Sequence Analysis of beta A3, beta B3, and beta A4 Crystallins Completes the Identification of the Major Proteins in Young Human Lens*

(Received for publication, October 9, 1996, and in revised form, November 2, 1996)

Kirsten J. Lampi Dagger , Zhixiang Ma §, Marjorie Shih Dagger , Thomas R. Shearer Dagger , Jean B. Smith §, David L. Smith § and Larry L. David Dagger

From the Dagger  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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as alpha A, alpha B, beta A1, beta A3, beta A4, beta B1, beta B2, beta B3, gamma S, gamma C, and gamma D-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human beta A1, beta A3, beta A4, and beta 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 beta 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.


INTRODUCTION

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 alpha A, alpha B, beta B1, beta B2, gamma S, gamma C, and gamma D-crystallins have already been determined and their presence in the human lens has been demonstrated (1-11). Furthermore, the positions where human alpha A, alpha B, beta B1, beta B2, and gamma 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 beta A1, beta A3, beta B3, and beta A4 to the list of major beta -crystallins in young, normal human lens. We also correct the sequence of human beta A3, complete the sequence determination of human beta B3, and for the first time, report the sequence of human beta A4. These deduced amino acid sequences were confirmed by mass spectrometry.


MATERIALS AND METHODS

Identification of Major Human Crystallins

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 Human beta A4, beta B3, and beta A3/A1 cDNAs

Lenses 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 beta 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 beta 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 beta 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 beta 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 beta 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 beta B3 cDNA, the same procedure described above for 5'-RACE of human beta A4 cDNA was followed. The gene-specific antisense primer, CAGACCACAAGCTGCATCTGT, complementary to nucleotides 8-28 in exon 5 of human beta 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 beta B3 (GenBank accession number X15144[GenBank]), was used to perform 5'-RACE of human beta 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 beta A3 protein suggested that a portion of the reported genomic sequence of human beta 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 beta 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 beta 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 beta 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 right-arrow 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 beta A4 cDNA, and 2 clones of the PCR product of the beta A3 cDNA were sequenced in both sense and antisense directions. The sequence from 4 antisense strands of the 5'-RACE product of beta B3 cDNA were sequenced. DNA sequences were edited and theoretical pI values of proteins calculated using Geneworks 2.5 software (IntelliGenetics, Mountain View, CA).

Confirmation of Deduced beta A3/A1, beta B3, and beta A4 Protein Sequences using Mass Spectrometry

The deduced amino acid sequences of beta A3/A1, beta B3, and beta 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 beta -crystallin aggregates of approximately 160,000 and 50,000 molecular weight (beta H- and beta 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.

beta -Crystallin subunits were partially purified from beta H and beta 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 beta -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 beta -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 beta 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 beta 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 beta A3/A1, beta B3, and beta 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 beta B3, beta A3, and beta A4 for tryptic digestion, respectively. Tryptic peptides of beta A4 and beta B3 were prepared from proteins isolated by gel filtration and reversed-phase HPLC. Peptides of beta 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.


RESULTS

Identification of Crystallin Subunits of Young Lens Separated by Two-dimensional Electrophoresis

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 beta B1 and beta B1 missing 15 residues from its N terminus (beta B1, residues 16-251) was previously reported (3). Due to blockage of the N termini, the identification of beta B2, beta A3, beta A4, gamma S, alpha A, and alpha 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 beta B1, beta B2, gamma S, alpha A, and alpha B on two-dimensional gels (12-14) and demonstrate the presence of beta A3 and beta A4 in human lens and their positions on two-dimensional gels.


Fig. 1. Two-dimensional electrophoresis of soluble lens protein from 3-day-old human donor. Identification of major crystallin species was performed by both Edman sequencing and mass spectrometric analysis as summarized in Table I and Figs. 2 and 3. The identification of beta B1-crystallin and its degradation product beta B1 (16-251) was previously reported (3). Note that gamma S and beta A1, as well as beta A3- and beta B3-crystallins co-migrate. The left side of the gel is basic and the right side is acidic. Only the region of the two-dimensional gel containing crystallins is shown.
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Table I.

Identification of peptides from major crystallins from young human donors


Crystallina Amino acid sequence Residue numbers

 alpha A HFSPEDLTVKb 79 -88
TLGPFYPSR 13 -21
 alpha B APSWFDTGLS 57 -66
 beta B2 DMQWHQR 191 -197
GAFHPSN 198 -204
 beta A3 EWGSHAQTSQIQSIR 197 -211
 beta A3 (23-215) PTPGSLGP 23 -30
 beta A4 MVVWDEDGFQGR 13 -24
GFQYVLEXDHHSGDYK 158 -173
EWGSHAPTFQVQSIR 177 -191
 gamma S ITFYEDK 7 -13
 gamma C GKITFYEDRAFQGRSYETTTDXPNL 1 -25
 gamma D GKITLYEDRG 1 -10

a  The following crystallin species labeled in Fig. 1 were identified by comparing the results of Edman sequencing to reported amino acid sequences of crystallins. The sequences of alpha A, alpha B, beta B2, beta A3, beta A4, and gamma S-crystallins were determined from tryptic fragments, and the sequences of beta A3/A1 (23-215), gamma C, and gamma D-crystallins by direct Edman sequencing of unfragmented proteins.
b  Several analyzed fractions of the reverse-phase purified tryptic fragments contained more than one peptide.

Due to their lack of acetylated N termini, the positions of gamma C and gamma D, as well as a truncated beta -crystallin, could be determined by direct sequence analysis of electroblotted proteins (Fig. 1, Table I). The truncated subunit contained a sequence matching that of beta A3/A1 and could have arisen from truncation of the first 22 amino acids of beta A3 or the first 4 amino acids of beta A1. beta A1 and beta A3 are identical, except beta A3 contains a longer N-terminal extension due to the use of an alternate start codon in the single beta A3/A1 transcript (20). For simplicity, the truncated beta -crystallin is referred to as beta A3 (23-215). The presence of beta A3 (23-215) in human lens was recently reported in another laboratory (21).

Although mRNAs coding for gamma A, gamma B, gamma C, and gamma D have been reported in fetal and neonatal human lenses, transcripts for gamma C and gamma D were more abundant (7). In the present study, gamma C and gamma D crystallins were detected, but no gamma A or gamma B crystallins were observed (Fig. 1, Table I). Earlier mass spectrometric and chromatographic analysis also detected gamma C and gamma D in human lenses, and found either small or undetectable amounts of gamma A and gamma B (10, 11). This suggested that little translation of either gamma A or gamma B mRNA may occur in human lens. The lower migration position of gamma D was not expected (Fig. 1), since its calculated molecular weight was nearly identical to gamma C. This is likely explained by anomalous migration of gamma -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 gamma S or beta A3. The region containing gamma S (as determined by Edman sequencing) yielded peptides with masses matching peptides expected from both gamma S and beta A1. Representative mass spectra showing two peptides with masses matching residues 84-94 and 148-153 of gamma S (Fig. 2A), and a peptide with a mass matching the acetylated N terminus of beta 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 gamma S was actually beta A1, and not a post-translationally derived form of beta A3.


Fig. 2. Fast-atom bombardment mass spectra of tryptic peptides isolated from the region of two-dimensional gels containing both gamma S- and beta A1-crystallin. A, mass spectrum of two tryptic peptides corresponding to the expected molecular masses of residues 84-94 (MH+ 1156.6) and 148-153 (MH+ 779.4) of human gamma S-crystallin. B, mass spectrum of a single tryptic fragment corresponding to the expected molecular mass of the acetylated N-terminal tryptic peptide of human beta A1-crystallin (residues 1-14, MH+ 1495.7).
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A protein corresponding to beta 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 beta A3 included masses corresponding to peptides from both beta A3 and beta B3. For example, masses matching the acetylated N-terminal peptide (residues 1-17) and peptide 96-109 of beta A3 (Fig. 3A), as well as peptide 110-122 of beta A3 and peptide 153-167 of beta B3 (Fig. 3B) were found.


Fig. 3. Electrospray ionization mass spectra of tryptic peptides isolated from the region of two-dimensional gels containing both beta A3- and beta B3-crystallin. A, the acetylated N terminus of human beta A3 produces a tryptic peptide with MH+ 2018.9 (the doubly charged peptide is MH2+2 1010). The peak at 846.5 corresponds to the doubly charged ion for peptide 96-109 of beta A3. The peak at 446.2 was unidentified. B, spectrum showing a peak at 865.8 attributed to the doubly charged peptide 153-167 of beta B3 (MH2+2 866.0). The peak at 787.5 was attributed to the doubly charged peptide 110-122 of beta A3. This beta A3 fragment included an acrylamide adduct on cysteine 117 which increased its mass by 71.
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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 beta A1 and gamma S, as well as beta A3 and beta 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 beta H- and beta L-crystallin aggregates from 3- and 7-day-old human lenses removed gamma S and allowed estimation of the relative amount of beta A1. beta A1 and beta A4 were found in nearly equal quantities in the young human lens (gels not shown).

Table II.

Relative amounts of each crystallin subunit in the soluble protein of young human lens


Crystallin subunit % of total

 alpha A 21.4  ± 1.0a
 gamma S/beta A1b 15.4  ± 1.2
 beta B2 14.3  ± 1.2
 gamma C 14.3  ± 0.8
 beta B1 8.8  ± 2.4
 alpha B 6.3  ± 0.8
 beta A3/beta B3b 6.2  ± 0.8
 beta A4 4.7  ± 1.4
 gamma D 2.5  ± 0.9
 beta B1 (16-251) 2.2  ± 0.8
 beta A3 (23-215) 1.6  ± 0.6
Total 97.7c

a  Mean ± 1 S.D. (n = 4), resulting from the densitometric analysis of individual two-dimensional electrophoretic gels of soluble protein of newborn, 3-, 4-, and 7-day old human donors.
b  The individual amounts of gamma S, beta A1, beta A3, and beta B3 could not be estimated because these subunits migrated to similar positions during two-dimensional electrophoresis.
c  The remaining 2.3% of the total crystallin was due to minor, unidentified species migrating to the acidic sides of beta A3/beta B3 and beta A4.

Human beta A4, beta B3, beta A3/A1-crystallin cDNAs

A proposed full-length human beta A4-crystallin cDNA was obtained by 3'- and 5'-RACE PCR because there was no previously published sequence for human beta 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 beta 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 beta A4 shared 92-94% sequence identity with rat2 and bovine (22) beta A4, and 65% identity with chicken beta A4 (23). The N-terminal extensions of human, rat, and chicken beta A4 were all 10 residues in length, supporting the previous suggestion that the N-terminal extension of bovine beta A4 is also 10 residues in length (23).



Fig. 4. The nucleotide sequences of human beta A4- (A), beta B3- (B), and beta A3-crystallin (C) cDNAs. Numbers to the right indicate the nucleotides, while numbers below indicate deduced amino acids. The entire coding region of beta A4 is given, while the beta B3 and beta A3/A1 cDNA sequences encode only amino acids 1-102 and 44-215 of each crystallin, respectively. The poly(A) adenylation signal is underlined in the beta A4-crystallin cDNA sequence. The nucleotides and deduced amino acids differing from the previously reported (20) sequence of human beta A3-crystallin are underlined.
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Since only exons 4-6 of human beta B3 were previously reported (24), the 5' end of human beta 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 beta B3 (GenBank accession number U71216[GenBank]). This sequence overlapped with the first 114 base pairs of the previously reported sequence for human beta B3 exon 4 (Genbank accession number X15144[GenBank]). Combining this sequence with the previously reported sequences of human beta 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 beta A3 determined by ESIMS (25,192) did not agree with the mass calculated from the published sequence (20), a portion of the human beta 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 beta A3 and beta A1 were 25,193 and 23,102, respectively, including the acetylation of their N termini. The pI values of beta A3 and beta A1 were 5.58 and 6.17, respectively.

Confirmation of the Deduced beta A4, beta B3, and beta A3/A1 Amino Acid Sequences

The molecular weights of the subunits in lens beta H- and beta L-crystallin aggregates from a 3-day-old donor were determined by ESIMS (Table III). Two of the components had masses corresponding to beta B1 and beta B2. Two other components had masses corresponding to previously identified partially degraded beta B1-crystallins (3). A spot corresponding to beta 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 beta B1 (3), may not have been detected by electrophoresis because they may co-migrate with other crystallins.

Table III.

Molecular masses of beta -crystallins in young human lens

All experimentally measured masses were determined by ESIMS from the major components of the beta H and beta L fractions from a 3-day-old donor lens, except beta A3 (23-215) and beta B3, which were determined from the beta H fraction of a 3-year-old donor lens and 32-week gestation lens, respectively. The accuracy of these determinations was ± 3 Da.
Probable identity Experimental Calculateda

Da
 beta A1 23,101 23,102
 beta A3 25,192 25,193
 beta A3 (23-215) 22,645 22,645
 beta A4 22,285 22,285
 beta B1 27,935 27,935
 beta B1 (16-251) 26,535 26,535
 beta B1 (40-251) 24,390 24,391
 beta B2 23,291 23,291
 beta B3 24,222 24,224

a  For the acidic beta -crystallins and beta B3 the molecular masses were calculated based on the deduced amino acid sequences in Fig. 4. For beta B1 and beta B2 the calculated molecular masses were based on previously confirmed sequences (3, 11).

Experimentally determined masses of other beta -crystallins agreed with the calculated masses of beta A1, beta A3, beta A3 (23-215), beta A4, and beta B3 (Table III), confirming the deduced amino acid sequences of beta A4, beta B3, and beta A3/A1 (Fig. 4). A spot corresponding to beta A3 (23-215) was consistently observed by two-dimensional electrophoresis of soluble protein from newborn lenses (Table II). However, analysis of beta -crystallin aggregates by reversed-phase HPLC and ESIMS barely detected a component with the mass of beta A3 (23-215) in lenses less than 3 years old. The presence of beta 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 beta 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 beta A3 (23-215) was 22,999 (Fig. 5), supporting the Edman sequencing results (Table I) and confirming the presence of beta A3 (23-215).


Fig. 5. Electrospray ionization mass spectrum of N-terminally degraded beta A3 (23-215) from a 4-day-old lens electroeluted from two-dimensional electrophoretic gels. The mass includes an acrylamide adduct (+71 Da) at each of the five cysteine residues. The mass of beta A3 (23-215) calculated from the sequence is 22,646; the calculated mass including the acrylamide adducts is 23,001.
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Further confirmation of the deduced sequences of human beta A4, beta B3, and beta 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 beta A4, beta B3, and beta A3 were found (Fig. 6). Analysis of the tryptic peptides also confirmed that the N termini of beta A4, beta B3, beta A3 (Fig. 6), and beta A1 (Fig. 2B) were all acetylated, and showed that the N-terminal methionine was removed from beta A4 and beta A1, but retained on beta B3 and beta 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 beta A4 is shown in Fig. 7. The spectrum shows the fragments formed by collisional activation of beta 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.


Fig. 6. Confirmation of deduced amino acid sequences of (A) beta A4-, (B) beta B3-, and (C) beta A3-crystallins by mass spectrometric analysis of tryptic digests. The numbers above each sequence give the amino acid residue in each protein, while the numbers below each sequence give the calculated molecular mass of the indicated tryptic peptide. Molecular ions corresponding to all the expected tryptic peptides were found by ESIMS analysis of the tryptic digests of each protein. The identities of tryptic peptides with asterisks were further confirmed by MS/MS analysis.
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Fig. 7. MS/MS spectrum of peptide 106-117 from beta A4. The expected fragmentation pattern is shown in the inset. When the charge on the peptide stayed with the N terminus, fragments from the b series were seen; when the charge stayed with the C terminus, fragments from the y" series were seen (26). Fragments labeled w were from cleavage of the side chains of the amino acid residues. Horizontal arrows labeled x2.00 and x150.00 indicate the magnification of the mass spectral response in these regions.
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DISCUSSION

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 beta A4, beta B3, and beta A3/A1 crystallins; and 3) demonstrated the presence of a truncated beta 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 alpha -, beta -, and gamma -crystallin subunits determined from two-dimensional gels were 27.7, 42.5, and 27.5%, respectively. This corresponds to previously published amounts of alpha -, beta -, and gamma -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 alpha - and gamma -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 beta -crystallin subunits in human lens. The relative amounts of the various beta -crystallins apparently differ greatly between species. No beta A2 subunits were detected in newborn human lens, or in rat lens (16), while bovine (29) and chicken lenses3 contained significant amounts of beta A2. beta 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 beta A3 and beta 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 beta A4, beta B3, and beta 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 beta A3/A1-crystallin. N-terminal degradation of beta B1 has previously been reported (3). Even in the lenses from donors of less than 1-week-old, partially degraded forms of both beta B1 and beta A3/A1 were present. beta B1 and beta A3/A1 may be the most proteolytically labile crystallin subunits in the human lens. The major truncated forms of beta A3/A1 and beta B1 result from cleavage between an Asn22-Pro23 of beta A3 and Asn15-Pro16 of beta B1. A similar site is also present in beta B2 at Asn15-Pro16. However, no cleavage of beta B2 was seen in the lenses examined in this study. The asparagine-proline sequence in beta 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 beta -crystallins have relatively greater flexibility than the Greek key containing motifs (31). Therefore, the proteolytically resistant Asn15-Pro16 region of beta B2 may be relatively inaccessible compared to the Asn-Pro regions of beta B1 and beta A3 which are cleaved. Of interest is the additional observation that beta B1 and beta A3 have longer N-terminal extensions than the proteolytically resistant beta B2 and beta 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 beta -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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants EY07755 (to L. L. D.) and EY07609 (to D. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].


   To whom correspondence should be addressed: 611 S.W. Campus Dr., Portland, OR 97201. Tel.: 503-494-8625; Fax: 503-494-8918; E-mail: davidl{at}ohsu.edu.
1    The abbreviations used are: FAB-MS, fast atom bombardment mass spectrometry; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; ESIMS, electrospray ionization mass spectrometry.
2    K. J. Lampi and L. L. David, unpublished results.
3    L. L. David and M. K. Duncan, unpublished results.

Acknowledgments

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.


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