(Received for publication, February 14, 1997, and in revised form, March 31, 1997)
From the Department of Biochemistry and Molecular
Biology, Medical College of Georgia, Augusta, Georgia 30912 and
§ Center for Vision Research, Department of Ophthalmology,
Case Western Reserve University, Cleveland, Ohio 44106
In the previous report we demonstrated that
B-crystallin is glycated predominantly at the N-terminal
-amino
group (Casey, E. B., Zhao, H. R., and Abraham, E. C. (1995)
J. Biol. Chem. 270, 20781-20786). To investigate the
possible role of
- and
-amino groups of
B-crystallin in
glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were
mutated to threonine by site-directed mutagenesis in bovine
B-crystallin cDNA. Wild type and mutant
B-crystallins were
expressed in Escherichia coli cells. Cross-linking studies
were performed by incubating wild type and mutant
B-crystallins with
glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines
of
B-crystallin were mutated to threonines (
B-K2T/K163T), the
quantity of cross-linked products was greatly reduced, indicating that,
despite the fact that the
-amino group is a major glycated site,
-amino groups play a predominant role in cross-linking. Therefore,
cross-linking ability depends not only upon the level of glycation but
also upon which amino group is glycated. Steric hindrance may decrease
the cross-linking ability of the
-amino group. Our results also show
that Lys-2 and Lys-163 play almost equal roles in cross-linking of
B-crystallin. By incubating carbonic anhydrase, a protein with a
blocked N terminus, and our novel "no lysine"
B (
B-K2T/K163T)
with sugar, we were able to show for the first time that significant
cross-linking occurs between lysines and non-lysine sites. The fact
that pentosidine and imidazolysine, formed from ribose and
methylglyoxal, respectively, were present in the cross-linked
B-crystallins revealed the existence of Lys-Arg and Lys-Lys
cross-linking.
Aldehyde or keto groups of reducing sugars react mainly with -
and
-amino groups on proteins forming unstable Schiff base adducts,
which rearrange to form more stable Amadori products (1). These Amadori
products undergo a series of chemical reactions, becoming brown,
fluorescent, and irreversibly cross-linked products. Vlassara et
al. (2) first used the term advanced glycosylation (glycation) end
products (AGEs)1 to describe these
cross-linked products formed during the late stage of the Maillard
reaction in vivo. AGE-mediated cross-linking is believed to
be important in the pathogenesis of aging and in the complications of
diabetes (3-5). Cross-linking of proteins is believed to occur mostly
among
-amino groups of lysines as well as guanidinium groups of
arginines. But the roles of these groups and of
-amino groups in
glycation-mediated cross-linking are still not clear because AGE
structure is largely unknown. At present, AGEs are thought to be
composed of diverse structures. However, studies with recently
developed monoclonal and polyclonal antibodies against AGEs have
suggested that they all have a common structure (6, 7). Elucidated AGE
structures include
N
-(carboxymethyl)lysine (8),
pentosidine (9), pyrraline (10), N
-(carboxymethyl)hydroxylysine (11),
crossline (12), and imidazolysine (13, 14). Among them, pentosidine
(Lys-Arg), crosslines (Lys-Lys), and imidazolysine (Lys-Lys) have
cross-linking properties.
-Crystallin is one of the major classes of lens-soluble proteins.
Bovine
B-crystallin, the major species of bovine
-crystallin, contains only two lysines (Lys-2 and Lys-163) among its 174 amino acid
residues (15).
-Crystallin is the only crystallin with a free N
terminus (16). It is synthesized at an early stage in the development
of the lens and is found mainly at the core region of the mammalian
lens (17). Since there is little or no protein turnover in the core
region (18), postsynthetic modifications of
B-crystallin such as
glycation will accumulate in the nucleus of the lens. Indeed, it is the
core region where nuclear cataract, the important form of cataract with
protein cross-linking and brunescense in humans, is formed.
In a previous report, we showed that the N-terminal -amino group of
bovine
B-crystallin is the predominant glycation site (19), as
confirmed recently by mass spectrometry analysis (20). In the present
communication, the two lysines of bovine
B-crystallin were mutated
to threonine; this "no lysine"
B-crystallin, as well as the
mutants containing only Lys-2 or Lys-163, was used to study the role of
the N-terminal
-amino group of Gly-1 and the
-amino groups of two
lysines in glycation-mediated cross-linking of
B-crystallin.
The bovine B-crystallin
cDNA in pBluescript (15) was kindly provided by Dr. Regine Hay
(Department of Ophthalmology, Washington University).
B-crystallin
cDNA was subcloned into the NcoI/HindIII cloning site of expression vector pMON5743 (pMON
B), and
site-directed mutagenesis of Lys-2 to Thr (pMON
B-K2T) was carried
out using polymerase chain reaction as described previously (19).
Creation of two new mutants, Lys-163 to Thr (K163T) as well as Lys-2
and Lys-163 to Thr (K2T/K163T), was accomplished with synthetic
oligonucleotides. The desired base changes of Lys-163 to Thr were
incorporated into the oligonucleotides (AACCAACTGTGGCATTC
and AATGCCACAGTTGGTTC), and mutagenesis was performed by
two-stage polymerase chain reaction-based overlap extension (21). Using
pMON
B as a template led to the generation of a
B-K163T mutant,
whereas when pMON
B-K2T was used as the template, the double mutant
B-K2T/K163T was generated. The nucleotide sequences were checked
using the sequencing kit from U. S. Biochemical Corp. Other methods
used in subcloning were described previously (19).
Clones containing pMONB, pMON
B-K2T,
pMON
B-K163T, and pMON
B-K2T/K163T were used to express recombinant
B-crystallins of wild type, mutant K2T (
B-K2T), mutant K163T
(
B-K163T), and mutant K2T/K163T (
B-K2T/K163T), respectively.
Expression of wild type and mutant
B-crystallins was performed
according to the method of Olins and Rangwala (22). Wild type and
mutant
B-crystallins in the cell lysates were purified using
cation-exchange HPLC according to the method of Siezen et
al. (23) with minor modifications (19). Since
B-K2T/K163T has a
lower pI, buffers used in cation-exchange HPLC were adjusted to pH 5.5 instead of pH 6.0, and bacterial proteins found in the
B-K2T/K163T
fraction were removed by molecular sieve chromatography on a 2 × 100-cm Sephadex G-100 (Sigma) column.
SDS-PAGE was performed according
to the method of Laemmli (24) using a 12% separating gel and a 4%
stacking gel under reducing conditions. Gels were routinely stained
with Coomassie Blue, but in the case of galactose incubation a silver
stain kit (Bio-Rad) was used to visualize small amounts of cross-linked
products. Western blotting was carried out using a monoclonal antibody
against rat -crystallin (19, 25). Bovine
-crystallin was prepared as described previously (19).
Fifty microliters of wild type or mutant B-crystallin
(2 mg/ml) were incubated with or without sugar in darkness at 37 °C in phosphate-buffered saline (pH 7.4) containing 0.02% sodium azide.
Since the cross-linking ability of different sugars varies (26),
different sugar concentrations were used: glyceraldehyde (5 mM), ribose (100 mM), and galactose (1 M) (all from Sigma). To investigate the ability of lysine
to cross-link with non-lysine sites (e.g.
-amino group as
well as arginine and histidine residues), equal concentrations of
carbonic anhydrase (CA) and
B-K2T/K163T or wild type
B were
incubated with 5 mM glyceraldehyde. After incubation for 10 days (glyceraldehyde and ribose) or 20 days (galactose), the samples
were analyzed by SDS-PAGE under reducing conditions, and the gels were
scanned on a CS-9301 PC dual wavelength Fly Spot scanning densitometer
(Shimadzu Corp., Tokyo, Japan). Protein concentration was determined by
the method of Lowry et al. (27).
Pentosidine (Lys-Arg cross-link) was synthesized according
to the method of Sell and Monnier (9). Imidazolysine (Lys-Lys cross-link) was synthesized as described previously (14). Wild type and
mutant B-crystallin (2 mg/ml) were incubated with either 25 mM D-ribose (for pentosidine) or methylglyoxal
(for imidazolysine) in phosphate-buffered saline at 37 °C. Aliquots
of 0.3 ml were withdrawn on days 0, 5, and 10. Proteins incubated
simultaneously without the added carbohydrate served as controls. After
incubation, the protein was hydrolyzed with 6 N HCl at
110 °C for 20 h. The acid was evaporated in a Speed Vac
concentrator (Savant Instruments, Inc., Farmingdale, NY), and the amino
acids were reconstituted in water followed by centrifugation at
14,000 × g for clarification. Pentosidine was
estimated by reversed-phase HPLC on a C18 column (Vydac, 218TP, 25 × 0.46 cm, 10 µm, Separations Group, Hesperia, CA). The mobile phase
consisted of water (A) and 60% acetonitrile in water (B) with 0.01 M heptafluorobutyric acid (Sigma). The gradient program
used was 16-28% B in 35 min. The effluent from the column was
monitored for fluorescence at 385 nm (excitation, 335 nm). Pentosidine
eluted at ~29.0 min. For quantification, the peak area in the samples
was compared with the peak area of a known amount of purified
pentosidine. Other details of the HPLC settings are described elsewhere
(28). Imidazolysine was estimated by reversed-phase HPLC on a C18
column (Vydac, 201HS54, 25 × 0.46 cm, 5 µm). The mobile phase
consisted of water (A) and 70% methanol in water (B) with 0.1%
heptafluorobutyric acid. The gradient program was 0-100% B in 35 min.
The column effluent was allowed to mix with o-phthalaldehyde
for postcolumn derivatization, and the fluorescent derivatives were
detected by an on-line fluorescence detector (Jasco International Co.
Ltd., Toyko, Japan) set at excitation/emission of 340/455 nm as
described by Nagaraj et al. (14). Under these conditions
imidazolysine eluted at ~22 min. For quantification, the peak area in
the samples was compared with the peak area of a known amount of
purified imidazolysine.
Wild type and mutant B-crystallins were
expressed in JM101 Escherichia coli cells. The supernatants
of cell lysates were concentrated and analyzed on 12% SDS-PAGE. Heavy
bands with molecular mass equal to calf
-crystallin were found in
all cell lysates except in the lysate of the cells containing the
vector pMON5743 without
B-crystallin cDNA (Fig.
1A). The recombinant proteins were purified,
and the purity of the proteins was checked on SDS-PAGE (Fig.
1B). Western blotting with a monoclonal antibody to rat
-crystallin (19, 25) confirmed the expression of
B-crystallin (Fig. 1B).
Glycation-mediated Cross-linking
To cross-link
B-crystallins in a realistic in vitro time span, purified
wild type and mutant
B-crystallins were reacted with different
concentrations of sugars that are known to glycate and cross-link much
faster than glucose and subjected to SDS-PAGE. After a 10-day
incubation with DL-glyceraldehyde, dimer (~40 kDa), trimer (~60 kDa), and higher molecular mass cross-linked products were seen in wild type
B (Fig. 2A). Fewer
cross-linked products were found in
B-K2T and
B-K163T, but heavy
dimer bands could still be seen in both the mutants. If
B-K2T and
B-K163T are compared, the former seems to have slightly less
cross-linked protein than the latter. Only a faint band of cross-linked
product was observed with the double mutant
B-K2T/K163T despite the
fact that the
-amino group of Gly-1 is the predominant glycation
site of
B-crystallin. Gel scanning showed that formation of the
dimer of
B-K2T/K163T decreased by 73% as compared with that of wild type
B. When other slower reacting sugars, such as ribose and galactose, were incubated with wild type and mutant
B-crystallins, similar results were obtained (Fig. 2, B and C),
but no significant difference in cross-linking was seen among the wild
type
B,
B-K2T, and
B-K163T, and no trimer or higher
cross-linking product was observed. Again, only faint cross-linked
dimer bands were found in the incubation of
B-K2T/K163T, which
confirmed that
-amino groups of lysine are important in
glycation-mediated cross-linking.
To investigate possible cross-linking between -amino groups and
non-lysine cross-linking sites (e.g.
-amino group as well as arginine and histidine residues), CA was incubated with
B-K2T/K163T or wild type
B in the presence of 5 mM
glyceraldehyde for 10 days. CA was chosen because it has a blocked N
terminus, 18 lysines (29), and a molecular mass of about 29 kDa, which
facilitates distinguishing the different cross-linked dimers,
e.g.
B-K2T/K163T-
B-K2T/K163T,
B-K2T/K163T-CA, and
CA-CA, on SDS-PAGE. Fig. 3 shows that there are
substantial cross-linked products consisting of
B-K2T/K163T and CA
(lane 2), and the quantity of the cross-linked product is
much higher than that made up of two
B-K2T/K163T mutants (lane 5) but less than that comprised of wild type
B-crystallin and CA (lane 3).
Formation of Pentosidine and Imidazolysine
To confirm the
existence of Lys-Arg and Lys-Lys cross-linking, pentosidine and
imidazolysine were analyzed after cross-linking recombinant B with
sugars (Fig. 4). Our data show that pentosidine and
imidazolysine do exist in the cross-linking of
B-crystallin and the
amounts of pentosidine and imidazolysine decrease along with the
decrease of lysine content of
B. Fig. 4 also shows that approximately the same amount of pentosidine or imidazolysine was found
from
B-K2T and K163T. This finding agrees with our cross-linking
data, which show that
B-K2T and
B-K163T incubations contain about
the same quantities of cross-linking products (Fig. 2A).
Some pentosidine and imidazolysine detected in
B-K2T/K163T could
have come from a small amount of bacterial protein contamination. Since
B-crystallin is a low lysine content protein, small amounts of high
lysine content bacterial proteins may cause significant increases of
pentosidine and imidazolysine even though the cross-linking of
bacterial proteins cannot be seen on the SDS-PAGE of the protein cross-linking (Fig. 2).
Determination of the roles that specific glycated amino acid
residues play in cross-linking and insolubilization of crystallins will
shed light on the mechanism by which diabetic and senile cataracts
form. From our results, it is clear that lysine, although only weakly
glycated, is important for glycation-mediated cross-linking, and the
-amino group of
B-crystallin, despite being a major glycation
site (19, 20, 30), has much less ability to cross-link with itself or
with other residues. Therefore, cross-linking ability depends not only
upon the level of glycation but also upon which amino group is
glycated. Furthermore, by using our novel "no lysine"
B-crystallin (
B-K2T/K163T) and another protein having no free N
terminus, we were able to show for the first time that significant cross-linking between lysine and non-lysine cross-linking sites exists
(Fig. 3). Noticeable loss of arginine and lysine after incubating
lysozyme with glucose suggests that lysine and arginine are important
candidates for cross-linking (31). Earlier studies (9) have shown that
lysine and arginine can be cross-linked by a pentose to form a Lys-Arg
cross-link, namely pentosidine. Pentose could be formed from hexose
through sugar fragmentation or from triose, tetrose, and ketose by
condensation and/or reverse aldose reactions (32). Recently, Nagaraj
et al. (14) showed that a three-carbon metabolite,
methylglyoxal, which increases in diabetic lenses, reacts with lysines
of proteins forming a Lys-Lys cross-link, namely imidazolysine. Thus,
pentosidine and imidazolysine were analyzed in cross-linked
B-crystallin. The fact that pentosidine and imidazolysine are
present in the cross-linking of
B-crystallin reveals the existence
of Lys-Arg and Lys-Lys cross-linking. The high levels of imidazolysine
(5-9 nmol/23 nmol of lysine) may have resulted from the possible
lysine-rich protein contamination on crystallin preparation.
Experiments with ultrapure preparations of proteins may shed light on
this issue.
According to Prabhakaram and Ortwerth (33), [14C]lysine
was more readily incorporated into lysozyme than
[14C]leucine during glycation-mediated cross-linking
studies. This finding was interpreted as evidence for cross-linking
principally at the -amino group rather than at the
-amino group.
This hypothesis was confirmed by our protein-protein cross-linking
model. Okitani et al. (31) show that acetylation of free
amino groups of lysozyme before incubation with glucose prevents
cross-linking of the protein and loss of lysine and arginine. This
finding strengthens the importance of the amino group in cross-linking.
By using site-directed mutagenesis, we were able to differentiate
between the roles of
- and
-amino groups and show that the
-amino group is more important than the
-amino group in
cross-linking. The present study shows for the first time that an
engineered "no lysine" protein is useful in assessing the role of
- and
-amino groups in glycation-mediated cross-linking. The
advantage of site-directed mutagenesis over the chemical modification
is that the roles of individual amino groups of proteins in
cross-linking can be ascertained.
The reason the readily glycated -amino group has a weaker ability to
cross-link with itself or other non-lysine sites than lysine is not
clear. The N-terminal
-amino group is, in fact, on the surface of
B-crystallin (34) and is thus accessible to other cross-linking
sites. One possible explanation is that cross-linking at this site is
greatly decreased by steric hindrance. Interestingly, an earlier study
by Beswick and Harding (35) shows that changes in the conformation of
-crystallin take place after glycation with glucose 6-phosphate, and
this may prevent cross-linking at the
-amino group. Prabhakaram and
Ortwerth (33) observe that incorporation of [14C]leucine
into protein is ~20% that obtained with lysine. We also noted that
although
-amino groups have much less cross-linking ability than
-amino groups, cross-linking studies with
B-K2T/K163T suggest
that the
-amino group of Gly-1 could form some low level cross-links
with itself or with arginine and histidine residues (Fig. 2).
By incubating CA, used as an -amino group provider, with
B-K2T/K163T, we were able to show that there was significant
cross-linking between the
-amino groups of CA and the non-lysine
cross-linking sites of
B-K2T/K163T (Fig. 3). We noted that the
quantity of cross-linking products found in the incubation containing
CA and wild type
B (lane 3) is higher than that found in
CA and
B-K2T/K163T incubation (lane 2). This increased
quantity could come from the cross-linking of lysine to lysine and/or
lysines of wild type
B to non-lysine sites of CA. When CA alone is
incubated with glyceraldehyde, a clear dimer band can be observed on
SDS-PAGE, but when CA is incubated with wild type
B or
B-K2T/K163T, the CA-CA dimer bands almost disappear (Fig. 3). CA
seems to cross-link more readily with
B-crystallin than with itself,
and thus the major cross-linking product is CA-
B rather than
CA-CA.
Arginines and histidines are thought to be involved in
glycation-mediated cross-linking (9, 31, 36). Even though bovine B-crystallin contains 16 arginines and 5 histidines, when both lysines were mutated to threonine, cross-linking was greatly reduced, suggesting that they act only as cross-linking "acceptors" of the
glycated
-amino group.
We express our appreciation to Dr. Regine Hay
for the gift of bovine B-crystallin cDNA and to Monsanto Company
for the gift of pMON5743 expression vector.