(Received for publication, January 10, 1997, and in revised form, February 10, 1997)
From the Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
The aggregates formed by the interactions of the
human lens -crystallins have been particularly difficult to
characterize because the
-crystallins comprise several proteins of
similar structure and molecular weight and because their sequences were not known until recently. Previously, it could not be ascertained whether the species of various acidities were different proteins or
modifications of the same proteins. The recent determination of the
sequences permits calculation of molecular weights and unambiguous
identification of the various
-crystallins and their modified forms
by mass spectrometry. In this investigation, the components of the
three sizes of
-crystallin aggregates,
1
(~150,000),
2 (~92,000), and
3
(~46,000), were determined. The principal differences among the
different
-crystallin aggregates was the presence of
A4 in
1 and
2, but not
3, and
the length of the N-terminal extension of
B1. The size of the
-crystallin aggregate correlated with the length of the N-terminal
extension of
B1, indicating that the flexible N terminus of
B1 is
critical to the formation of higher molecular weight aggregates of
-crystallins. Separation of the components by ion exchange under
non-denaturing conditions showed that
B2 occurs as homo-dimers and
homo-tetramers as well as contributing to hetero-oligomers. Other
-crystallins were present only as hetero-oligomers.
The lens is a transparent avascular tissue with a high
concentration of proteins closely packed to give a refractive index that will focus light on the retina (1). The lens proteins, which have
monomeric molecular weights of approximately 20,000-30,000, form
aggregates with molecular weights up to 1 million. It is believed that
the clarity of the lens depends on the proper assembly of these
aggregates (2). In the mammalian lens there are three primary groups of
crystallins, -,
-, and
-crystallins, each composed of proteins
homologous within the group but differing from the proteins in other
groups. The
-crystallins have aggregate molecular weights of
180,000-1,000,000, the
-crystallins are aggregates of
40,000-150,000, and the
-crystallins do not aggregate. Within the
- and
-crystallin subgroups, the proteins have been named
according to their acidities. For example, the
-crystallins include
A-crystallin, the more acidic, and
B-crystallin, the more basic.
Within the
-crystallins of various mammalian species, seven
-crystallins,
A1,
A2,
A3,
A4,
B1,
B2, and
B3,
have been identified; the
-crystallins include
S,
A,
B,
C,
D, and
E. Not all the genes are expressed in each species.
In human lenses, expressed genes include those coding for
A (3),
B (4),
A1,
A3,
A4 (5),
B1 (6),
B2 (7),
B3 (5),
S (8),
C, and
D (9, 10). The
- and
-crystallins have considerable homology, both being composed of two globular domains forming "Greek key motifs" joined by a linker segment (11-14). All
of the
- and
-crystallins as well as
S-crystallin are
acetylated at the N terminus.
The -crystallins of the mammalian lens form aggregates that
fractionate by size exclusion chromatography into two or three subgroups of varying proportions depending on the chromatographic conditions (10, 15, 16). When they separate into two subgroups they are
referred to as
HIGH (
H) and
LOW (
L); when they separate into three
subgroups they are
H,
L1, and
L2 (17) or
1,
2, and
3 (15). The unique structural features of the
-crystallins that distinguish them from the
-crystallins, which
do not aggregate, include N- and C-terminal extensions from the
globular domains and a distinctive linker sequence. There is evidence
supporting the importance of both of these features in the formation of
aggregates. Investigations of mutant rat
-crystallins have
demonstrated that replacing the linker sequence of
B2-crystallin
with the
-crystallin linker prevents dimer formation, whereas
deletion of the N- and C-terminal extensions does not affect folding
of the domains or dimer formation (18, 19). On the other hand, in
studies of bovine
-crystallins, it has been noted that the largest
of the
-crystallins,
B1, which has a long N-terminal extension,
is a component only of the highest molecular weight aggregates
(
1), implying that the long N terminus is vital to
formation of large aggregates (16, 20, 21).
Six human -crystallin sequences are now known:
B1 (6),
B2 (7),
and
B3,
A1,
A3, and
A4 (5). Mass spectrometric determinations of the molecular weights of the crystallins confirmed the cDNA-determined sequences and identified three of the
crystallins,
B1,
A1, and
A3, in forms with truncated N
termini.
B1 missing 15, 34, 39, and 40 residues of the N terminus
were identified;
A3 was found in one truncated form, missing the
first 22 residues. Because
A3 and
A1 have the same sequence
except that the N terminus of
A1 begins with residue 19 of the
A3
sequence, truncated
A3-(23-215) is the same as
A1-(5-197) (5).
The previous studies of the
-crystallins did not describe which
-crystallins were present in the various size aggregates. The goal
of this study was to use molecular weights determined by mass
spectrometry to identify the
-crystallins and their truncated forms
in each of the subgroups and, from these data, derive an understanding
of the crystallin features contributing to aggregate formation.
Lenses were obtained from the National Disease Research Interchange (Philadelphia, PA). Trypsin was purchased from Worthington, BDH Aristar grade urea was from Gallard-Schlesinger Industries (Carle Place, NY), and all other chemicals were from Sigma. All chemicals were of reagent grade and used without further purification.
The lenses examined in this study were from donors 16, 27, 37, 56, and
70 years old. All lenses were clear; the donors had no diseases known
to affect lens opacity. Each lens, analyzed individually, was
homogenized in 2.5 ml of a buffer (0.05 M
NaHSO3, 0.05 M Tris, 0.02 M EDTA,
0.02% NaN3, pH 7.4). The water-soluble crystallins,
removed as the supernatant after centrifugation at 27,000 × g for 15 min, were fractionated into -,
1-,
2-,
3-, and
-crystallins by size exclusion chromatography (70 × 2 cm Sephacryl S-300HR column) using the homogenizing buffer as the mobile
phase at a flow rate of 15 ml/h. The column was calibrated using
thyroglobulin (Mr 669,000), alcohol
dehydrogenase (Mr 150,000), bovine serum albumin
(Mr 66,000), and ovalbumin
(Mr 45,000). The absorbance of the eluate was
monitored at 280 nm. Because there was some overlap between the peaks
for
3 and
-crystallins, the
3-crystallins were separated from the
-crystallins by
further size exclusion chromatography (Sephadex G-75) using the above buffer. The size exclusion buffer was replaced with the ion exchange buffer by passing each of the
-crystallin fractions through Sephadex G-25 (1 × 5 cm column) at a flow rate of 1.5 ml/min.
After
isolation by size exclusion chromatography, each of the -crystallin
fractions was further fractionated by anion exchange chromatography
using a Mono-Q HR column (Pharmacia Biotech Inc.) connected to an
analytical HPLC system (Rainin Instrument Co. Inc., Woburn, MA)
operating at flow rate of 1.0 ml/min. Elution of the crystallins was
monitored by the absorbance at 280 nm. Buffer A was 0.01 M
Tris at pH 7.63; buffer B was the same as buffer A plus 1.0 M NaCl. The sample was eluted following a gradient of 0% B
for 3 min, 0-11% B for 17 min, 11-50% B for 14 min, and 100% B in
1 min. The Mono-Q column was washed between runs with 2 ml of 2.0 M NaCl dissolved in 1:1 water and methanol.
The -crystallin fractions from anion
exchange chromatography were further fractionated by reversed phase
HPLC (4.6 × 150 mm Vydac C4 column, 300 Å) using a
gradient of acetonitrile and water, both solvents with 0.1%
trifluoroacetic acid. The proteins were eluted using a gradient of
10-30% acetonitrile for 5 min, 30-50% for 30 min, and 50-98% for
2 min. The absorbance was monitored at 280 nm; the fractions were
collected manually, lyophilized to dryness, and stored at
80 °C
before analysis.
The molecular weights of the isolated proteins were determined using a quadrupole electrospray ionization mass spectrometer (Micromass Platform II, Manchester, United Kingdom) calibrated with horse heart myoglobin (Mr 16,951). The sample was introduced in a solution of 1:1 acetonitrile:water with 2% formic acid at a flow rate of 5 µl/min. Molecular weight determinations of proteins were ±0.01%, yielding an uncertainty of ± 2 Da for a protein with a molecular mass of 20 kDa.
The data presented were obtained with the 56-year-old lens. Not
all molecular weight determinations were performed on all lenses, but
the chromatograms for the size exclusion, ion exchange, and reversed
phase fractionation for the different lenses were similar, suggesting
that there were only minor age-related differences in the water-soluble
-crystallins from ages 16 to 70. For the size exclusion
chromatographic conditions used in this study,
-crystallins gave
three peaks with aggregates of approximate molecular weights of 150,000 (
1), 92,000 (
2), and 46,000 (
3) (Fig. 1). By employing several
chromatographic techniques, size exclusion, non-denaturing anion
exchange HPLC, and reversed phase HPLC, along with mass spectrometric
determination of molecular weights of the fractionated proteins, it was
possible to unambiguously determine the components of the
-crystallin aggregates (Table I). Each
-crystallin
was identified by matching its molecular weight as determined by mass
spectrometry with a molecular weight calculated from the known
sequences of the
-crystallins. For all identifications, the
agreement between the experimentally determined and calculated
molecular weights was within 2 atomic mass units.
|
Anion exchange chromatography was performed without urea in the buffer
to observe which components were associated in the -crystallin
aggregates. Under these non-denaturing conditions, three peaks labeled
A, B, and C (Fig. 2)
were evident. The intensities of A, B, and C varied considerably among
the different groups of aggregates. For example, peak A from
1 was small (Fig. 2a), but it was
approximately one-third of
2 (Fig. 2b) and
3 (Fig. 2c). Peak B was a relatively minor
component of
1 but a major component of
2
and
3. Peak C was a major component of all three size
exclusion fractions. Although the chromatograms were generally similar
for all lenses, the relative intensities of peaks B and C derived from
2 and
3 varied somewhat, from about 3:2
to 2:3, among the lenses.
The reversed phase chromatogram of proteins eluting in peak A had only
one peak, with an elution time corresponding to B2 (Fig.
3a) (7). ESIMS analysis of this peak showed
the presence of one protein with a molecular weight
(Mr 23,291) (Fig. 4) that confirmed its identity as
B2 (Mr 23,291) (7).
The peak labeled A
(Fig. 2c) also had a
molecular weight matching
B2. Since deamidation of a protein causes
it to elute later on anion exchange but changes the molecular weight by
only 1 mass unit and therefore cannot be distinguished by ESIMS
determination of its molecular weight, it seemed likely that A
is a
deamidated form of
B2.
Reversed phase analysis of ion exchange peaks B and C gave similar
chromatograms, each with two major peaks (Fig. 3, b and c). One peak had a retention time of B2 (peak
1 of Fig. 3), and the other had a retention time corresponding to
B1 (peak 3 of Fig. 3) (6) and
A4 (5), which co-elute
with these reversed phase conditions. Identification of
B2,
B1,
and
A4 was confirmed by ESIMS analysis (Table I). Finding the same
masses for the proteins in B and C suggested that B and C contain the
same proteins but perhaps with the proteins in C modified by
deamidation. The
B2-crystallins in peaks B and C, whether from
1,
2, or
3, were all
intact (Mr 23,291) (Fig. 4).
A4-crystallin,
which also was present only as the intact protein
(Mr 22,282), was found in size exclusion
fractions
1 and
2; a mass corresponding
to
A4 was not detected in
3. In contrast,
B1-crystallin was identified by ESIMS in several forms (Fig.
5) with molecular weights corresponding to
B1 and its
truncated products at the N terminus (6). The relative amounts of
B2:
B1/
A4, based on their intensities in the reversed phase
chromatograms, were approximately 3:1 in peak B and 1:1.5 in peak C. A
minor component, labeled 2 in Fig. 3, present in both ion exchange
peaks B and C from
1 and
2, was identified by its reversed phase elution time as
A3/
A1 (5).
The electrospray mass spectra for the proteins with reversed phase
elution times appropriate for B1 were remarkably different for
1,
2, and
3 (Fig. 5,
a, b, and c, respectively). The
B1-crystallins showed progressive N-terminal truncation from
1 to
2 to
3. For all the
lenses, intact
B1 (Mr 27,933) was a major
component only of
1, which also contained
B1 missing
the first 15 residues (Mr 26,535) and sometimes
minor amounts with as many as the first 39 residues (Fig.
5a). In contrast, all the
B1 in
2 was
truncated at the N terminus with 15-41 residues missing (Fig.
5b), and most of
B1 in
3 was even further
truncated at the N terminus, with at least 34-41 residues missing
(Fig. 5c).
Since non-denaturing conditions were used in the anion exchange
chromatography, the proteins isolated in this separation could be
expected to retain their native associations. Finding only B2 in
peak A of the ion exchange chromatograms of
2 and
3 indicated that some dimers of
3 and
some tetramers of
2 are
B2 homo-oligomers. The small
peak A of
1 indicated that
B2 homo-oligomers are a very minor component of
1. Neither
B1 nor
A4 was
found alone in the ion exchange fractions, implying that these
-crystallins do not form homo-oligomers.
In previous investigations the components of the various
aggregates of human -crystallins were identified primarily by
SDS-polyacrylamide gel electrophoresis (15, 16, 22). Because the
sequences of the human
-crystallins were not known and because there
appeared to be several proteins with similar molecular weights, the
exact composition of each of the aggregates could not be determined. Data from fetal lenses showed the presence of a 29-kDa protein, later
identified as
B1 in both
1 and
2 (16),
whereas data from lenses more than 5 years old indicated the presence
of
B1 only in
1 (15, 16). From the SDS-polyacrylamide
gel electrophoresis data presented in those studies, it appeared that
B1 was not a component of the lowest molecular weight aggregates. A
protein of 24-26 kDa, first called
Bp then renamed
B2, was a
major component of
1,
2, and
3 from lenses of all ages (15, 16, 22). The other
-crystallins were not identified.
Results from our mass spectrometric investigation of -crystallins,
separated by ion exchange and reversed phase HPLC, have led to
unambiguous identification of the components of each of the subgroups
of
-crystallins (Table I). The data show that
B1 in a variety of
forms truncated at the N terminus is present in the aggregates of
1,
2, and
3 and that the
size of the aggregates correlates with the length of the N terminus of
B1. The largest aggregates,
1, are composed primarily
of
B1 (both intact and with the first 15 residues missing), intact
B2, and intact
A4. The principal forms of
B1 present in
1,
B1-(1-251) and
B1-(16-251), correspond to
bovine proteins previously identified as
B1a and
B1b (23). These
two forms of
B1 are the major
B1-crystallins found in newborn
human lenses (6). The ion exchange chromatography performed under
non-denaturing conditions indicated that the oligomers of
1 are hetero-oligomers of
B1,
A4, and
B2.
Because
B1 and
A4 co-elute on reversed phase HPLC, the relative
amounts of each component were not ascertained. A very minor component
in
1 had the correct retention time for
A3. Intact
A3 has previously been detected only in fetal lenses; in adult
lenses it was found missing the first 22 residues of the N terminus. An
insufficient amount of this minor component was isolated from the
56-year-old lens for molecular weight determination, but it was
presumed to be
A3 truncated at the N terminus based on its reversed
phase HPLC elution time. A molecular weight corresponding to
A3-(23-215), which is the same as
A1-(5-197), was determined
for this protein isolated from other adult lenses.
The tetrameric aggregates of the -crystallins,
2,
included the same proteins as
1 except that
B1 was
found with further N-terminal truncation. Although no intact
B1 was
present in
2, the following forms, truncated at the N
terminus, were found:
B1-(16-251),
B1-(35-251),
B1-(40-251), and
B1-(41-251). As in
1,
B2 and
A4 were found only as intact proteins. The non-denaturing ion
exchange of the aggregates in
2 indicated that the
tetramers were homo-oligomers of
B2 and hetero-oligomers of
B1,
B2, and
A4.
The dimers of -crystallin,
3, included homo-oligomers
of
B2 and hetero-oligomers of
B1 and
B2. The
B1 in
3 was degraded at the N terminus even further than in
2, with most of
B1 missing 34 or more residues from
its N terminus. Finding
B1, although only in forms truncated at the
N terminus, in
3 is in opposition to previous reports
that
B1 exists only in larger aggregates (15, 17, 20, 21). These
differing observations are easily explained by the fact that the
B1
products without the N-terminal 34-41 residues have molecular weights
of 24,192-24,834, similar to the molecular weight of
B2 (23,291)
and may not have been recognized as
B1 by SDS-polyacrylamide gel
electrophoresis analysis. Identification would have been further
complicated by the fact that
B1 minus these residues has a pI (6.38)
similar to the pI of
B2 (6.33).
B3, which has previously been
isolated only from fetal or newborn lenses (5), was not detected in any
of the subgroups of the
-crystallins from these adult lenses.
For bovine lens crystallins, it has been demonstrated that the various
aggregates of -crystallins are in a dynamic equilibrium with the
size of aggregates affected by concentration, temperature, and ionic
strength (24). Hydrophilic interactions appeared to be the main factor
affecting the association-dissociation equilibrium (24). Even though
these data may not be directly applicable to human lenses because the
composition of bovine
-crystallins differs considerably from human
-crystallins, it is interesting to consider the effect
hydrophilicity might have on the stability of the various human
-crystallin aggregates. The hydrophilicities of the
-crystallin
or portions of them can be calculated from the Bull and Breese indices
(25). The long N-terminal extension of
B1, which appears to play a
unique role in the formation of large aggregates as demonstrated by the
presence of intact
B1 only in
1-crystallins, has a
Bull and Breese index (BB) of +365. The hydrophilicity indices for
B1 in the middle size aggregates of
2 and the dimers
of
3, where 15-41 residues are missing from the N
terminus, range from BB
20 to
57. (The more positive numbers indicate a more hydrophilic sequence). Further evidence of the likelihood that hydrophilicity is important in aggregate formation is
demonstrated by the fact that
B2 forms both homo-dimers and tetramers, but
B1 and
A4 do not form homo-oligomers. Overall,
B2 is a more hydrophilic protein (BB +62) than either
A4 (BB +30)
or
B1(BB +5).
Comparison of the sequence of the first 34 residues of human B1 with
the N terminus of bovine
B1 shows that this region is not as rich in
alanine and proline as bovine
B1 (23), but it does include 8 alanines, 5 prolines, and 4 glycines and no bulky amino acids, giving
this region considerable flexibility. These characteristics, along with
its hydrophilicity, may allow the N terminus to be flexible for
easy interaction with charged portions of the other
-crystallins,
stabilizing high molecular weight aggregates.
Previous investigations using mutant forms of B2 have demonstrated
that the linker portion (residues 80-88, PIKVDSQEH) that joins the two
domains of
B2 is critical to the formation of dimers (19), whereas
the N- and C-terminal extensions of
B2 are not essential (18). If
this linker region were the sole determinant of dimer formation, it
might be expected that
B1, which contains a region highly homologous
with the
B2 linker (residues 140-148, PIKMDAQEH), would also form
homo-oligomers. Failure to detect homo-oligomers of
B1 in any of the
fractions suggests that the sequence of the linker portion is not the
only factor that determines the stability of dimers.
The fact that A4 is in
1 and
2, but
not in
3, suggests that it too may contribute to the
formation of higher aggregates. If so, its interactions are different
from those proposed for other
-crystallins. The sequence features
previously proposed to be favorable to aggregation, the long N terminus
and the
B2 linker sequence, are missing in
A4.
A4 has a short
N terminus and the sequence of the linker region, PAACANHRD, is very
different from the sequence of
B2 that favors dimer formation.
In contrast to B1, which was found in many truncated forms at the N
terminus,
B2 and
A4 were present only as intact proteins. None of
the fractions of
-crystallins contained proteins with molecular
weights consistent with truncated products of either
B2 or
A4.
These results do not support the presence of truncated
B2 at the N
terminus in older lenses, a modification that has been proposed to
explain a lack of reactivity of older human lenses with an antisera to
residues 1-12 of
B2 (26).
Despite the similarity between the sequences of human and bovine
-crystallins, there are striking differences between the composition
of the aggregates of the human and bovine
-crystallins (27). In both
species, intact
B1 is found only in
1 (17). Slingsby
and Bateman (27) also reported finding no
B1 in
2 and
3 of bovine lenses and identified the lower molecular
weight proteins in
2 and
3 as
A3 and
B3. Because their techniques may not have recognized truncated
B1, it is possible that some truncated
B1-crystallins were
present in
2 and
3 but incorrectly identified. One similarity between bovine and human lenses is that
B2 is present as homo-dimers as well as being a major component of
all three sizes of aggregates in the lenses of both species. In their
recombination studies, Slingsby and Bateman (27) showed that bovine
B2 could form homo-dimers as well as hetero-dimers with other acidic
and basic
-crystallins. Our results for human lens
-crystallins showed that, in addition to forming homo-dimers and hetero-dimers,
B2 also formed homo- and hetero-tetramers.
In bovine lenses, both B3 and
A3 are major components, whereas in
humans
B3 is produced only in fetal and newborn lenses, and
A3 is
a very minor component. Furthermore, the
A3 that is present in adult
lenses is truncated at the N terminus (5). Slingsby and Bateman (27)
concluded that, for bovine
-crystallins, oligomers larger than
dimers required the presence of an acidic
-crystallin with a long N
terminus, such as
A3. This conclusion was supported by studies of
mutant rat
A3, showing that
A3 without the first 29 residues of
the N terminus formed smaller aggregates than
A3 with only 6 residues missing (28). Such a role for
A3 in human lenses is
improbable because very little
A3 is present in adult lenses and
that which is present lacks the first 22 residues of the N terminus. It
is much more likely that the long N terminus of
B1 is the major
determinant of the size of the
-crystallin aggregates in human
lenses.