Size of Human Lens beta -Crystallin Aggregates Are Distinguished by N-terminal Truncation of beta B1*

(Received for publication, January 10, 1997, and in revised form, February 10, 1997)

M. Saleh Ajaz , Zhixiang Ma , David L. Smith and Jean B. Smith Dagger

From the Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The aggregates formed by the interactions of the human lens beta -crystallins have been particularly difficult to characterize because the beta -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 beta -crystallins and their modified forms by mass spectrometry. In this investigation, the components of the three sizes of beta -crystallin aggregates, beta 1 (~150,000), beta 2 (~92,000), and beta 3 (~46,000), were determined. The principal differences among the different beta -crystallin aggregates was the presence of beta A4 in beta 1 and beta 2, but not beta 3, and the length of the N-terminal extension of beta B1. The size of the beta -crystallin aggregate correlated with the length of the N-terminal extension of beta B1, indicating that the flexible N terminus of beta B1 is critical to the formation of higher molecular weight aggregates of beta -crystallins. Separation of the components by ion exchange under non-denaturing conditions showed that beta B2 occurs as homo-dimers and homo-tetramers as well as contributing to hetero-oligomers. Other beta -crystallins were present only as hetero-oligomers.


INTRODUCTION

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, alpha -, beta -, and gamma -crystallins, each composed of proteins homologous within the group but differing from the proteins in other groups. The alpha -crystallins have aggregate molecular weights of 180,000-1,000,000, the beta -crystallins are aggregates of 40,000-150,000, and the gamma -crystallins do not aggregate. Within the alpha - and beta -crystallin subgroups, the proteins have been named according to their acidities. For example, the alpha -crystallins include alpha A-crystallin, the more acidic, and alpha B-crystallin, the more basic. Within the beta -crystallins of various mammalian species, seven beta -crystallins, beta A1, beta A2, beta A3, beta A4, beta B1, beta B2, and beta B3, have been identified; the gamma -crystallins include gamma S, gamma A, gamma B, gamma C, gamma D, and gamma E. Not all the genes are expressed in each species. In human lenses, expressed genes include those coding for alpha A (3), alpha B (4), beta A1, beta A3, beta A4 (5), beta B1 (6), beta B2 (7), beta B3 (5), gamma S (8), gamma C, and gamma D (9, 10). The beta - and gamma -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 alpha - and beta -crystallins as well as gamma S-crystallin are acetylated at the N terminus.

The beta -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 beta HIGH (beta H) and beta LOW (beta L); when they separate into three subgroups they are beta H, beta L1, and beta L2 (17) or beta 1, beta 2, and beta 3 (15). The unique structural features of the beta -crystallins that distinguish them from the gamma -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 beta -crystallins have demonstrated that replacing the linker sequence of beta B2-crystallin with the gamma -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 beta -crystallins, it has been noted that the largest of the beta -crystallins, beta B1, which has a long N-terminal extension, is a component only of the highest molecular weight aggregates (beta 1), implying that the long N terminus is vital to formation of large aggregates (16, 20, 21).

Six human beta -crystallin sequences are now known: beta B1 (6), beta B2 (7), and beta B3, beta A1, beta A3, and beta A4 (5). Mass spectrometric determinations of the molecular weights of the crystallins confirmed the cDNA-determined sequences and identified three of the crystallins, beta B1, beta A1, and beta A3, in forms with truncated N termini. beta B1 missing 15, 34, 39, and 40 residues of the N terminus were identified; beta A3 was found in one truncated form, missing the first 22 residues. Because beta A3 and beta A1 have the same sequence except that the N terminus of beta A1 begins with residue 19 of the beta A3 sequence, truncated beta A3-(23-215) is the same as beta A1-(5-197) (5). The previous studies of the beta -crystallins did not describe which beta -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 beta -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.


EXPERIMENTAL PROCEDURES

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 alpha -, beta 1-, beta 2-, beta 3-, and gamma -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 beta 3 and gamma -crystallins, the beta 3-crystallins were separated from the gamma -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 beta -crystallin fractions through Sephadex G-25 (1 × 5 cm column) at a flow rate of 1.5 ml/min.

Ion Exchange HPLC1

After isolation by size exclusion chromatography, each of the beta -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.

Reversed Phase HPLC

The beta -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.

Electrospray Ionization Mass Spectrometry (ESIMS)

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.


RESULTS

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 beta -crystallins from ages 16 to 70. For the size exclusion chromatographic conditions used in this study, beta -crystallins gave three peaks with aggregates of approximate molecular weights of 150,000 (beta 1), 92,000 (beta 2), and 46,000 (beta 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 beta -crystallin aggregates (Table I). Each beta -crystallin was identified by matching its molecular weight as determined by mass spectrometry with a molecular weight calculated from the known sequences of the beta -crystallins. For all identifications, the agreement between the experimentally determined and calculated molecular weights was within 2 atomic mass units.


Fig. 1. Chromatogram of the size exclusion separation of the water-soluble crystallins from a 56-year-old human lens. The fractions labeled beta 1, beta 2, and beta 3 were further fractionated by anion exchange (Fig. 2) and reversed phase HPLC (Fig. 3).
[View Larger Version of this Image (11K GIF file)]


Table I.

Components of beta -crystallin aggregates

Aggregates were identified by agreement of the experimentally determined molecular weight and a molecular weight calculated from the sequence to within 2 atomic mass units.


 beta -Crystallin  beta 1-Aggregates (Mr ~ 150,000)  beta 2-Aggregates (Mr ~ 92,000)  beta 3-Aggregates (Mr ~ 46,000)

 beta A3/beta A1  beta A3-(23-215) and/or beta A1-(5-197) (minor)  beta A3-(23-215) and/or beta A1-(5-197) (minor) NDa
 beta A4  beta A4-(1-195)  beta A4-(1-195) ND 
 beta B1  beta B1-(1-251)  beta B1-(16-251)  beta B1-(35-251)
 beta B1-(16-251)  beta B1-(35-251)  beta B1-(36-251)
 beta B1-(40-251) (minor)  beta B1-(36-251)  beta B1-(37-251)
 beta B1-(37-251)  beta B1-(40-251)
 beta B1-(40-251)  beta B1-(41-251)
 beta B1-(41-251)  beta B1-(42-251)
 beta B1-(42-251)
 beta B2  beta B2-(1-204)  beta B2-(1-204)  beta B2-(1-204)
 beta B3 ND ND ND 

a ND, not detected.

Anion exchange chromatography was performed without urea in the buffer to observe which components were associated in the beta -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 beta 1 was small (Fig. 2a), but it was approximately one-third of beta 2 (Fig. 2b) and beta 3 (Fig. 2c). Peak B was a relatively minor component of beta 1 but a major component of beta 2 and beta 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 beta 2 and beta 3 varied somewhat, from about 3:2 to 2:3, among the lenses.


Fig. 2. Chromatograms showing anion exchange separation of beta -crystallin fractions under non-denaturing conditions. Fractions collected from size exclusion chromatography (Fig. 1) are shown: a, beta 1; b, beta 2; and c, beta 3. The dotted line indicates the concentration of NaCl in the eluting buffer.
[View Larger Version of this Image (15K GIF file)]


The reversed phase chromatogram of proteins eluting in peak A had only one peak, with an elution time corresponding to beta 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 beta B2 (Mr 23,291) (7). The peak labeled A' (Fig. 2c) also had a molecular weight matching beta 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 beta B2.


Fig. 3. Chromatograms showing the reversed phase separation of the components of each of the peaks from anion exchange chromatography (a, peak A (Fig. 2); b, peak B (Fig. 2); and c, peak C (Fig. 2)). The reversed phase retention times of the ion exchange components, peaks A, B, and C were the same for the three beta -crystallin oligomers, beta 1, beta 2, and beta 3. The relative amounts of each component differed, as discussed in the text. The dotted line indicates the %CH3CN in the eluting solvent.
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Fig. 4. Reconstructed electrospray mass spectrum of peak 1 of the reversed phase chromatogram identifying this peak as beta B2 (Mr 23,291). The same molecular weight was obtained for peak 1 in the reversed phase chromatograms of all ion exchange fractions.
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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 beta B2 (peak 1 of Fig. 3), and the other had a retention time corresponding to beta B1 (peak 3 of Fig. 3) (6) and beta A4 (5), which co-elute with these reversed phase conditions. Identification of beta B2, beta B1, and beta 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 beta B2-crystallins in peaks B and C, whether from beta 1, beta 2, or beta 3, were all intact (Mr 23,291) (Fig. 4). beta A4-crystallin, which also was present only as the intact protein (Mr 22,282), was found in size exclusion fractions beta 1 and beta 2; a mass corresponding to beta A4 was not detected in beta 3. In contrast, beta B1-crystallin was identified by ESIMS in several forms (Fig. 5) with molecular weights corresponding to beta B1 and its truncated products at the N terminus (6). The relative amounts of beta B2:beta B1/beta 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 beta 1 and beta 2, was identified by its reversed phase elution time as beta A3/beta A1 (5).


Fig. 5. Reconstructed electrospray mass spectra showing beta B1 and its N-terminally truncated forms in peak 3 of the reversed phase separation from beta 1 (a), beta 2 (b), and beta 3 (c). The observed masses can be attributed to beta B1 either intact (Mr 27,933) or with various N-terminal truncations, beta B1-(16-251) (Mr 26,534), beta B1-(35-251) (Mr 24,834), beta B1-(36-251) (Mr 24,777), beta B1-(37-251) (Mr 24,676), beta B1-(40-251) (Mr 24,391), beta B1-(41-251) (Mr 24,294), and beta B1-(42-251) (Mr 24,193). The spectra for beta 1 and beta 2 also had a mass at 22,284, corresponding to beta A4, indicating that it was present in comparable amounts to beta B1.
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The electrospray mass spectra for the proteins with reversed phase elution times appropriate for beta B1 were remarkably different for beta 1, beta 2, and beta 3 (Fig. 5, a, b, and c, respectively). The beta B1-crystallins showed progressive N-terminal truncation from beta 1 to beta 2 to beta 3. For all the lenses, intact beta B1 (Mr 27,933) was a major component only of beta 1, which also contained beta 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 beta B1 in beta 2 was truncated at the N terminus with 15-41 residues missing (Fig. 5b), and most of beta B1 in beta 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 beta B2 in peak A of the ion exchange chromatograms of beta 2 and beta 3 indicated that some dimers of beta 3 and some tetramers of beta 2 are beta B2 homo-oligomers. The small peak A of beta 1 indicated that beta B2 homo-oligomers are a very minor component of beta 1. Neither beta B1 nor beta A4 was found alone in the ion exchange fractions, implying that these beta -crystallins do not form homo-oligomers.


DISCUSSION

In previous investigations the components of the various aggregates of human beta -crystallins were identified primarily by SDS-polyacrylamide gel electrophoresis (15, 16, 22). Because the sequences of the human beta -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 beta B1 in both beta 1 and beta 2 (16), whereas data from lenses more than 5 years old indicated the presence of beta B1 only in beta 1 (15, 16). From the SDS-polyacrylamide gel electrophoresis data presented in those studies, it appeared that beta B1 was not a component of the lowest molecular weight aggregates. A protein of 24-26 kDa, first called beta Bp then renamed beta B2, was a major component of beta 1, beta 2, and beta 3 from lenses of all ages (15, 16, 22). The other beta -crystallins were not identified.

Results from our mass spectrometric investigation of beta -crystallins, separated by ion exchange and reversed phase HPLC, have led to unambiguous identification of the components of each of the subgroups of beta -crystallins (Table I). The data show that beta B1 in a variety of forms truncated at the N terminus is present in the aggregates of beta 1, beta 2, and beta 3 and that the size of the aggregates correlates with the length of the N terminus of beta B1. The largest aggregates, beta 1, are composed primarily of beta B1 (both intact and with the first 15 residues missing), intact beta B2, and intact beta A4. The principal forms of beta B1 present in beta 1, beta B1-(1-251) and beta B1-(16-251), correspond to bovine proteins previously identified as beta B1a and beta B1b (23). These two forms of beta B1 are the major beta B1-crystallins found in newborn human lenses (6). The ion exchange chromatography performed under non-denaturing conditions indicated that the oligomers of beta 1 are hetero-oligomers of beta B1, beta A4, and beta B2. Because beta B1 and beta A4 co-elute on reversed phase HPLC, the relative amounts of each component were not ascertained. A very minor component in beta 1 had the correct retention time for beta A3. Intact beta 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 beta A3 truncated at the N terminus based on its reversed phase HPLC elution time. A molecular weight corresponding to beta A3-(23-215), which is the same as beta A1-(5-197), was determined for this protein isolated from other adult lenses.

The tetrameric aggregates of the beta -crystallins, beta 2, included the same proteins as beta 1 except that beta B1 was found with further N-terminal truncation. Although no intact beta B1 was present in beta 2, the following forms, truncated at the N terminus, were found: beta B1-(16-251), beta B1-(35-251), beta B1-(40-251), and beta B1-(41-251). As in beta 1, beta B2 and beta A4 were found only as intact proteins. The non-denaturing ion exchange of the aggregates in beta 2 indicated that the tetramers were homo-oligomers of beta B2 and hetero-oligomers of beta B1, beta B2, and beta A4.

The dimers of beta -crystallin, beta 3, included homo-oligomers of beta B2 and hetero-oligomers of beta B1 and beta B2. The beta B1 in beta 3 was degraded at the N terminus even further than in beta 2, with most of beta B1 missing 34 or more residues from its N terminus. Finding beta B1, although only in forms truncated at the N terminus, in beta 3 is in opposition to previous reports that beta B1 exists only in larger aggregates (15, 17, 20, 21). These differing observations are easily explained by the fact that the beta B1 products without the N-terminal 34-41 residues have molecular weights of 24,192-24,834, similar to the molecular weight of beta B2 (23,291) and may not have been recognized as beta B1 by SDS-polyacrylamide gel electrophoresis analysis. Identification would have been further complicated by the fact that beta B1 minus these residues has a pI (6.38) similar to the pI of beta B2 (6.33). beta B3, which has previously been isolated only from fetal or newborn lenses (5), was not detected in any of the subgroups of the beta -crystallins from these adult lenses.

For bovine lens crystallins, it has been demonstrated that the various aggregates of beta -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 beta -crystallins differs considerably from human beta -crystallins, it is interesting to consider the effect hydrophilicity might have on the stability of the various human beta -crystallin aggregates. The hydrophilicities of the beta -crystallin or portions of them can be calculated from the Bull and Breese indices (25). The long N-terminal extension of beta B1, which appears to play a unique role in the formation of large aggregates as demonstrated by the presence of intact beta B1 only in beta 1-crystallins, has a Bull and Breese index (BB) of +365. The hydrophilicity indices for beta B1 in the middle size aggregates of beta 2 and the dimers of beta 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 beta B2 forms both homo-dimers and tetramers, but beta B1 and beta A4 do not form homo-oligomers. Overall, beta B2 is a more hydrophilic protein (BB +62) than either beta A4 (BB +30) or beta B1(BB +5).

Comparison of the sequence of the first 34 residues of human beta B1 with the N terminus of bovine beta B1 shows that this region is not as rich in alanine and proline as bovine beta 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 beta -crystallins, stabilizing high molecular weight aggregates.

Previous investigations using mutant forms of beta B2 have demonstrated that the linker portion (residues 80-88, PIKVDSQEH) that joins the two domains of beta B2 is critical to the formation of dimers (19), whereas the N- and C-terminal extensions of beta B2 are not essential (18). If this linker region were the sole determinant of dimer formation, it might be expected that beta B1, which contains a region highly homologous with the beta B2 linker (residues 140-148, PIKMDAQEH), would also form homo-oligomers. Failure to detect homo-oligomers of beta 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 beta A4 is in beta 1 and beta 2, but not in beta 3, suggests that it too may contribute to the formation of higher aggregates. If so, its interactions are different from those proposed for other beta -crystallins. The sequence features previously proposed to be favorable to aggregation, the long N terminus and the beta B2 linker sequence, are missing in beta A4. beta A4 has a short N terminus and the sequence of the linker region, PAACANHRD, is very different from the sequence of beta B2 that favors dimer formation.

In contrast to beta B1, which was found in many truncated forms at the N terminus, beta B2 and beta A4 were present only as intact proteins. None of the fractions of beta -crystallins contained proteins with molecular weights consistent with truncated products of either beta B2 or beta A4. These results do not support the presence of truncated beta 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 beta B2 (26).

Despite the similarity between the sequences of human and bovine beta -crystallins, there are striking differences between the composition of the aggregates of the human and bovine beta -crystallins (27). In both species, intact beta B1 is found only in beta 1 (17). Slingsby and Bateman (27) also reported finding no beta B1 in beta 2 and beta 3 of bovine lenses and identified the lower molecular weight proteins in beta 2 and beta 3 as beta A3 and beta B3. Because their techniques may not have recognized truncated beta B1, it is possible that some truncated beta B1-crystallins were present in beta 2 and beta 3 but incorrectly identified. One similarity between bovine and human lenses is that beta 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 beta B2 could form homo-dimers as well as hetero-dimers with other acidic and basic beta -crystallins. Our results for human lens beta -crystallins showed that, in addition to forming homo-dimers and hetero-dimers, beta B2 also formed homo- and hetero-tetramers.

In bovine lenses, both beta B3 and beta A3 are major components, whereas in humans beta B3 is produced only in fetal and newborn lenses, and beta A3 is a very minor component. Furthermore, the beta A3 that is present in adult lenses is truncated at the N terminus (5). Slingsby and Bateman (27) concluded that, for bovine beta -crystallins, oligomers larger than dimers required the presence of an acidic beta -crystallin with a long N terminus, such as beta A3. This conclusion was supported by studies of mutant rat beta A3, showing that beta A3 without the first 29 residues of the N terminus formed smaller aggregates than beta A3 with only 6 residues missing (28). Such a role for beta A3 in human lenses is improbable because very little beta 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 beta B1 is the major determinant of the size of the beta -crystallin aggregates in human lenses.


FOOTNOTES

*   This work was supported by National Institutes of Health Research Grant EY RO1 07609 and National Science Foundation Grant 9413023.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.
Dagger    To whom correspondence should be addressed. Tel.: 402-472-1684; Fax: 402-472-9862; E-mail: jbsmith{at}unlinfo.unl.edu.
1   The abbreviations used are: HPLC, high performance liquid chromatography; ESIMS, electrospray ionization mass spectrometry; BB, Bull and Breese hydrophilicity index.

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