(Received for publication, October 9, 1996, and in revised form, November 2, 1996)
From the Departments of Oral Molecular Biology and
Ophthalmology, Oregon Health Sciences University, Portland, Oregon
97201 and the § Department of Chemistry, University of
Nebraska, Lincoln, Nebraska 68588
A combination of Edman sequence analysis and mass
spectrometry identified the major proteins of the young human lens as
A,
B,
A1,
A3,
A4,
B1,
B2,
B3,
S,
C, and
D-crystallins and mapped their positions on two-dimensional
electrophoretic gels. The primary structures of human
A1,
A3,
A4, and
B3-crystallin subunits were predicted by determining
cDNA sequences. Mass spectrometric analyses of each intact protein
as well as the peptides from trypsin-digested proteins confirmed the
predicted amino acid sequences and detected a partially degraded form
of
A3/A1 missing either 22 or 4 amino acid residues from its
N-terminal extension. These studies were a prerequisite for future
studies to determine how human lens proteins are altered during aging
and cataract formation.
Elucidating the structure and function of crystallins, the major proteins of human lens, is important, because alterations in these proteins may contribute to cataract formation. The goal of our laboratories is to determine what changes occur in human crystallins during aging and cataract formation. To accomplish this goal it is first necessary to perform the following in young, normal human lenses: 1) determine which crystallin subunits are present in the young lens; 2) map the positions where these crystallins migrate on two-dimensional electrophoretic gels; and 3) deduce and confirm the amino acid sequences of these proteins.
The sequences of human A,
B,
B1,
B2,
S,
C, and
D-crystallins have already been determined and their presence in the human lens has been demonstrated (1-11). Furthermore, the positions where human
A,
B,
B1,
B2, and
S-crystallins migrate on
two-dimensional gels have been determined (12-14). In the present
study, we completed the identification of all major crystallins
resolved by two-dimensional electrophoresis, and add
A1,
A3,
B3, and
A4 to the list of major
-crystallins in young, normal
human lens. We also correct the sequence of human
A3, complete the
sequence determination of human
B3, and for the first time, report
the sequence of human
A4. These deduced amino acid sequences were
confirmed by mass spectrometry.
The posterior
poles of human eyes from organ donors 7-months of age or less were
obtained from the Lions Eyebank of Oregon within 48 h post-mortem.
Following decapsulation, the lenses were homogenized in 1.0 ml of 20 mM phosphate buffer (pH 7.0), and 0.1 mM EGTA.
Water-soluble proteins were isolated by centrifugation at 10,000 × g for 30 min. Protein content was assayed by the BCATM assay (Pierce) according to the manufacturer's protocol using bovine
serum albumin as a standard. Water-soluble fractions were then dried by
vacuum centrifugation and stored at 70 °C prior to electrophoretic
or chromatographic separation.
Two-dimensional electrophoresis of water-soluble lens proteins, transfer to polyvinylidene difluoride membranes, and direct sequence analysis were carried out as described previously (15). Non-equilibrium pH gradient electrophoresis using pH 3.5-10 ampholine was used in the first dimension and SDS-polyacrylamide electrophoresis in the second dimension. However, most crystallins were blocked on the N terminus and could not be identified by direct sequence analysis. Therefore, electroblots of two-dimensional gels were reversibly stained with Ponceau S, individual proteins digested with trypsin, and peptides isolated from the resulting mixtures for sequencing as described previously (16), except that peptides were separated using a 250 × 2.1-mm C18 column (Vydac, Hesperia, CA), and linear 100-min 0-35% acetonitrile gradient containing 0.1% trifluoroacetic acid. The tryptic peptides derived from regions of electroblots thought to contain more than one crystallin species were additionally analyzed by mass spectrometry using either FAB-MS1 or ESIMS as described below.
The identified crystallins were quantified after staining two-dimensional gels with Coomassie Brilliant Blue R-250, or colloidal Coomassie Brilliant Blue G-250 (Instaview Universal Stain, Gallard-Schlesinger Industries, Inc., Carle Place, NY). Each of the stained gels contained 100 µg of total protein. The density of each species was determined from images obtained with a Gel Doc 1000 camera and analysis using Molecular Analyst Software (Bio-Rad).
Cloning and Sequencing of HumanLenses from organ donors 19-months of age or less were
obtained as described above. Total lens RNA was isolated by
homogenation of dissected lenses in TRIzol reagent according to the
manufacturer's protocol (Life Technologies, Inc.). To amplify the 3
end of the human lens
A4 cDNA, reverse transcription was
performed on total RNA from human lens using an oligo(dT) containing
adapter primer as described previously (3). The resulting cDNA was
then subjected to 3
-RACE PCR according to the manufacturer's protocol
(3
-RACE System for Rapid Amplification of cDNA Ends, Life
Technologies Inc.), using a 3
-RACE universal adapter primer, and the
gene-specific sense primer AGGCTGACCATCTTCGAGCA matching residues
390-410 of bovine
A4 cDNA (GenBank accession number M60328[GenBank]).
Cycling conditions for PCR were as described previously (3), except an
annealing temperature of 61 °C was used. The amplification resulted
in a single PCR product of approximately 550 base pairs.
To amplify the 5 end of the human lens
A4 cDNA, reverse
transcription was performed on total RNA from human lens using the gene-specific antisense primer GTCATCGCTCAGCTCTCCTT complementary to
residues 392-411 of the final human
A4 cDNA sequence. The cDNA was then homopolymer tailed according to the manufacturer's protocol (5
-RACE System for Rapid Amplification of cDNA Ends, Life
Technologies Inc.). The resulting cDNA was then PCR amplified using
a second gene-specific antisense primer, TCTTGCCCAGGAAGTTCTCT, complementary to residues 371-391 of the final human
A4 cDNA and a 5
-RACE anchor primer supplied by the manufacturer (Life Technologies, Inc.). Cycling conditions for PCR were as described previously (3), except an annealing temperature of 55 °C was used.
The amplification resulted in a single PCR product of approximately 450 base pairs.
To amplify the unknown 5 end of human
B3 cDNA, the same
procedure described above for 5
-RACE of human
A4 cDNA was
followed. The gene-specific antisense primer, CAGACCACAAGCTGCATCTGT,
complementary to nucleotides 8-28 in exon 5 of human
B3 (GenBank
accession number X15145[GenBank]), was used for reverse transcription of total
lens RNA. A second gene-specific antisense primer, CCTCCGGCCTCTGAATATT, complementary to nucleotides 115-133 in exon 4 of human
B3 (GenBank accession number X15144[GenBank]), was used to perform 5
-RACE of human
B3
cDNA. Cycling conditions were as described previously (3), except
an annealing temperature of 60 °C was used. A PCR product of
approximately 400 base pairs was obtained.
Preliminary mass spectrometric analysis of human A3 protein
suggested that a portion of the reported genomic sequence of human
A3 contained several errors in nucleotide identification in exons
4-6 (GenBank accession numbers M14304[GenBank], M14305[GenBank], and M14306[GenBank]). Therefore,
a corresponding region of human
A3 cDNA was amplified and
sequenced for comparison with the earlier genomic sequence. Total
human lens RNA was reversed transcribed using the gene-specific
antisense primer, GCAAGGTCTCATGCTTGAGG, complementary to residues
185-204 of exon 6 of human
A3 (GenBank accession number M14306[GenBank]).
After treating with RNase H, the cDNA was PCR amplified using the
gene-specific sense primer, TGATCAGGAGAACTTTCAGG, matching residues
366-385 of exon 3 of human
A3 (GenBank accession number M14303[GenBank]) and
the antisense primer used above in the reverse transcription reaction.
Cycling conditions for PCR were as described previously (3), except an
annealing temperature of 55 °C was used. A PCR product near the
expected size of 589 base pairs was produced.
PCR products were cloned with either the Prime PCR ClonerTM Cloning
System (5 Prime 3 Prime, Inc., Boulder, CO) or with the Original TA
Cloning® Kit (Invitrogen, San Diego, Ca). Plasmid DNA was isolated
using either a FlexiPrepTM Kit (Pharmacia-Biotech, Inc., Piscataway,
NJ), or Quantum PrepTM kit (Bio-Rad). Following screening to confirm
the presence of the correct sized insert, sequencing was performed
using either the AutoRead sequencing kit and automated laser
fluorescence DNA analysis system (Pharmacia Biotech), or a Cycle
Sequencing Kit and Model 373A DNA Sequencer (Applied Biosystems, Inc.,
Foster City, CA). Depending on the plasmid utilized, sequencing was
performed using either M13/pUC forward and reverse primers or T7 and
Sp6 primers. Three clones of each of the 3
- and 5
-RACE products of
the
A4 cDNA, and 2 clones of the PCR product of the
A3
cDNA were sequenced in both sense and antisense directions. The
sequence from 4 antisense strands of the 5
-RACE product of
B3
cDNA were sequenced. DNA sequences were edited and theoretical pI
values of proteins calculated using Geneworks 2.5 software
(IntelliGenetics, Mountain View, CA).
The deduced amino acid
sequences of A3/A1,
B3, and
A4 crystallins were confirmed by:
1) determining the molecular mass of intact proteins; 2) determining
the molecular masses of tryptic peptides; and 3) confirming the
sequences of tryptic peptides. The types of mass spectrometry employed
were ESIMS, FAB-MS, and tandem mass spectrometry (MS/MS).
Soluble lens protein was isolated from 32-week gestation, 3-7-day-old,
and 42-year-old human donors as described above. Proteins were
dissolved in gel filtration buffer containing 50 mM
phosphate (pH 7.0), 150 mM NaCl, and 5-mg portions injected
onto a Superose 6 HR 10/30 gel filtration column (Pharmacia Biotech) at
a 0.2 ml/min flow rate. Protein elution was monitored at 280 nm and -crystallin aggregates of approximately 160,000 and 50,000 molecular weight (
H- and
L-crystallin fractions,
respectively) were collected. These aggregates were then concentrated
and desalted using CentriconTM 10 microconcentrators (Amicon, Inc.,
Beverly, MA) in preparation for mass analysis.
-Crystallin subunits were partially purified from
H
and
L aggregates using a Vydac 4.6 × 150-mm
C4 reversed phase column and 25-60% acetonitrile gradient
containing 0.1% trifluoroacetic acid over 35 min and 1 ml/min flow
rate. Reversed phase HPLC purified
-crystallins were then injected
into the mass spectrometer with a 50:50 solution of acetonitrile and
water at a flow rate of 5 µl/min. The masses of the isolated
-crystallins were determined using a Micromass Platform II
electrospray ionization mass spectrometer with a quadrupole analyzer
and Mass Lynx software (Micromass, Manchester, UK). For protein
analysis, the instrument was calibrated with horse skeletal muscle
myoglobin over the range of 700-1600 Da. Accuracy of protein molecular
mass determinations was ±3 Da.
As an alternative to reversed phase HPLC, two-dimensional
electrophoresis was used as a preparative tool to isolate the truncated A3/A1 species. Total soluble proteins from a 4-day-old human donor
were separated by two-dimensional electrophoresis, proteins visualized
by precipitation of SDS-protein complexes with ice-cold 0.25 M KCl (17), and the trucated
A3 species excised and
electroeluted from 12 gels. Proteins were electroeluted into a
CentriconTM 10 microconcentrator using the method recommended by the
manufacturer (Amicon, Inc.). Electroelution into this device
facilitated the concentration and desalting of the sample for analysis
by ESIMS as described above.
The deduced amino acid sequences of A3/A1,
B3, and
A4 were
confirmed from the masses of peptides in tryptic digests of the
proteins (50:1 substrate:trypsin, pH 8.2, 4 h). Lenses of 32-week-gestation, 4-day-old, and 42-year-old donors were used to
isolate
B3,
A3, and
A4 for tryptic digestion, respectively. Tryptic peptides of
A4 and
B3 were prepared from proteins
isolated by gel filtration and reversed-phase HPLC. Peptides of
A3
were prepared from protein separated by two-dimensional
electrophoresis, transfer to polyvinylidene difluoride membrane, and
digestion from the membrane surface with trypsin. The peptides were
fractionated by C18 reversed-phase HPLC with a gradient of
2-50% acetonitrile in water, containing 0.1% trifluoroacetic acid.
For peptide analysis, the mass spectrometers used included a Kratos
MS-50 fast atom bombardment mass spectrometer (Kratos Analytical,
Manchester, UK), a Micromass Platform II electrospray ionization mass
spectrometer and a Micromass Autospec mass spectrometer with an
orthogonal TOF analyzer for the MS/MS analyses. Some analyses employed
a microbore column on-line to the electrospray ionization mass
spectrometer (with a flow rate of 50 µl/min), with 5 µl/min
entering the mass spectrometer and 45 µl/min monitored by a UV
detector and collected for further analysis. The instruments were
calibrated over the range of 200-3000 with NaI; the accuracy of the
determinations was ±0.3 Da.
Sequences of tryptic peptides were also confirmed by MS/MS. This technique consists of one mass spectrometer to isolate the peptide of interest, a chamber where the peptide is fragmented by collision with xenon, and a second mass analyzer which determines the masses of the resulting fragments (18). The tandem mass spectrometer used in this investigation (Micromass Autospec oa-TOF) consisted of a conventional magnetic sector instrument as the first mass spectrometer and an orthogonal acceleration time-of-flight analyzer as the second mass spectrometer (19). These analyses were performed in the fast atom bombardment mode of ionization.
Two-dimensional electrophoresis
of the constituent proteins in the water-soluble fraction from lenses
of young human donors revealed 11 major proteins (Fig.
1). The identification of B1 and
B1 missing 15 residues from its N terminus (
B1, residues 16-251) was previously
reported (3). Due to blockage of the N termini, the identification of
B2,
A3,
A4,
S,
A, and
B subunits was performed by
trypsinization of electroblotted proteins, separation of tryptic
fragments, and 6-15 cycles of Edman sequencing of isolated tryptic
fragments (Table I). These results confirm the
previously reported positions of human
B1,
B2,
S,
A, and
B on two-dimensional gels (12-14) and demonstrate the presence of
A3 and
A4 in human lens and their positions on two-dimensional gels.
|
Due to their lack of acetylated N termini, the positions of C and
D, as well as a truncated
-crystallin, could be determined by
direct sequence analysis of electroblotted proteins (Fig. 1, Table I).
The truncated subunit contained a sequence matching that of
A3/A1
and could have arisen from truncation of the first 22 amino acids of
A3 or the first 4 amino acids of
A1.
A1 and
A3 are
identical, except
A3 contains a longer N-terminal extension due to
the use of an alternate start codon in the single
A3/A1 transcript
(20). For simplicity, the truncated
-crystallin is referred to as
A3 (23-215). The presence of
A3 (23-215) in human lens was
recently reported in another laboratory (21).
Although mRNAs coding for A,
B,
C, and
D have been
reported in fetal and neonatal human lenses, transcripts for
C and
D were more abundant (7). In the present study,
C and
D crystallins were detected, but no
A or
B crystallins were
observed (Fig. 1, Table I). Earlier mass spectrometric and
chromatographic analysis also detected
C and
D in human lenses,
and found either small or undetectable amounts of
A and
B (10,
11). This suggested that little translation of either
A or
B
mRNA may occur in human lens. The lower migration position of
D
was not expected (Fig. 1), since its calculated molecular weight was
nearly identical to
C. This is likely explained by anomalous
migration of
-crystallins during SDS-PAGE and not by
post-translational modification.
Many of the crystallins described above were identified from the
sequence of one peptide in each tryptic digest. This did not exclude
the possibility that some spots observed on two-dimensional gels
actually contained more than one protein species. Therefore, mass
spectrometry was used to determine the masses of all peptides in
tryptic digests from each of the two regions previously thought to
contain only S or
A3. The region containing
S (as determined by Edman sequencing) yielded peptides with masses matching peptides expected from both
S and
A1. Representative mass spectra showing two peptides with masses matching residues 84-94 and 148-153 of
S
(Fig. 2A), and a peptide with a mass matching
the acetylated N terminus of
A1 (residues 1-14) (Fig.
2B) are shown. The presence of the acetylated N-terminal
tryptic peptide (Fig. 2B) indicated that the species
comigrating with
S was actually
A1, and not a
post-translationally derived form of
A3.
A protein corresponding to B3 was not identified during limited
Edman sequencing of the major protein species of young human lens
(Table I). However, the ESIMS determined masses of the tryptic peptides
derived from the region containing
A3 included masses corresponding
to peptides from both
A3 and
B3. For example, masses matching the
acetylated N-terminal peptide (residues 1-17) and peptide 96-109 of
A3 (Fig. 3A), as well as peptide 110-122 of
A3 and peptide 153-167 of
B3 (Fig. 3B) were
found.
Densitometric Analysis of Two-dimensional Gels
Once the major
crystallin subunits in young human lenses were identified, the relative
amount of each subunit was determined by densitometric analysis of
two-dimensional electrophoretic gels of the total soluble lens protein
from newborn, 3-, 4-, and 7-day old human donors (Table
II). The relative amounts of A1 and
S, as well as
A3 and
B3, could not be determined from two-dimensional gels of
whole soluble protein, because these proteins did not resolve from one
another. However, two-dimensional electrophoresis of isolated
H- and
L-crystallin aggregates from 3- and 7-day-old human lenses removed
S and allowed estimation of the
relative amount of
A1.
A1 and
A4 were found in nearly equal
quantities in the young human lens (gels not shown).
|
A proposed
full-length human A4-crystallin cDNA was obtained by 3
- and
5
-RACE PCR because there was no previously published sequence for
human
A4. Cloning and sequencing of these PCR products resulted in
the 809-base pair sequence shown in Fig. 4A
(GenBank accession number U59057[GenBank]). An ATG start codon was found at
nucleotide 37 and a stop codon at nucleotide 625. A putative polyadenylation signal was also found at nucleotides 788-793. Human
A4 cDNA encoded a protein of 195 amino acids, excluding the
N-terminal methionine. This gave a calculated molecular mass of 22,285 including the acetylation of the N terminus (see mass spectrometric
analysis below). The predicted pI was 5.63. Excluding the N-terminal
extensions, the amino acid sequence of human
A4 shared 92-94%
sequence identity with rat2 and bovine (22)
A4, and 65% identity with chicken
A4 (23). The N-terminal
extensions of human, rat, and chicken
A4 were all 10 residues in
length, supporting the previous suggestion that the N-terminal
extension of bovine
A4 is also 10 residues in length (23).
Since only exons 4-6 of human B3 were previously reported (24), the
5
end of human
B3 cDNA was amplified by 5
-RACE PCR and
sequenced (Fig. 4B). The resulting sequence was 379 nucleotides in length, contained a 71-nucleotide untranslated region at
its 5
end, and coded for amino acids 1-102 of human
B3 (GenBank accession number U71216[GenBank]). This sequence overlapped with the first 114 base pairs of the previously reported sequence for human
B3 exon 4 (Genbank accession number X15144[GenBank]). Combining this sequence with the
previously reported sequences of human
B3 exons 4-6 (GenBank
accession numbers X15144[GenBank], X15145[GenBank], and X15146[GenBank]) resulted in a deduced
sequence for a 211-amino acid protein with a calculated mass of 24,224, including the acetylated N-terminal methionine. The calculated pI was
5.77.
Because the mass of A3 determined by ESIMS (25,192) did not agree
with the mass calculated from the published sequence (20), a portion of
the human
A3 cDNA containing the 3
coding region was amplified
by PCR and sequenced. The 549-base pair sequence with a stop codon at
nucleotide 517 is shown in Fig. 4C (GenBank accession number
U59058[GenBank]). This sequence differs from the earlier sequence at two
positions within exon four (Genbank accession number M14304[GenBank]), and 4 positions within exon six (Genbank accession number M14306[GenBank]). Each of
these nucleotide differences resulted in a change in the deduced amino acid sequence. The calculated masses for the new sequences of
A3 and
A1 were 25,193 and 23,102, respectively, including the acetylation
of their N termini. The pI values of
A3 and
A1 were 5.58 and
6.17, respectively.
The molecular weights of the subunits in lens
H- and
L-crystallin aggregates from a
3-day-old donor were determined by ESIMS (Table III).
Two of the components had masses corresponding to
B1 and
B2. Two
other components had masses corresponding to previously identified
partially degraded
B1-crystallins (3). A spot corresponding to
B1
(40-251) was not identified during Edman sequencing of proteins
separated by two-dimensional electrophoresis (Fig. 1). This protein, as
well as other partially degraded forms of
B1 (3), may not have been
detected by electrophoresis because they may co-migrate with other
crystallins.
|
Experimentally determined masses of other -crystallins agreed with
the calculated masses of
A1,
A3,
A3 (23-215),
A4, and
B3
(Table III), confirming the deduced amino acid sequences of
A4,
B3, and
A3/A1 (Fig. 4). A spot corresponding to
A3 (23-215)
was consistently observed by two-dimensional electrophoresis of soluble
protein from newborn lenses (Table II). However, analysis of
-crystallin aggregates by reversed-phase HPLC and ESIMS barely detected a component with the mass of
A3 (23-215) in lenses less than 3 years old. The presence of
A3 (23-215) was confirmed in newborn lenses after soluble protein from a 4-day-old lens was separated by two-dimensional electrophoresis and the region containing
A3 (23-215) eluted and analyzed by ESIMS. A molecular mass of 23,001 was expected, because acrylamide reacts with cysteinyl residues, adding
71 Da to each of 5 cysteines (25). The ESIMS determined mass of eluted
A3 (23-215) was 22,999 (Fig. 5), supporting the Edman
sequencing results (Table I) and confirming the presence of
A3
(23-215).
Further confirmation of the deduced sequences of human A4,
B3,
and
A3 was obtained from the molecular weights of the peptides in
tryptic digests of the proteins. Peptides with masses corresponding to
all portions of the sequences of
A4,
B3, and
A3 were found (Fig. 6). Analysis of the tryptic peptides also
confirmed that the N termini of
A4,
B3,
A3 (Fig. 6), and
A1
(Fig. 2B) were all acetylated, and showed that the
N-terminal methionine was removed from
A4 and
A1, but retained on
B3 and
A3. The peptides marked with an asterisk in
Fig. 6 were additionally analyzed by MS/MS to confirm the proposed
sequence. An example of an MS/MS spectrum of a peptide from
A4 is
shown in Fig. 7. The spectrum shows the fragments formed
by collisional activation of
A4 peptide 106-117, which has the
sequence, LTIFEQENFLGK. Collisional activation causes the peptide to
fragment, primarily along the backbone. Fragments formed with the
charge remaining with the C terminus are called the a, b, and c series;
fragments formed with the charge remaining with the N terminus are
called x, y, and z series (26). Masses of expected fragments due to the
b and y" series (the " indicates an additional H on the fragment) are
shown in the diagram at the top. The presence of many peaks in the
spectrum with masses corresponding to the expected fragments confirmed
that the peptide analyzed has the given sequence.
This investigation: 1) identified and quantified the major protein
species in the young human lenses following separation by
two-dimensional electrophoresis; 2) determined and confirmed the
sequences of human A4,
B3, and
A3/A1 crystallins; and 3) demonstrated the presence of a truncated
A3/A1 crystallin. The present report completes the mapping of the major human crystallin species separated by two-dimensional electrophoresis first initiated by
De Vries et al. (12) and Datiles et al. (13).
Furthermore, the work demonstrates how an analysis of human crystallins
combining two-dimensional electrophoresis, Edman sequencing, and mass
spectrometry can rapidly identify and characterize the primary
structure of crystallins and detect their post-translational
modifications. The information in this report will be used as a
reference point for a thorough characterization of modifications
occurring during lens maturation, aging, and opacification.
The relative amounts of the total -,
-, and
-crystallin
subunits determined from two-dimensional gels were 27.7, 42.5, and
27.5%, respectively. This corresponds to previously published amounts
of
-,
-, and
-crystallin aggregates isolated by gel filtration
from lenses of similar age to the newborn lenses used in this study
(27, 28). The percent contribution of individual
- and
-crystallin subunits to the total lens protein was also consistent
with the literature (8, 10). The present report for the first time
estimates the relative amounts of various
-crystallin subunits in
human lens. The relative amounts of the various
-crystallins apparently differ greatly between species. No
A2 subunits were detected in newborn human lens, or in rat lens (16), while bovine (29)
and chicken lenses3 contained significant
amounts of
A2.
B3 is also a major protein of rat lens (30), but
its relative amount in human lens is much lower. Changes in the
relative concentrations of various crystallins in lens may alter its
properties. Therefore, future studies will further characterize the
relative amounts of crystallins and how this changes during lens
maturation.
While two-dimensional electrophoresis was capable of resolving the
majority of human crystallins, several crystallins did not resolve
completely from one another. Initially Edman sequencing identified just
one of the crystallins because only a single tryptic peptide from a
complex mixture was analyzed. In these cases, the presence of
co-migrating crystallins was detected by mass spectrometric analysis.
The co-migration of human A3 and
B3 was unexpected, since these
two proteins migrate to different positions during two-dimensional
electrophoresis of rat or bovine crystallins (16, 29), and have
theoretical pI values which differ by 0.2 pH units.
Independent confirmation of the cDNA determined sequences of human
A4,
B3, and
A3/A1 crystallins by mass spectrometric analysis
can detect errors in the deduced sequences and characterize post-translational modifications. A major finding of the present paper
was the truncation of
A3/A1-crystallin. N-terminal degradation of
B1 has previously been reported (3). Even in the lenses from donors
of less than 1-week-old, partially degraded forms of both
B1 and
A3/A1 were present.
B1 and
A3/A1 may be the most
proteolytically labile crystallin subunits in the human lens. The major
truncated forms of
A3/A1 and
B1 result from cleavage between an
Asn22-Pro23 of
A3 and
Asn15-Pro16 of
B1. A similar site is also
present in
B2 at Asn15-Pro16. However, no
cleavage of
B2 was seen in the lenses examined in this study. The
asparagine-proline sequence in
B2 occurs at the interface between
the N-terminal extension and the first "Greek key" containing motif
(22). 1H NMR spectroscopy has confirmed that the N-terminal
extensions of
-crystallins have relatively greater flexibility than
the Greek key containing motifs (31). Therefore, the proteolytically resistant Asn15-Pro16 region of
B2 may be
relatively inaccessible compared to the Asn-Pro regions of
B1 and
A3 which are cleaved. Of interest is the additional observation that
B1 and
A3 have longer N-terminal extensions than the
proteolytically resistant
B2 and
A4 (57 and 30 residues
versus 15 and 10 residues, respectively). The protease(s)
responsible for these cleavages in newborn human lens remain
unknown.
In conclusion, two-dimensional electrophoresis combined with Edman
sequencing and mass spectrometry successfully identified the major
crystallins in the young human lens and confirmed their predicted
sequences and masses. This combination of techniques permitted
separation and identification of proteins and modifications not
otherwise possible. Knowledge of the sequences of all the human
-crystallins and their exact molecular weights obtained in this
investigation will facilitate identification of the various modified
forms which become more numerous with age. The ability to electroelute
a modified crystallin species from two-dimensional gels and carry out
mass spectrometric measurements on the recovered protein was noteworthy
(Fig. 5). This technique should speed progress in identifying the
numerous protein modifications occurring in human lenses with age.
Post-translational modifications of crystallins may alter the way they
interact with one other and affect the highly ordered protein
structures required to maintain lens clarity. Determining the
modifications of crystallins in human lenses from donors of different
ages and stages of cataract will contribute to our understanding of
cataractogenesis. Such information is important, because cataract is
still a major cause of blindness.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U59057[GenBank], U59058[GenBank], and U71216[GenBank].
Mass spectral analyses were performed by the Nebraska Center for Mass Spectrometry. DNA sequencing was performed by the Department of Microbiology Core Facility at the Oregon Health Sciences University. GenBank data was accessed through the National Center for Biotechnology Information, Bethesda, MD.