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Effect of Homocysteinylation on Structure,

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932 Protein & Peptide Letters, 2013, 20, 932-941

Effect of Homocysteinylation on Structure, Chaperone Activity and Fibril-

lation Propensity of Lens Alpha-crystallin

Reza Yousefi*1, Sima Khazaei1, Ali-Akbar Moosavi-Movahedi2

1
Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran;
2
Institute of Biochemistry and Biophysics (IBB), the University of Tehran, Tehran, Iran

Abstract: Various chemical modifications can reduce chaperone activity of
-crystallin (
-Cry) and the loss of which has
been implicated in the development of cataract diseases. The side chains of lysine residues are the target of both glycation
and homocysteinylation, and lysine modification by the two reactions may similarly affect the structure and function of
-
Cry. In this study,
-Cry was incubated with homocysteine thiolactone (HCTL), resulting in significant protein homocys-
teinylation, as determined with Ellman's assay.
Homocysteinylation of
-Cry resulted in the reduction in surface hydrophobicity and alpha-helix to beta-sheet transition,
as observed respectively with fluorescence and circular dichroism (CD) spectroscopy. The structural alteration of homo-
cysteinylated
-Cry was accompanied by protein aggregation, including the formation of amyloid fibrils as detected by
thioflavin T (ThT) fluorescence and Congo red (CR) absorption spectroscopy. The mobility shifts of homocysteinylated

-Cry on reducing and non-reducing SDS-PAGEs suggest that disulfide cross-linking in addition to lysine modification,
also plays a role in aggregation of this protein. The chaperone activities of
-Cry, namely to prevent aggregation, to assist
refolding and to restore activity of thermally stressed
-glucosidase (
-Gls) were reduced after homocysteinylation. Over-
all, this study suggests that similar to non-enzymatic glycation, homocysteinylation of
-Cry is a risk factor for the devel-
opment of cataract disorders, for instance during hyperhomocysteinemia which is linked to the various ocular pathological
disorders.
Keywords: Aggregation,
-crystallin, cataract, chaperone activity, homocysteine thiolactone, fibrillation.

1. INTRODUCTION
B subunit is expressed extensively in other tissues, includ-

The transparency and refractive power of eye lens de-
pends on the native state of the - and -crystallins, which is
maintained by the chaperone action of
-Cry [1]. Eye lens
-
Cry subunits belong to the family of small heat shock pro-
teins (sHsps). They consist of the 20 kDa subunits A (173
AA) and B (175 AA), which are encoded by the CRYAA
and CRYAB genes. The two subunits share 57% sequence
identity [2] and exist as a polydisperse heteropolymer with
an average molecular mass of 600-800 kDa which contains
30-50 subunits [3, 4]. The subunit secondary structure con-
sists of approximately 14%
-helix, 35% -sheet and the rest
being random coils and turns [5]. Each
-Cry subunit has a
hydrophobic N-terminal domain which is necessary for
subunit interaction/multimerization and a hydrophilic C-
terminal domain (the so called
-Cry domain), which adopts
an immunoglobulin-like -sheet fold [6]. The later domain
follows by a flexible/electropositive C-terminal extension
which maintains the solubility and can bind to opposing
charged residues of the unfolding target proteins [7]. The
molar ratio of
A and
B subunits in the lenticular tissue
varies, depending on age and species [8]. Apart from lens,
*Address correspondence to this author at the Protein Chemistry Laboratory
(PCL), Department of Biology, College of Sciences, Shiraz University,
Shiraz, Iran; Tel: ++987116137617, ++987116137665;
E-mail: ryousefi@shirazu.ac.ir
1875-5305/13 $58.00+.00
ing lung, spleen, cardiac, skeletal muscle and brain, and it is
over-expressed in many neuropathological states [9-12]. The
subunits exist in a dynamic equilibrium between chaperone-
active dimeric and chaperone-inactive oligomeric states. The
constant subunit exchange between the two states at the
physiological temperatures is highly important for chaperone
function of
-Cry [3, 13,14]. In addition to the subunit ex-
change, other factors also influence chaperone-activity of
this protein; properties such as surface hydrophobicity, oli-
gomeric size/state, quaternary structure, stability and ionic
interactions between the chaperone and target proteins
[1,6,15]. Moreover,
-Cry undergoes a variety of post-trans-
lational modifications including phosphorylation, deamida-
tion, oxidation, glycation and C-terminal truncation which
accumulate with increasing age, resulting in loss of its chap-
erone activity, leading to the development of cataract disor-
der [16]. Homocysteinylation is known as a novel example
of protein modification, causing protein damages and in-
duces immune responses [17]. Homocysteinylation of lysine
residues generates additional thiol groups, that can form ex-
tra disulfide bridges, and such change the tertiary structure
and the aggregation propensity of the modified proteins [18-
20]. Moreover formation of intersubunit disulfide bridges
could induce and stabilize the interactions between crystal-
lins, leading to protein aggregates which have been observed
in the eye lens during cataractogenesis [21]. There are sev-
eral evidences suggesting a relationship between hyperho-
2013 Bentham Science Publishers
Homocysteinylation of
-crystallin Results in its Aggregation
Scheme 1. A possible mechanism for the relationship between hyperhomocysteinimia and cataract diseases.
mocysteinemia and various ocular disorders, such as cata-
ract, ectopic lenses, open-angle glaucoma, pseudoexfoliation
glaucoma, central retinal vein occlusion, maculopathy, optic
atrophy and diabetic retinopathy [22-29]. This study was
performed with the aim to evaluate the effect of homocyste-
inylation by homocysteine thiolactone (HCTL) which is cy-
clic thioether of homocysteine (Hcy), on structure, chaperone
activity and aggregation/fibrillation propensity of -Cry.
This study suggests that homocysteinylation of -Cry causes
structural alterations, leading to aggregation and fibrillation
of this protein. The induction of -Cry aggregation by HCTL
is of medical importance because it is believed that slow
aggregation and precipitation of crystallins which accumu-
late over years is the molecular basis of cataract diseases
[30].
2. MATERIALS AND METHODS
2.1. Materials
Sephacryl S-300 HR, HCTL, 1-anilino-8-naphthalene
sulfonate (ANS), thioflavin-T (ThT), Congo red (CR), bo-
vine pancreatic insulin, yeast -glucosidase (-Gls) and other
chemicals were purchased from Sigma.
2.2. Methods
2.2.1. Purification of
- and -crystallins
Purification of lens - and -crystallins were performed
according to previously published protocol [15]. In brief, the
bovine lenses were obtained from a local slaughterhouse and
dissected from the eyeballs. Then a 10% homogenate
(weight/volume) of the lenses was prepared in buffer A (25
mM Tris (pH 8.0), containing 0.5 mM EDTA, 0.1 M NaCl,
10 mM -mercaptoethanol and 0.01% NaN3), and centri-
fuged at 14,000 rpm, for 30 min at 4 C. The supernatant
(total soluble lens proteins, TSP) was collected and applied
on a Sephacryl S-300 HR (100 cm
1.5 cm) gel filtration
column equilibrated with buffer A (4 C, flow rate 0.25
mL/min, fraction size 2 mL). The protein concentration was
determined with the Bradford assay, and purity of the protein
was assessed by SDS-PAGE (12% acrylamide) [31]. While
- and -crystallins respectively form oligomers of large and
smaller sizes, -Cry exists essentially in the monomeric state
[32]. Therefore, these proteins were eluted from the column
as three major peaks, in order of their sizes. While -Cry
Protein & Peptide Letters, 2013, Vol. 20, No. 8 933
fractions were essentially pure, fractions containing -Cry
were concentrated and applied a second time to the same
column to obtain a pure sample of this protein. The highly
pure samples of - and -Cry were lyophilized and their
powders stored at -20 C until further use.
2.2.2. N-homocysteinylation of
-Cry
-Cry (3 mg/mL) was incubated at 37 C in buffer B
(100 mM NaPi containing 0.01% NaN3, pH 7.4) for 3 days
with and without 10 mM HCTL. To remove unreacted
HCTL, the treated sample was dialyzed against buffer B for
24 h. To evaluate extent of the protein N-homocys-
teinylation, free thiol groups were assayed, using Ellman's
reagent. To perform this experiment, a small portion of
HCTL treated -Cry was solubilized in 8 M urea in the pres-
ence of 5 mM dithiothreitol (DTT), and incubated for 10 min
at room temperature to ensure complete reduction of all di-
sulfide groups. The modified, reduced -Cry was separated
from DTT and Hcy (released from -Cry-Hcy mixed disul-
fides) on a Sephadex G25 desalting column. The extent of N-
homocysteinylation was determined by quantification of the
free thiol groups with Ellman's reagent (5, 5-dithio-bis (2-
nitrobenzoic acid) [33]. The reaction of one mole of thiol
group with the Ellman's reagent releases one mole of 5-
nitrothiobenzoate (NTB2-) which was measured by its ab-
sorption at 412 nm (molar extinction coefficient 13,700 M-1
cm-1) [19].
2.2.3. Measurement of Optical Density Changes and Ag-
gregation Process
The progress of -Cry aggregation in the presence of 10
mM HCTL in buffer B was measured as the light scattering
dependent increase of optical density between 300 and 600
nm. The experiments performed with a T90+ UV-Vis spec-
trophotometer (PG Instrument Ltd., UK) equipped with
Peltier temperature controller (Model PCT-2).
2.2.4. Thioflavin-T and Congo red Binding Assays
The formation of amyloid fibers was quantified with the
fluorescent dye ThT and with CR. The enhanced
fluorescence emission of ThT between 450-600 nm was re-
corded with a Cary-Eclipse spectrofluorimeter (Model Var-
ian, Australia), using an excitation wavelength of 440 nm
and slit bandwidths of 5 nm in both channels [19]. Freshly
prepared ThT solution (final concentration 20 M) was
934 Protein & Peptide Letters, 2013, Vol. 20, No. 8
added to -Cry (0.15 mg/mL), in buffer B and incubated for
5 min before the fluorescence measurements. Also, CR bind-
ing to amyloid structures was quantified by the spectral shift
of the absorption spectrum [34]. CR (final concentration 20
M) was added to -Cry (0.15 mg/mL) in buffer B and incu-
bated for 15 min at room temperature in the dark. The ab-
sorption between 400 and 700 nm was measured with a T90+
UV-Vis spectrophotometer instrument (PG Instrument Ltd.,
UK) equipped with peltier temperature controller (Model
PCT 2).
2.2.5. ANS and Trp Fluorescence Measurements
The Trp fluorescence of -Cry (0.1 mg/mL in buffer B,
37 C) was recorded between 300-500 nm at an excitation
wavelength of 295 nm. The slit widths were set to 5 nm in
the emission and excitation pathways. The ANS fluorescence
(0.12 mg/mL protein and 100 M ANS in buffer B at 37 C)
was recorded, between 400-600 nm with excitation at 365
nm, and slit bandwidths of 10 nm in both channels.
2.2.6. Far-UV CD Measurements
The far-UV CD spectra (190-260 nm, 25 C) were re-
corded with a CD spectrophotometer instrument (Aviv,
Model-215, USA), equipped with a thermoelectric sample
holder. The protein samples were diluted to 0.2 mg/mL in
buffer B. The results are expressed as mean residue elliptic-
ity at wavelength , []mrw (deg cm2 dmol-1) which is given
by the following equation:
[]mrw,=MRW /10d c (1)
where MRW stands for the mean residue weight of the pep-
tide bond,  is the observed ellipticity (degrees) at wave-
length , d is the path length (cm), and c is the protein con-
centration (g/mL) [35]. MRW=M/ (N-1), where M is the
molecular mass of the polypeptide chain (Da) and N is the
number of amino acids of the chain. The spectrum of buffer
B without protein was subtracted from each recorded spec-
trum. The secondary structure content was calculated with
the CDNN CD spectra deconvolution software [36].
2.2.7. Gel Electrophoresis Experiments
-Cry was incubated with and without 10 mM HCTL for
3 days at 37 C, in buffer B. To detect the formation of high-
molecular-weight (HMW) protein aggregates and the in-
volvement of disulfide bridges in the aggregates, HCTL
treated proteins and controls were separated on 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE), under non-reducing (sample buffer without -
mercaptoethanol) and reducing (with -mercaptoethanol)
conditions [37]. 12
g of each protein sample were loaded
per lane. The protein bands were stained with Coomassie
Brilliant Blue (CBB) [38].
2.2.8. Assaying of Anti-aggregation Ability of
-Cry
Concentration dependent chaperone activity (anti-
aggregation ability) of -Cry was measured with the target
proteins bovine pancreatic insulin (0.3 mg/mL) and -Cry
(0.3 mg/mL) in 25 mM NaPi pH 7.4. Aggregation of insulin
was induced with 20 mM dithiothreitol (DTT) and thermal
aggregation of -Cry by incubation at 65C. The progress of
aggregation was recorded as light scattering at 360 nm in a
T90+ UV-Vis spectrophotometer instrument (PG Instrument
Yousefi et al.
Ltd, UK) equipped with Peltier temperature controller
(Model PCT-2).
2.2.9. Thermal Inactivation and Refolding of
-Gls in the
Presence of
-Cry.
The kinetics of thermal unfolding of -Gls (0.2 unit/mL,
equal to 16.5 nM) was studied at 46 C in the absence and
presence of -Cry (0.05 mg/mL, equal to 2.5
M) [39]. Ali-
quots were withdrawn at regular intervals between 0 and 30
minutes and assayed for residual -Gls activity [39]. Refold-
ing of urea denatured -Gls was measured as follows: sam-
ples in 8 M urea, 20 mM DTT, 1 mM EDTA and 100 mM
NaPi, pH 7.0 were diluted a 100-fold dilution to a final con-
centration of 12 M in 100 mM NaPi, pH 7.0 in the presence
and absence of -Cry (260 nM). Then the recovery of en-
zyme activity was measured in aliquots withdrawn in inter-
vals [40].
2.2.10. Protein Assay
Protein (insulin, - and -crystallins) concentration was
measured from the UV absorption on a T90+ Uv/vis spectro-
photometer instrument. The concentration of insulin was
determined, using an extinction coefficient of 1.08 for 1
mg/mL at 276 nm [41]. Also, the extinction coefficients of
0.85 and 2.1 at 280 nm were used respectively for 1mg/mL
of - and -crystallins [42].
3. RESULTS AND DISCUSSION
3.1. Evaluation of N-homocysteinylation of
-Cry
Under physiological conditions, HCTL displays high
reactivity toward -amino group of Lys residues to form a
stable amide (isopeptide) bond (N-homocysteinylation).
Moreover, HCTL may react with cysteine thiols, forming
mixed Hcy-S-protein disulfides (S-homocysteinylation).
However, the rate of S-homocysteinylation is significantly
slower than N-homocysteinylation [43, 44]. While -A Cry
contains only one Cys and 7 Lys residues, -B Cry lacks Cys
and possesses 10 Lys residues [15], suggesting this protein
as a more feasible substrate for N-homocysteinylation, rather
than S-homocysteinylation. In view of the low content (1
Cys per 10 Lys assuming an A:B chain ratio of 3:1) and slow
reactivity of Cys, the effect - if any - of S-homocysteiny-
lation is not considered in this study.
-Cry was incubated
with HCTL for three days and N-homocysteinylation was
determined by the number of free thiols reacting with Ell-
man's reagent. N-homocysteinylation results in the incorpo-
ration of additional thiol groups into the primary structure of
-Cry which may affect both physicochemical properties and
biological activity of this protein. In two -Cry samples
which incubated with and without HCTL, the average quan-
tities of free thiol groups were 140 and 20
M respectively,
indicating a significant level of N-homocysteinylation. Also,
small quantity of the aggregates remained insoluble even
after dissolving in a solution of 8 M urea; therefore, the en-
tire extent of this modification cannot be analyzed by this
method. Since in the protein structure, side chains of Lys
residues are target of modification by both glucose and
HCTL, therefore, homocysteinylation of -Cry may have
similar structural and functional consequences to its non-
enzymatic glycation. As reported before, a variety of factors,
including glycation [45], crosslinking [46], proteolysis [47],
Homocysteinylation of
-crystallin Results in its Aggregation
oxidation and UV radiation [48] can reduce chaperone abil-
ity of
-Cry. Moreover, all of these modifications have been
already implicated in the development of cataract diseases
[49]. Therefore, homocysteinylation which induces
-Cry
aggregation via disulfide cross linking may have similar
pathological consequences.
3.2. HCTL Induces Aggregation/fibrillation of
-Cry
During ageing and cataract development, various post-
translational modifications of
-Cry may reduce its chaper-
one ability, leading to an increase in aggregation of other
crystallins and inactivation of some lenticular enzymes. The
aggregates will increase light scattering, leading to the lens
opacification [50]. Incubation of
-Cry with HCTL for three
days resulted in partial aggregation as evidenced by the ap-
pearance of turbidity (Fig. 1). As shown in this figure, visi-
ble light was scattered by HCTL treated
-Cry, but not by
the protein sample that was incubated for three days without
HCTL (control experiment). For assessment of the amount of
protein aggregates in the solution, at the end of incubation,
aliquots of the test and control samples were centrifuged at
10,000 rpm for 30 min.
Figure 1. Aggregation of
-Cry in the presence of HCTL.
-Cry
(3 mg/mL) incubated for 3 days with HCTL (dotted line) and with-
out (solid line). The protein samples were diluted to 0.2 mg/mL
before recording of the absorption spectra. For details see Materials
and Methods.
Once the aggregate in the test sample precipitated, pro-
tein concentration of the supernatant was determined by the
Bradford assay. The amount of protein in the aggregate
phase was determined by calculating the difference between
protein concentration of the control sample and that of su-
pernatant. The protein content in the supernatant of HCTL
treated protein was about 80% of the untreated control, indi-
cating that 20% of homocysteinylated
-Cry was lost in the
precipitate (pellet).
Since chaperone function of
-Cry to prevent aggregation
of other proteins is very important for the maintenance of
lens transparency, and because protein aggregation causes
light scattering in eye lens, leading to cataract disease;
HCTL induced aggregation of this protein might be consid-
ered as an important parameter contributing to the develop-
Protein & Peptide Letters, 2013, Vol. 20, No. 8 935
ment of cataract disorder. Also, there is increasing evidence
that cataract may be an amyloid fibril based disease [30].
Therefore, to characterize the type of structural change in-
duced by homocysteinylation, the protein sample incubated
with HCTL was stained with both ThT fluorescence- and CR
absorption assays which are diagnostic of diverse types of
amyloid fibrils.
After incubation of
-Cry with HCTL, a significant in-
crement in the ThT fluorescence was observed. Moreover,
the ThT fluorescence emission of the pellet was markedly
higher than that of either crude sample or the control ex-
periment (Fig. 2).
Figure 2. ThT fluorescence experiment of
-Cry fibrillation.
Homocysteinylated and control samples of
-Cry were incubated
with 20 M ThT, and the ThT fluorescence emission between 450-
600 nm was recorded.
-Cry without HCTL (thick line, control),
crude sample of homocysteinylated
-Cry (dotted line), supernatant
of (soluble) homocysteinylated
-Cry (dashed line), pellet of (in-
soluble) homocysteinylated
-Cry (dashed-dotted line), ThT alone
(solid circles). For details see Materials and Methods.
In contrast, the fluorescence intensity of protein-bound
ThT of the
-Cry supernatant incubated with HCTL was
similar to that of the control sample. Overall, the obtained
results revealed that only proteins of crude sample and ag-
gregate phase (pellet) were capable to interact with ThT,
suggesting the existence of significant amount of protein
fibrils in these two samples (Fig. 2). Also, incubation of CR
with the crude sample and resuspended pellet of
-Cry re-
sulted in significant increment in the absorption spectra, ac-
companying with red shift of absorbance maxima from 485
to 496 nm and 485 to 511 nm respectively. In addition, the
difference spectrum obtained by subtracting the free CR
spectrum without protein from the spectrum of CR with the
aggregated protein (pellet) had a maximum absorbance at
561 nm. In agreement with results of the ThT binding, CR
spectra of the treated supernatant and of the untreated control
sample were indistinguishable (Fig. 3).
In conclusion, both ThT fluorescence and CR absorption
results confirmed the formation of
-Cry amyloid entities
which incubated in the presence of HCTL. Consequently,
induction of amyloid fibril formation in
-Cry by HCTL
936 Protein & Peptide Letters, 2013, Vol. 20, No. 8 Yousefi et al.

Figure 3. The CR binding assay of
-Cry fibrillation. Both homocysteinylated and control samples of
-Cry were incubated with 20 M
CR, and the absorption/spectral shift of CR were recorded. A-D, CR alone (larger circles). A,
-Cry incubated without HCTL (solid line); B,
supernatant of homocysteinylated
-Cry (dashed line); C, crude sample of homocysteinylated
-Cry (dotted line); D, pellet of (insoluble)
homocysteinylated
-Cry (dashed & dotted line). The background absorption spectrum of the NaPi buffer was subtracted from the CR spec-
tra. The difference spectrum (D, inset) was obtained by subtracting the spectrum of the protein alone and that of CR alone from spectra of the
protein samples in the presence of CR. For details see Materials and Methods.

Figure 4. SDS-PAGE of different
-Cry samples.
-Cry and HCTL were incubated at 37 C for 3 days and aliquots were analyzed under
(A) non-reducing and (B) reducing conditions. Lanes: 1, non-incubated fresh
-Cry; 2,
-Cry incubated without HCTL; 3, crude sample of
homocysteinylated
-Cry; 4, supernatant of (soluble) homocysteinylated
-Cry; 5, pellet of (insoluble) homocysteinylated
-Cry. The pro-
teins are stained with Coomassie Brilliant Blue. For details see Materials and Methods.

might be involved in the pathomechanism of cataract dis-
ease.
3.3. Gel Electrophoresis for the Assessment of
-Cry Ag-
gregation by HCTL
N-homocysteinylation of proteins introduces Hcy thiol
groups, a theoretical maximum of 17 in the A and B chains
of
-Cry. These thiols could form intra- and intersubunit
disulfide bonds - at least under oxidizing conditions. To find
out whether intersubunit disulfide crosslinks between the
Hcy residues are involved in the aggregation of homocyste-
inylated
-Cry, the soluble and insoluble forms were charac-
terized by polyacrylamide gel electrophoresis under non-
reducing and reducing conditions (Fig. 4). As shown in (Fig.
4A), under non-reducing condition, both crude sample and
Homocysteinylation of
-crystallin Results in its Aggregation Protein & Peptide Letters, 2013, Vol. 20, No. 8 937

pellet of
-Cry treated with HCTL, comprising HMW pro-
tein species which detained inside of the wells of electropho-
resis gel (Lanes 3 and 5). In both conditions the bulk of
-
Cry has an electrophoretic mobility corresponding to the
monomer molecular mass of 20 kDa. Under non-reducing
conditions a faint band with the mobility corresponding to
the 40 kDa dimer is visible. This band disappears under re-
ducing condition, and is stronger in the samples not treated
with HCTL. This band is stronger in the samples not treated
with HCTL, and it disappears from all samples, under
reducing condition. This suggests that the dimer is caused by
cysteine disulfide-crosslinks between two A chains, and not
by crosslinks between Hcy residues of homocysteinylated
-
Cry.
As the experiment repeated under reducing conditions
(Fig. 4B), a large quantity of the aggregates was disappeared,
indicating role of disulfide bond in formation of HMW
protein species. However, in both conditions, a small amount
of HMW proteinaceous (Coomassie stained) material was
retained in the pockets of the stacking gel (Fig. 4A), suggest-
ing the involvement of other factors additional to disulfide
cross-linking, in the formation of
-Cry aggregates. The di-
sulfide cross-linking in eye lens proteins is believed to be
involved during cataractogenesis [21]. Consequently, disul-
fide bridge formation which facilitates aggregation of homo-
cysteinylated
-Cry might be highly relevant to the cataract
pathomechanism.
3.4. N-homocysteinylation Reduces Chaperone Activity
of
-Cry
In the cortex of lens,
-Cry demonstrates higher level of
chaperone activity than that located in the lens nucleus [51],
reflecting different degrees of posttranslational modifications
and ageing of the protein. Because of the limited protein
turnover at the center of eye lens, crystallins in the nucleus
are as old as the individual, and due to the accumulation of
chemical modifications, they demonstrate a reduced degree
of both water solubility and chaperone activity. In this study,
chaperone activity (anti-aggregation ability) of the modified-
and unmodified
-Cry were examined and compared in both
chemical and thermal-induced aggregation systems. In the
former system, DTT was used to induce insulin aggregation
at 37 C, and in the later one, -Cry aggregation performed
at 65 C. As shown in (Fig. 5), homocysteinylated
-Cry
exhibits significantly lower anti-aggregation activity than
unmodified protein counterpart in both systems.
In the chemical induced aggregation system, anti-aggre-
gation ability of untreated
-Cry was increased moderately in
a concentration dependent manner. To the contrary, anti-
aggregation activity disappears with increasing concentration
of homocysteinylated
-Cry (Fig. 5A1-A3). This observation
can be explained as follows:
-Cry occurs in a dimeric,
chaperone-active form and in an oligomeric, inactive form.
The two forms are in constant equilibrium. N-homocys-
teinylation may shift the equilibrium in favor of the oli-
gomeric state and thus reduce the percentage of the chaper-
one-active, dimeric form at a given total concentration of

Figure 5. Chaperone activity of homocysteinylated and non-homocysteinylated
-Cry: Protection against aggregation of insulin and -Cry.
A, Aggregation of insulin was initiated by reduction of disulfide bridges with DTT and the progress of aggregation monitored by light-
scattering (absorbance): without
-Cry (dotted), with
-Cry (solid), with homocysteinylated
-Cry (dashed). The
-Cry concentrations were
0.05, 0.15 and 0.2 mg/mL (A1-A3). B, Aggregation of -Cry was initiated by shifting the temperature to 65 C. Progress of aggregation at 65
C without
-Cry (solid circles), with
-Cry (solid), with homocysteinylated
-Cry (dashed). The
-Cry concentrations were 0.025, 0.05 and
0.1 mg/mL (B1-B3). For details see Materials and Methods.
938 Protein & Peptide Letters, 2013, Vol. 20, No. 8 Yousefi et al.

Figure 6. Chaperone activity of homocysteinylated and non-homocysteinylated
-Cry: Assisting the refolding and protection against thermal
unfolding of
-Gls. A, Refolding of urea-denatured yeast
-Gls was initiated by 100 fold dilution in a
-Cry containing refolding buffer. The
progress of folding was monitored as time-dependent recovery of enzymatic activity, without
-Cry (triangle), with
-Cry (solid circle), with
homocysteinylated
-Cry (open circle). B, The progress of thermal unfolding of
-Gls in the presence of
-Cry was measured as the time-
dependent loss of
-Gls activity. The residual activity was determined in diluted aliquots. Heat inactivation without
-Cry (triangle), with
-
Cry (solid circle), with homocysteinylated
-Cry (open circle). For details see Materials and Methods.

homocysteinylated
-Cry. The observed increase of light
scattering and dye-binding of homocysteinylated
-Cry, de-
scribed above, provide direct biophysical evidence for the N-
homocysteinylation induced aggregation of this protein. Dif-
ferent results were observed with thermally stressed -Cry as
the substrate protein (Fig. 5 B1-B3). In this case, protection
against heat induced aggregation was concentration depend-
ent for both native
-Cry and the homocysteinylated protein
(Fig. 5B). These results may support the evidences suggest-
ing that rate of subunit exchange increases with elevated
temperature, contributing to the enhancement of
-Cry chap-
erone ability [52]. However, under normal physiological
conditions and at the chemical induced aggregation system,
the subunit exchange is mainly restricted by the high activa-
tion energy of 60 kcal/mol [52]. As a molecular chaperone,

-Cry not only protects target proteins against unfolding but
it also supports their refolding from the denatured back to the
native state and restores enzyme activity under both chemi-
cal and physical stresses[40]. As reported before,
-Cry can
specifically protect several enzymes through complex forma-
tion against both chemically- and thermally-induced inacti-
vation. Refolding was assayed with
-Gls that was unfolded
with 8 M urea. Refolding was initiated by 100-fold dilution
in a buffer containing
-Cry and homocysteinylated
-Cry.
The time course of refolding was measured as recovery of
the enzyme activity. To analyze the refolding yield, aliquots
were taken at indicated times and the kinetics of enzyme
reactivation examined. In the absence of
-Cry, 31% of the
initial activity was recovered within less than 20 min. How-
ever, when the experiments repeated in the presence of ho-
mocysteinylated and control
-Cry, refolding yields were 38-
and 60% respectively (Fig. 6A). From this result, it appears
that N- homocysteinylation significantly compromises the
chaperone/refolding activity of
-Cry.
Yeast
-Gls is a temperature-sensitive enzyme, with an
inactivation half time of 15 min at 46 C [39,53]. In this
study the protective effect of both homocysteinylated and
control
-Cry on the time course of thermal inactivation of

-Gls was compared (Fig. 6B). The protective effect of na-
tive
-Cry was spurious while the effect was even less pro-
nounced for the homocysteinylated chaperone. As reported
before, complex formation between
-Cry and denatured
enzymes is important in protection against enzyme thermal
inactivation. The weaker thermal protection ability of homo-
cysteinylated
-Cry compared to non-modified counterpart
can be explained with the lesser level of solvent exposed
hydrophobicity of this protein. The surface hydrophobicity is
necessary for interaction with aggregation-prone or dena-
tured (non-native) unfolded enzyme. Overall, as mentioned
above, the chaperone function of
-Cry to prevent aggrega-
tion of other proteins or enzyme inactivation is very impor-
tant for the maintenance of lens transparency. Consequently,
loss of chaperone action of this protein as a result of homo-
cysteinylation may suggest this modification as one of the
possible contributing factors in the development of cataract
disease. It is likely to happen, because cataract is known as a
disease of essentially Cry protein aggregation and subse-
quent precipitation, occurring over the time frame of years.
3.5. Homocysteinylation Causes the Structural Altera-
tions in
-Cry
As a major cause of blindness, cataract is associated with
conformational changes and unfolding of lens proteins, aris-
ing directly as a result of post-translational modifications
[54]. The structural alteration after homocysteinylation of
-
Cry was measured by Trp fluorescence study.
A- and
B-
Cry respectively have 1 and 2 Trp residues in their primary
structures [15]. As shown in (Fig. 7A), the Trp fluorescence
of homocysteinylated
-Cry is significantly reduced com-
pared to the unmodified control sample, suggesting that these
residues in homocysteinylated
-Cry are in a more polar
microenvironment than in the control protein. The movement
of Trp residues to an environment with different polarity
indicates structural alteration of the homocysteinylated pro-
tein. A characteristic feature of chaperones is surface ex-
Homocysteinylation of
-crystallin Results in its Aggregation Protein & Peptide Letters, 2013, Vol. 20, No. 8 939

Table. 1. The Percentage of Secondary Structure Composition of Different Samples of -Cry

-Cry -Helix -Sheet -Turn Random coil

Fresh sample 11 35 15 39

-HCTL 12 34 15 39

+ HCTL (supernatant) 10 36 16 38

+ HCTL (crude sample) 7 41 16 36

The secondary structural content was calculated by deconvolution of the CD spectra. For details see Materials and Methods.

posed hydrophobic patches, which are important for the in-

teraction with (partially) unfolded target proteins.
-Cry
could prevent the aggregation of target proteins by the inter-
action between the hydrophobic patches on its surface and
the exposed hydrophobic sites of partially unfolded target
proteins The dye ANS binds to external sites of proteins,
which are exposed to the aqueous phase and the binding re-
sults in an increase of its fluorescence emission intensity and
a blue shift of the emission maximum [57, 58]. As shown in
(Fig. 7B), the homocysteinylated
-Cry displayed signifi-
cantly lower level of ANS fluorescence intensity than non
modified control sample which is mainly due to the hiding of
surface-exposed hydrophobic sites upon aggregation. Also
the supernatant of homocysteinylated
-Cry demonstrates a
reduction in ANS fluorescence emission compared to the
control protein sample, indicating homocysteilyaltion results
in significant structural alteration which is accompanied with
the changes in hydrophobic surfaces of
-Cry. Although, a
quantifiable relationship between hydrophobicity and chap-
erone activity of
-Cry remains a matter to be concerned
about, it should be noted that both oligomerization and
subunit exchange are interconnected and mediated by hydro-
phobic interactions. However, the reduction of surface hy-
drophobicity was correlated with the reduced chaperone ac-
tivity (Fig. 6). The result of this study is another example,
where chaperone activity and hydrophobicity of
-Cry run in
the same directions.
As mentioned before, amyloid fibrils contribute to the
pathology of several diseases, including cataract. CD is an
averaging technique, which can be used also to characterize
properties of protein amyloid fibril [59]. According to the
previous report, native
-Cry has a maximum negative ellip-
ticity at approximate wavelength of 217 nm (Fig. 7C), which
is characteristic of a -sheet structure [60, 61]. To analyze
changes in the secondary structure contents of
-Cry upon
homocysteinylation, the far-UV CD spectra in the range of
190-260 nm were obtained. As shown, HCTL induces sig-
nificant changes in the secondary structures of
-Cry, both at
208 and 222 nm. Deconvolution of the spectra indicates a
slight reduction in
-helical content; along with an increment
in the -sheet content of homocysteinylated
-Cry. The in- Figure 7. Fluorescence- and far-UV CD analysis of different
-Cry
crease in -sheet content of
-Cry together with the decrease samples. A, Trp fluorescence. B, ANS fluorescence. C, Far-UV
in
-helical percentage is characteristic of amyloid-like pro- circular dichroism (CD):
-Cry incubated without HCTL (thick
tein transitions, occurring upon homocysteinylation of this line), crude sample of homocysteinylated
-Cry (dotted line), su-
protein (Fig. 7C and Table 1) [62]. pernatant of homocysteinylated
-Cry (dashed line), fresh sample
of
-Cry (dashed & double dotted line), ANS (larger circle). The
background of the protein free buffer has been subtracted from the
CD spectrum as shown. For details see Materials and Methods.
940 Protein & Peptide Letters, 2013, Vol. 20, No. 8
CONCLUSION
Homocysteinylation of
-Cry causes structural altera-
tions, leading to aggregation and fibrillation. Results of SDS-
PAGE suggest that disulfide cross-linking mediates aggrega-
tion of this protein. Also homocysteinylation results in a re-
duction of both surface hydrophobicity and chaperone activ-
ity of
-Cry. Since side chains of Lys residues are modified
not only by non-enzymatic glycation which has been impli-
cated in the pathomechanism of cataract diseases, but also by
homocysteinylation; the two modifications may have similar
structural and functional consequences. This proposal can be
further supported by the relationship between various ocular
disorders and hyperhomocysteinimia.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
We would like to thank the financial supports of Iran
National Science Foundation (INSF)/Grant no. 88001578.
Also the financial supports of the research council of Shiraz
University are gratefully acknowledged. Moreover, the
authors would like to thank Professor Bernhard Erni (De-
partment of Chemistry and Biochemistry, Bern University,
Switzerland) for his invaluable contribution in improvement
of the manuscript.
ABBREVIATIONS
HCTL = Homocysteine thiolactone
Hcy = Homocysteine
ThT = Thioflavin T
CR = Congored
ANS = 1-anilino-8-naphthalene sulfonate
CD = Circular dichroism

-Cry = Alpha-crystallin
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