat shock proteins, acts as a
molecular chaperone by preventing the aggregation of other proteins (13-19).
The mechanism of the chaperone action of ct-crystallin is not understood,

ct-

crystallin is shown to be efficient in preventing the thermal aggregation o f proteins (13).

There is little information regarding the ability of this putative chaperone to interact with
non-native states formed during protein folding reactions at low temperatures. One group
of workers (14,17,18) claimed that a conformational transition above 30 o C converted it to
an active form and below 30 ~ C it was inactive. In a previous study (15) conformational
changes were not detected in c~-crystallin in this temperature range. In an investigation of
the chaperone activity of o~-crystallin at a number of temperatures below and above 30~
it is reported that ct-crystallin does not require temperature activation for its function as a
molecular chaperone.
MATERIALS AND METHODS

Materials: Calf eye lenses were obtained from a local slaughter house and stored
at -80~ until use. Sephacryl S-300HR, Sephadex G-10, Sephadex G-50 and insulin were
purchased from Sigma Chemical Co., USA. Phenylmethyl-sulfonyl fluoride (PMSF)
used as a protease inhibitor was from E.Merck, Germany. Fluorescein isothiocyanate
(FITC - FI43 isomer) was obtained from Molecular Probes, USA.
Preparation of a- and y-crystallin: ~-crystallin was purified from calf lenses
according to (19). Briefly, lenses were homogenized in l0 mM Tris-HC1 buffer pH 7.2
containing 100 mM NaC1, 1 mM EDTA, 0.02% sodium azide and 0.2 mM PMSF and
then centrifuged at 4~ at 12,000 rpm for 45 min. The supernatant fraction was applied to
a Sephacryl S-300 HR column (95 x 1.5 cm). Fractions of the first peak containing both
heavy (ctH) and light (ORE)crystallin were again rechromatographed on the same column.
The major peak contained ct-crystallin. SDS-PAGE showed two closely spaced bands
corresponding to 20 KDa.
,/-crystallin corresponding to the third peak of the first column was further purified
through Sephacryl S-100 HR (95 x 1.5 cm) column at 25~
A single peak was obtained.
SDS-PAGE showed a single band corresponding to a molecular weight of 20 KDa.
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FITC labeling of insulin: FITC labeled insulin was prepared according to (20). Briefly,
insulin in 10 mM sodium phosphate buffer pH 7.2 was incubated with 5-fold excess of
FITC with constant stirring at 27~ for 3 hr. The reaction was stopped by the addition of
100 gl of 1.5 M glycine to the mixture. The reaction mixture was then dialyzed for 12 hr
at 4~ in benzyloated dialysis bags (molecular weight cut off 3000) against the same
buffer. Complete removal of free FITC was done by passing the dialyzed reaction mixture
through Sephadex G-10 colunm (25 x 2.0 cm). FITC tagged to insulin was obtained in
the void volume. Percentage incorporation of FITC to insulin was calculated from the UV
spectral peaks of insulin and FITC at 275 nm and 495 nm. To ensure complete removal of
free FITC from the insulin, it was again dialyzed overnight and the constancy in
percentage incorporation value revealed the removal of free FITC.

Assay of chaperone activity: Chaperone activity of ct-crystallin using insulin (B-chain)

as substrate was assayed by the method of (16). 0.4 mg of insulin in 1 ml of 10 mM
sodium phosphate buffer, pH 7.2, in the absence or presence of 0.4 mg a-crystallin was
equilibrated for 10 rain in a black spacer filled quartz cuvett maintained at a constant
temperature in a Hitachi U-2000 Spectrophotometer. Insulin was reduced with freshly
prepared dithiothreitol (DTT) to a final concentration of 20 mM and kinetics of B-chain
aggregation was followed by measuring the scattering at 360 nm. The assay was
performed at 18~ 27~ 30~ and 37~
Chaperone activity of ct-crystallin against UV-light induced aggregation of 7crystallin was assayed according to (14) using a Perkin Elmer MPF 44B
spectrofluorimeter. 0.2 mg of 7-crystallin with or without 0.2 mg c~-crystallin was taken
in 1 ml of 10 mM phosphate buffer, pH 7.2, containing 100 mM NaC1. The cuvett was
continuously irradiated at 295 nm with excitation band pass 10 nm and emission band
pass 2 nm. Emission intensity (scattering) was measured at 295 nm for 60 rain.

Isolation of ct-crystallin-insulin complex: ct-crystallin and FITC-insulin in 1:1 molar

ratio was incubated at various temperatures in the presence of 20 mM of DTT (16). The
complexes formed between ct-crystallin and insulin B-chain at different temperatures
were isolated using Sephadex G-50 (27 x 1.5 cm) size exclusion chromatography. The
presence of insulin B-chain co-eluted with ct-crystallin was detected by the 495 nm
absorbance of the FITC. The presence of bound FITC-insulin B chain was also confirmed
by fluorescence spectroscopy.
RESULTS
Although complete protection of insulin B-chain aggregation by ct-crystallin was
reported by (16) at 1:6 weight ratio of insulin : ct-crystallin, (17) reported that at a 1:1
weight ratio, the percentage protection against aggregation was zero at and below 30~
In an assay of chaperone activity of ct-crystallin against insulin using method (16) it was

251

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BIOCHEMISTRYond
MOLECULAR BIOLOGYINTERNATIONAL

shown that there was a detectable and non-negligible amount of chaperone activity of cccrystallin at room temperature (27~

This result prompted the study of the chaperone

activity of a-crystallin at other temperatures.

Figure 1 shows the kinetic traces of aggregation of insulin B-chain at four different
temperatures 18~

23~

27~ and 37~ in the absence and presence of cc-crystallin. At

all four temperatures, less aggregation of insulin was observed in the presence of cccrystallin than in its absence. Two points are made from the results. (1) Considerable
chaperone activity ( 2 5 % ) of ot-crystallin was detected even at 18~

(2) Chaperone

activity was increased with increasing temperature. The temperature dependence of the
protection against insulin aggregation is shown in Table 1. This results indicate that
thermal activation is not an absolute requirement for the chaperone activity of acrystallin, although such activity is enhanced by an increase in temperature. Complete
protection of insulin B chain is obtained at 12 : 1 (w/w) ratio of ct-crystallin at 18~
(Table 2).
It is of note that in the assay protocol, the chaperone activity is measured by the
prevention of aggregation, which is measured by light scattering. Scattering (as detected
by apparent absorbance) is often measurable when aggregate size grows beyond a limiting
value. The lag time observed in the kinetic traces (Figure 1) approximately indicates the
time taken for each system to grow to aggregate sizes that lead to visible scattering. Under
these conditions, the percentage protection calculated using the data does not
quantitatively indicate the amount of substrate bound to the chaperone. Complexes of cccrystallin-insulin have been isolated and the UV spectra of the complexes formed at
different temperatures are shown in Figure 2. The presence of a distinct peak at 495 nm
indicates the presence of FITC labeled insulin bound to cc-crystallin. All the chaperonesubstrate complexes formed at 20~

27~

and 37~

produced typical FITC emission

spectra in the 490-570 nm region (Figure 3a) and tryptophan emission spectra in the 310390 nm region (Figure 3b) confirming the presence of both FITC labeled insulin B-chain
and cc-crystallin in the complexes. Since the excitation wavelength was chosen to be 295
nm, the tryptophan emission spectra shown in Figure 3b do not contain any contribution
from the tyrosines of the insulin B chain. The results again show that chaperone-substrate
binding took place at all temperatures below and above 30~

252

This again confirms the

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Vol. 46, No. 2, 1 9 9 8

0120!/

(1

f, - o . o s

.o
L.

400

800

1 2 0 0 1601

'
400

8'00

'
1200

'
'
1600

<

1"20
C

C
L,
0
O.

<
-0-05
0

400

800

Time

Figure 1 :

1200

1600

400

800

1 2 0 0 1600

in s e c o n d s

Aggregation of insulin B chain at (a) 37~ (b) 27~ (c) 23~ (d) 18~
Aggregation was monitored by measuring scattering at 360 nm. (-) 0.4
mg/ml insulin alone, and at an insulin to ct-crystallin weight ratio of 1 : 1

(--).

TABLE I
Temperature dependent prevention of aggregation of insu]in by ~crystallin
at 1 : 1 (w/w) ratio.
Temperature
(~

Percentage
Protection*

18

26+0.32

23

28 + 0.13

27

28 + 0.51

37

52 + 0.83

The results are mean + sem o f five observations.

*Percentage protection is calculated using the equation Io-Ia / Io x 100; where Io is the
light scattering in the absence and Iot in the presence of a-crystallin at a fixed time (1800
sec) after initiation of the reaction with DTT.

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TABLE II
Dose dependent prevention of aggregation of insulin by
ot-crystallin at 18~

Ratio of
Insulin : c~-crystallin

Percentage
protection

1:1

26 + 0.32

1:6

74+0.11

1:12

95+0.23

The results are mean + sem of five observations.

0-7,5I/~

o.s
240

340

440

540
b

~0.25

240

340

< 0-4 ~,.~

240
Figure 2 :

440

540

340
440 540
Wavelength(nm)

UV absorption spectra of c~-crystallin bound to FITC-insulin B chain at (a)

37~ (b) 27~ (c) 20~

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Vol. 46, No. 2, 1998

BIOCHEMISTRY
and MOLECULAR BIOLOGYINTERNATIONAL

80

37oc

s~c

80

4.-,
4--,

I-,

60

60

tt..

.O

I.-

40

4O
e"

-i--,

4-"
r

r"

U
r

Ga

20

20
U
1/1
k..
O
h
0

Figure 3 :

;o

490
335
570
Wovetength (nm)

390
310
340
Wovetength (nm)

C
Ga
ta

O
t.t.

Fluorescence emission spectra of ct-crystallin FITC-insulin B complex at

different temperature. Excitation and emission band passes were 5 nm
each. (a) Emission of FITC chromophore bound to the FITC-insulin-otcrystallin complex; excitation wavelength -- 490 nm. (b) Emission of
tryptophan moiety of ct-crystallin bound to the FITC-insulin-ct-crystallin
complex; excitation wavelength = 295 nm.

observation that there is no need to invoke a thermal transition induced activation at 30~
as

suggested by (17) for ct-crystallin to be active as a chaperone.

Raman et. al. (14) also studied the chaperone activity of ct-crystallin against UV-

light induced aggregation of y-crystallin further emphasizing a thermal activation of etcrystallin for the expression of its chaperone activity. This system was reinvestigated at
different temperatures and the results are shown in Table 3. Even at 25~

approximately

20% protection was obtained. However, the photoaggregetion process of 7-crystallin itself
was found to be highly temperature dependent. In fact no measurable aggregation of UVlight induced q,-crystallin was observed at temperatures at and below 20~

and therefore

chaperone activity of ct-crystallin could not be assessed. In this system also increased
temperature was found to enhance the chaperone activity of ot-crystallin.

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MOLECULAR BIOLOGY INTERNATIONAL

DISCUSSION
Chaperone activity of c~-crystallin has been confirmed by various workers using
different substrates (14-18). Lack of substrate specificity of ct-crystallin is similar to the
bacterial chaperone GroEL which has been studied extensively (21).

Like GroEL, ot-

crystallin binds to substrate proteins in a conformation in between native and fully

extended form, often designated as a molten-globule state (22). However, conformational
features of ct-crystallin, essential

for recognition

of the

non-native

substrate

conformations are not yet established.

It is commonly believed that such recognition occurs through non-specific
hydrophobic interactions between already existing hydrophobic groups in the molecular
chaperone and the nascent or just exposed hydrophobic groups in the substrate proteins
(21). It was suggested that either freshly exposed hydrophobic sites of ct-crystallin or one
of its conformer having slightly enhanced hydrophobicity was essential for its chaperonelike activity (14,17). Present results clearly demonstrate that no such hypothesis needs to
be invoked to explain chaperone activity of ct-crystallin. It may be mentioned here that
contrary to the conformational transitions of ot-crystallin at 30~ reported by (14,17), a
subsequent study by (15) could not detect any conformational transition between 25~ 37~

range using highly conformation sensitive hydrophobic probe bis-ANS. In this

TABLE III

Temperature dependent prevention of photoaggregation of,/-crystallin by

ct-crystallin at 1 : 1 (w/w) ratio.

Temperature
(~

Percentage
protection

25

21 + 0.23

27

23 + 0.42

37

48 _+0.37

The results are mean + sem of three observations.

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temperature range, increase of temperature was found to lead to a gradual increase of

surface hydrophobicity and this exposure of hydrophobicity correlated well with increase
of chaperone activity (15).
The chaperone-like activity of a-crystallin is believed to be responsible for
maintenance of transparency of eye lens. Lens is a thermally insulated organ and
temperature fluctuations are generally small. Therefore the need for thermal activation for
ct-crystallin to be active is not physiologically relevant. Besides et-crystallin is present in
the lenses of cold blooded animals also. If ~x-crystallin were not active below 30~

large number of vertebrates inhabiting in cold weather conditions would have lost their
sight early! Proposition of temperature activation of (x-crystallin is thus physiologically
inconceivable.

Present results clearly eliminate one suggested mechanism for the

chaperone activity of a-crystallin.

ACKNOWLEDGEMENT
This work was supported in part by Department of Biotechnology (Govt. of India)
post-doctoral program, and also by Council of Scientific & Industrial Research (Govt. of
India) Project No: 37 (0943)/97/EMR-II.
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