©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reduced Chaperone-like Activity of A-crystallin, an Alternative Splicing Product Containing a Large Insert Peptide (*)

Ronald H. P. H. Smulders , Ingrid G. van Geel , Will L. H. Gerards , Hans Bloemendal , Wilfried W. de Jong (§)

From the (1) Department of Biochemistry, University of Nijmegen, NL-6500 HB Nijmegen, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, -crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major A- and B-subunits, -crystallin of rodents contains an additional minor subunit resulting from alternative splicing, A-crystallin. This polypeptide is identical to normal A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat A- and A-crystallin in Escherichia coli. The multimeric particles formed by A are larger and more disperse than those of A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with A, the A-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of -crystallin. Our experiments indicate that A-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant A-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.


INTRODUCTION

-Crystallins are members of the small heat shock protein family and are highly expressed as multimeric structural proteins in the vertebrate eye lens (1) . There are two types of homologous 20-kDa subunits, A- and B-crystallin, which are 173 and 175 amino acids in length, respectively (2) . A-crystallin is the more lens-specific one, occurring only in trace amounts in some other tissues, including brain, spleen, liver, and retina (3, 4, 5) . In contrast, B-crystallin is constitutively expressed at considerable levels in many non-lens tissues, notably in heart and striated muscle (6) , and is induced in various neurodegenerative diseases and brain tumors (7, 8, 9, 10) . -Crystallins and other small heat shock proteins (hsps) have chaperone-like properties, suppressing heat- or chemically induced aggregation of other proteins (11, 12) . Like for other small hsps, increased expression of A- and B-crystallin make cells thermotolerant (13, 14) . -Crystallins and other small hsps normally occur as large complexes, both homo- and heteromultimeric, with molecular masses of up to 800 kDa. The tertiary structure of these proteins is unknown, although experimental evidence supports the proposal by Wistow (15) that the subunits consist of two similarly folded domains and a short exposed and flexible C-terminal arm (16-18). Likewise, the arrangement of subunits in the multimeric complex is still unsolved, probably due to its polydisperse, noncompact, and dynamic nature (19, 20) . Conflicting models for the quaternary structure of -crystallin have been postulated, including three-layered structures (21, 22) , a micelle-like arrangement (23) , a combination of these two (24) , a rhombododecahedric structure (25) , and most recently a cylindrical structure (26) . This lack of structural information seriously hampers the understanding of their chaperone-like behavior.

-Crystallins of rodents and some other mammals contain a minor product resulting from alternative splicing (27, 28, 29, 30) . This subunit, A, is identical to normal A-crystallin, except for an insertion of 23 amino acids between residues 63 and 64, at the junction of the two putative domains (28, 31, 32) . This insertion is encoded by the optional exon 2 of the A gene, which is only spliced into 10-20% of the mature A mRNA (33) . Structural analysis has demonstrated that A is, next to A and B, an integral part of the rodent -crystallin complex, suggesting that the insert peptide does not seriously perturb the tertiary or quaternary structure (32) . It is an intriguing question how this variant could arise and be maintained for over 70 million years in evolution (34) and how the insertion influences the properties of -crystallin. Information on such a natural insertion mutant might shed light on the structure-function relation of -crystallins in general. We therefore report in this paper a comparison of recombinant rat A- and A-crystallin. We show that both subunits are able to form homogeneous multimeric particles which display a similar thermostability and morphology. Furthermore, we describe heat protection experiments revealing that A homomultimers have considerably less chaperone-like activity than A homomultimers. Our results thus indicate that, in spite of the structural similarities between A- and A-multimers, the insertion imposes considerable restraints on the functional potential of the A-subunit.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, T4 DNA polymerase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase were from Life Technologies, Inc. Ampicillin, chloramphenicol, isopropyl-1-thio--D-galactopyranoside, trypsin, and protease inhibitors were obtained from Sigma. AatII and 5-bromo-4-chloro-3-indolyl--D-galactoside were purchased from Promega. [-P]dCTP was from Amersham Corp. Oligonucleotides were synthesized by Eurogentec. Escherichia coli strains used as recipients during cloning and expression experiments were TG-1 (Amersham Corp.) and B BL21(DE3)pLysS (35) .

DNA Probe Preparations

In order to get DNA probes for cDNA library screening, a Puc18 construct containing the complete hamster A gene sequence (29) was digested with HindIII/BamHI and BamHI/StuI. These double digestions resulted in a 242-base pair fragment containing exon 1 and a 553-base pair fragment containing exon 2, respectively. Both fragments were radioactively labeled using a random primers labeling system (Life Technologies, Inc.).

gt11 Library Screening

A rat lens gt11 library was screened using standard techniques (36) . Briefly, plaques were lifted in duplicate onto Hybond-N filters (Amersham Corp.), which were denatured, neutralized, and exposed to UV light for 3 min. Replicate filters were prehybridized in 6 SSC, 0.05 Blotto, and 100 µg/ml salmon sperm DNA at 68 °C for 1 h. The duplicate filters were hybridized in the same solution with either the ``exon 1'' probe or the ``exon 2'' probe at 68 °C overnight. Filters were successively washed at least two times with each 2 SSC, 0.1% SDS (5 min); 1 SSC, 0.1% SDS (1 h); and 0.02 SSC, 0.1% SDS (1 h) at 68 °C. Subsequently, the filters were dried and subjected to autoradiography on Kodak XAR-5 films. Three clones positive for exon 1 (A) and three clones positive for both exon 1 and exon 2 (A) were plaque-purified using secondary and tertiary screening procedures.

DNA Subcloning

phage lysates generated from tertiary screening dishes were used to obtain sufficient recombinant phage DNA (36) . The EcoRI fragments of the cDNA inserts were isolated from gel using an electro-elution method (37) and ligated into the multiple cloning site of the pGEM3Zf(+) vector (Promega). To be sure that the cloned cDNAs were full length, 5` ends of the coding sequences were verified using the kilobase sequencing system (Life Technologies, Inc.). To get the required expression constructs, pGEM3Zf(+)A and pGEM3Zf(+)A were digested with AatII/StuI, and the resulting fragments were treated with T4 DNA polymerase to remove the AatII protruding 3` termini. The 519- and 588-base pair fragments, containing the A and A coding sequence, respectively, were isolated from agarose gel and ligated to a synthetic linker. This linker was obtained by cooling down a mixture of an unphosphorylated CATGGACGT oligonucleotide and a T4 polynucleotide kinase phosphorylated ACGTC oligonucleotide from 75 °C to room temperature in a water bath during 2 h. The ligation products were purified using low melting temperature agarose gel electrophoresis (36) and cloned into the NcoI site of the pET8c expression vector (35) .

Expression and Purification of Recombinant Proteins

The A and A expression constructs were transformed in the host E. coli B BL21(DE3)pLysS. Induction, cell lysis, and fractionation were essentially performed as described by Merck et al.(17) . The water-soluble fraction of the A cell lysate was dialyzed against DEAE starting buffer (50 mM NaCl, 1 mM EDTA, 20 mM Tris/HCl, pH 7.5) and applied onto a Fast Flow DEAE-Sepharose column (Pharmacia-LKB). A linear gradient from 50 to 500 mM NaCl at pH 7.5 was used to elute the proteins at a linear flow rate of 0.8 cm/min at 4 °C. The peak fractions containing A were pooled and successively dialyzed against demineralized water and DE52 starting buffer (6 M urea, 0.02% -mercaptoethanol (v/v), 5 mM Tris/HCl, pH 8.0). Isolation of pure A was carried out by ion-exchange chromatography under denaturing conditions on a DE52 column (Whatman) (38) . With regard to the purification of A, the DEAE-Sepharose step used for A was omitted. The water-insoluble fraction of the A cell lysate was stirred directly in DE52 starting buffer at 4 °C for 1.5 h, centrifuged at 15,000 g for 30 min, dialyzed briefly against DE52 starting buffer, and applied onto the DE52 column. Selected DE52 fractions of A and A were pooled, dialyzed against demineralized water, and lyophilized.

Reconstitution

For structural and functional studies, A and A were refolded under identical conditions: 6 mg of lyophilized protein was dissolved in 1 ml of denaturing buffer (6 M urea, 0.1 M NaSO, 0.02% -mercaptoethanol (v/v), 20 mM NaP, pH 6.9), stored for 2 h at 4 °C, and diluted with phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9) (39) to a concentration of 1 M urea and 1 mg/ml of protein. Subsequently, the solution was dialyzed against phosphate buffer and stored at -20 °C.

Gel Permeation Analysis

An LKB Bromma HPLC() system was used in conjunction with a Superose 6 HR 10/30 prepacked size exclusion column (Pharmacia-LKB) for analysis of aggregate sizes. Samples contained 100 µg of protein in 1 ml of phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9). The mobile phase was phosphate buffer at a flow rate of 0.5 ml/min. Absorbance was monitored at 225 nm. High molecular mass standards (Pharmacia-LKB) were used for calibration.

Tryptophan Fluorescence

Samples containing 100 µg of protein in 1 ml of phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9) were centrifuged for 15 min at 15,000 g to reduce scattering from possible insoluble particles. Fluorescence spectra were measured as a function of temperature on a Hitachi F-3000 spectrofluorometer equipped with a four-cuvette holder accessory and a thermostated circulating water bath. The excitation wavelength was set to 295 nm with a 5 nm band pass. Fluorescence emission was detected over a range of 320-380 nm with a 3 nm band pass at right angles to the incident beam after passing through a 10-mm quartz cuvette. Sample temperature was measured by a thermometer present in one of the four cuvettes.

Electron Microscopy

A small drop of -crystallin in phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9) was applied onto a copper grid coated with collodion and carbon. After 5-10 min, excess liquid was removed using filter paper, and the sample was fixated by incubation with 1% formaldehyde for 5 min. The grid was extensively washed with 0.1% ammonium acetate and negatively stained with 1% uranyl acetate for 5 min. Dried grids were examined in a Jeol 1210 electron microscope at an acceleration voltage of 80 kV.

Heat Protection Assay

Various concentrations of recombinant A- and A-crystallin were preincubated in a 4 10-mm quartz cuvette for 3 min at 58 °C. A constant amount of bovine -crystallin (234 µg in 150 µl) was added, and scattering was monitored at 360 nm as a function of time using a Perkin-Elmer Lambda 2 UV/VIS spectrophotometer equipped with a thermostated circulating water bath and a thermocouple to register the sample temperature. Total volume was 1.0 ml, and all solutions were in degassed phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9). After 30 min of analysis, the content of the cuvette was centrifuged at 15,000 g for 30 min, resulting in a soluble and an insoluble fraction. Proteins in the soluble fraction were precipitated using 72% trichloroacetic acid. Protein precipitates obtained from both fractions were dissolved in an equal volume of SDS-sample buffer and analyzed by SDS-PAGE, immunoblotting, and densitometry. The reasons for us to use a heterogeneous substrate-like -crystallin were first of all the natural occurrence of this protein complex in lens, and secondly, suppressing the aggregation of -crystallin required relatively small amounts of highly purified recombinant -crystallins. The reproducibility of our results was checked by performing the assay three times with different batches of purified A- and A-crystallin.

Saturation Assay

Samples containing 50 µg of A- or A-crystallin in 425 µl of phosphate buffer (0.1 M NaSO, 20 mM NaP, pH 6.9) were preincubated for 3 min at 58 °C. Various concentrations of -crystallin (40-200 µg in 75 µl of phosphate buffer) were added, and the mixtures were incubated for 30 min at 58 °C. Subsequently, the mixtures were centrifuged for 30 min at 15,000 g resulting in a soluble and an insoluble fraction. Proteins in the soluble fraction were precipitated using 72% trichloroacetic acid. Protein precipitates obtained from both fractions were dissolved in an equal volume of SDS-sample buffer and analyzed by SDS-PAGE, Western blotting, and densitometry. The experiments were performed several times to check reproducibility.

Miscellaneous Methods

Bovine -crystallin was isolated from the water-soluble fraction of calf lens cortex by gel permeation chromatography over Ultrogel AcA-34 (Pharmacia-LKB) (40) . Bovine B2-crystallin was isolated from the -fraction following the method described by Horwitz et al.(41) . Native rat -crystallin was isolated from the water-soluble fraction of rat lens using a Superose 6 HR 10/30 column (see ``Gel Permeation Analysis''). Peptide mapping of tryptic digestions was performed on a C-18 reverse phase column (Merck). Amino acid compositions were determined on an LKB Alpha plus amino acid analyzer. Isoelectric focusing was performed as described by van den Oetelaar et al.(42) . SDS-PAGE was performed according to standard procedures (43) . Protein concentrations were determined in triplicate (44) . Immunoblot analysis was performed using a monoclonal antiserum raised against rat A-crystallin (34) at a 1:50,000 dilution. Positive bands were visualized using an alkaline phosphatase-conjugated second antibody according to the manufacturer's instructions (Promega). Scanning of A- and A-crystallin on immunoblot was performed using a Bio-Rad GS-670 imaging densitometer.


RESULTS

Cloning, Expression, and Isolation of A- and A-Subunits

A rat lens cDNA library in gt11 was screened for A- and A-crystallin using as probes two DNA fragments isolated from a hamster A gene construct (29) . The first probe contained exon 1, encoding residues 1-63 of hamster A crystallin, and the second one contained the optional exon 2, encoding the 23 amino acids of the insert peptide. Screening with these probes allowed discrimination between A and A cDNAs: clones hybridizing only to the exon 1 probe contained A cDNA, whereas clones hybridizing to both probes contained A cDNA. The ratio of identified A to A clones was about 5-10%, apparently reflecting the proportion of both types of mature mRNA present in the rat lens. To confirm that the isolated cDNAs were full length, we have sequenced their 5`-coding region corresponding to exon 1 and exon 2. The obtained nucleotide sequences showed 96% identity to the hamster sequence (29) and were in complete agreement with the rat protein sequence (32) .

The isolated cDNAs were cloned into the pET8c vector, resulting in pET8cA and pET8cA expression constructs. As can be seen from SDS-PAGE, induction of these constructs in E. coli B BL21(DE3)pLysS host cells gave expression of polypeptides up to 50% of total bacterial protein (Fig. 1A, lanes 3 and 6). The identity of the induced proteins was confirmed by Western blotting with a monoclonal anti-rat A antiserum (34) (Fig. 1B). The bacterial lysate was divided into a water-soluble fraction and a urea-soluble fraction. SDS-PAGE showed A to be predominantly present in the water-soluble fraction, whereas A only appeared in the urea-soluble fraction, indicating a different behavior of solubility or aggregation in the E. coli host cell (data not shown). The expressed proteins were purified by denaturing anion-exchange chromatography, resulting in 95-98% pure A and A as estimated by SDS-PAGE (Fig. 1A, lanes 4 and 7). The primary structure of these purified products was checked by amino acid analysis, isoelectric focusing, and peptide mapping (data not shown). To avoid isolation and purification artifacts, both products were refolded in urea under identical conditions before further comparison.


Figure 1: Bacterial expression and purification of A and A-crystallin. A, Coomassie Brilliant Blue-stained protein pattern after SDS-PAGE. Molecular mass markers (M in kDa) are indicated. B, corresponding Western blot probed with a monoclonal antiserum raised against rat A-crystallin. Samples are: native rat -crystallin (lane 1, 5.2 µg in A and 1 µg in B), cell lysate of E. coli expressing A before (lane 2) and after induction (lane 3), purified recombinant A (lane 4, 4 µg in A and 0.8 µg in B), cell lysate of E. coli expressing A before (lane 5) and after induction (lane 6), purified recombinant A (lane 7, 4 µg in A and 0.8 µg in B).



Multimerization of A and Asubunits

The refolded A and A subunits were analyzed by gel permeation chromatography in order to check whether they are able to form native-like multimeric structures. As can be seen from Fig. 2, A and B, the elution times for A and A homomultimers, kept at 20 °C, appear to be of the same order of magnitude as for native -crystallin isolated from rat lens. However, A displays a clearly smaller elution volume than A, revealing its higher molecular mass. Estimated molecular masses of the various multimers were obtained on basis of calibration with high molecular mass protein standards (). Although we are aware of the limitations of this calibration method, it seems that the estimated difference between the masses of A and A cannot solely be attributed to the mass of the insert peptide. Therefore, it may be possible that the A- and A-multimers have either a dissimilar number of subunits or a different hydrodynamic volume (45) .


Figure 2: The multimeric size of A and A with and without prior heating. Amounts of 100 µg A and A in 1 ml of phosphate buffer pH 6.9 were heated for 30 min at 58 °C and compared with room temperature controls on a Superose 6 gel permeation column. A, elution profiles of heated and unheated A compared with native -crystallin isolated from rat lens. B, elution profiles of heated and unheated A. Calibration was performed using the following high molecular mass standards: blue dextran (2000 kDa, 9.1 ml), thyroglobulin (669 kDa, 12.8 ml), ferritin (400 kDa, 15.1 ml), and catalase (232 kDa, 16.3 ml).



Thermostability of A and AHomomultimers

From a structural point of view it is interesting to know whether the insert peptide of A may interfere with its heat stability. In order to investigate the thermal properties of A and A we used gel permeation analysis and tryptophan fluorescence measurements.

To see whether the quaternary structure of A and A undergoes irreversible alteration at high temperatures, we incubated both proteins for 30 min at 58 °C and analyzed their multimeric size on an HPLC gel permeation column. As can be seen from Fig. 2 and , exposure to heat nearly doubles the size of A as well as A compared with room temperature controls. However, in spite of this increase, it should be emphasized that both A and A remain soluble. Some other studies have revealed the same behavior for native -crystallin (46, 47) , but there are also conditions known which preserve the complex size upon heating (48).

Tryptophan fluorescence spectroscopy can be used to study the conformational stability of tertiary protein structures. The insert peptide of A contains a tryptophan (position 69) in addition to the single one present in the N-terminal domain of A (position 9) (32) . Fig. 3shows the fluorescence emission maximum () of A-, A-, and B2-crystallin (positive control) as a function of temperature. The for A and A remains essentially unchanged, whereas for B2 a red shift from 333 to 348 nm is observed at about 55 °C. The sigmoidal transition found for B2 agrees with previous results (49) and indicates a conformational change of the tertiary structure. In general, the for tryptophan residues can vary from about 330 nm (completely buried) to about 353 nm (completely exposed) (50) . Therefore, the emission of A and A at 337 nm suggests all tryptophan residues of these subunits to be partially buried. Importantly, the relative constancy of this emission upon heating, which was found earlier for native -crystallin (49) , reveals that at least the N-terminal domains of A and A, and the insertion of A, retain their local conformation at high temperatures.


Figure 3: Tryptophan emission maximum of A, A, and B2 as a function of temperature. Proteins were dissolved in phosphate buffer pH 6.9 to a concentration of 100 µg/ml (see ``Experimental Procedures''). The excitation wavelength was set to 295 nm, and fluorescence emission spectra were recorded at increasing temperatures over a range of 320-380 nm.



Electron Microscopy

Electron microscopical analysis after negative staining (Fig. 4) reveals that recombinant A and A as well as freshly purified native rat -crystallin form similar more or less globular particles. Although these kind of spherical structures have been observed for many years (2, 51, 52), some recent reports indicate that -crystallin can also display a torus-like morphology (5, 53) . The reason for this variation is unclear. The A-particles have a larger diameter than the A-particles (). The A/A spherical volume ratio, calculated from the measured diameters, is in agreement with the molecular mass ratio as determined by gel permeation chromatography (). Heating A and A at 58 °C does not result in formation of differently shaped particles (data not shown). However, the electron micrographs of these heated samples do show a clear increase of the particle diameters corresponding to our gel permeation data.


Figure 4: Electron micrographs of A, A, and native rat -crystallin. Samples of A (A), A (B), and freshly purified native -crystallin (C) were negatively stained and shown at a magnification of 200,000. The bars are 50 nm.



Chaperone Activity of A and AHomomultimers

Using an in vitro assay, it has been shown that -crystallin from lens as well as reconstituted A and B homomultimers can prevent thermal aggregation of other proteins (11) . To determine whether the insert peptide of A influences this chaperone-like activity, we performed heat protection experiments with bovine -crystallin as a substrate. Fig. 5, A and B, show how the typical aggregation of at 58 °C (curve 1) is influenced by the presence of increasing amounts of A and A, respectively (curves 2-6). It is immediately clear that A displays a greatly reduced and altered chaperoning behavior. As can be seen from Fig. 5A, aggregation of can be prevented almost completely when the A to mass ratio is increased to 86%. In contrast, Fig. 5B shows that an A to mass ratio of up to 94% indeed delays aggregation, but eventually even leads to an increase of the final level of aggregation.


Figure 5: Ability of increasing amounts of A and A to prevent heat-induced aggregation of -crystallin. A, aggregation curves of 234 µg of in the presence of increasing amounts of recombinant A-crystallin: curve 1, none; curve 2, 22 µg (A to mass ratio = 9.6%); curve 3, 67 µg (29%); curve 4, 90 µg (38%); curve 5, 135 µg (58%); curve 6, 202 µg (86%). Curve 7 shows the behavior of 202 µg of A without . B, aggregation curves of 234 µg of in the presence of increasing amounts of recombinant A-crystallin: curve 1, none; curve 2, 25 µg (A to mass ratio = 10%); curve 3, 74 µg (31%); curve 4, 98 µg (42%); curve 5, 147 µg (63%); curve 6, 221 µg (94%). Curve 7 shows the behavior of 221 µg A without . All protein concentrations were determined in triplicate using the Bradford assay. The proteins were dissolved in a total volume of 1 ml of phosphate buffer, pH 6.9 (see ``Experimental Procedures'') and the incubation temperature was 58 ± 1 °C. The dashed lines represent the average of an irregular pattern caused by large insoluble aggregates disturbing the light path of the incident beam.



To determine the nature of the obtained aggregates, the heat protection samples were centrifuged at the end of the experiment and soluble and insoluble fractions were analyzed by SDS-PAGE (Fig. 6, A and C). As can be seen from Fig. 6C, the A band coincides with the lower -crystallin bands. Therefore, we have quantified relative amounts of A and A using Western blotting followed by densitometry (Fig. 6, B and D). Fig. 6B shows A to shift from the insoluble to the soluble fraction when its concentration is increased, whereas Fig. 6D shows A to remain in the insoluble fraction. This different behavior must be the result of interaction with -crystallin because neither A nor A precipitate significantly when incubated at 58 °C without a denaturing substrate (Fig. 5, curve 7). Therefore, the data presented in Fig. 5and Fig. 6indicate that A has a much lower capacity than A to remain soluble when binding unfolding -chains. To confirm this observation, we heated A and A with increasing amounts of -crystallin and tried to make a rough estimate at which -concentration A and A become insoluble using SDS-PAGE, Western blotting, and densitometry (Fig. 7). As can be seen from Fig. 7, B and D, A precipitates at a much lower molecular mass ratio than A. Taking into account the difference in molecular mass between A and A, we estimate that A has a three to four times lower substrate binding capacity than A.


Figure 6: Insolubilization of A and A during the heat protection assay as a function of the A to mass ratio. The heat protection samples of which the aggregation profiles are shown in Fig. 5 were centrifuged and soluble and insoluble fractions were subjected to SDS-PAGE and densitometric Western blot analysis. Left: Coomassie Brilliant Blue-stained SDS-gels of incubated with A (A) and A (C). The numbers refer to A to mass ratios. Native -crystallin () and heat-exposed A (A) are shown as references. Right, volume analysis of corresponding immunoblots to quantify relative amounts of A (B) and A (D) present in the soluble and insoluble fraction.




Figure 7: Insolubilization of A and A with increasing amounts of -crystallin. Amounts of 50 µg of A and A in 500 µl of phosphate buffer, pH 6.9, were incubated with increasing amounts of for 30 min at 58 °C. The reaction mixtures were centrifuged and soluble, and insoluble fractions were subjected to SDS-PAGE and densitometric Western blot analysis. Left: Coomassie Brilliant Blue-stained SDS-gels of A (A) and A (C) incubated with . The numbers refer to the to A mass ratios. Native -crystallin () is shown as a reference. Please note that A in C, bottom row, coincides with the lower -crystallin bands in the soluble fraction. Right: volume analysis of corresponding immunoblots to quantify relative amounts of A (B) and A (D) present in the soluble and insoluble fraction.




DISCUSSION

Our data demonstrate that the presence of 23 inserted residues in A-crystallin does not notably influence its multimerization, heat stability, and particle morphology. The A complexes are larger than those of A and appear to be somewhat more disperse. Furthermore, the size of both the A and A complexes increases approximately 2-fold at 58 °C.

However, the insert peptide of A has a major impact on its chaperoning behavior. From the combined data presented in Figs. 5-7, we can deduce the following sequence of events for the interaction of normal A with during thermal denaturation. We assume that denaturing of the subunits of -crystallin is a stochastic process, so they do not unfold all at once. The first unfolding -chains will noncovalently bind to an A-particle forming an unsaturated A- complex. The formation of this soluble complex prevents the unfolded -chains from aggregation between themselves, explaining the delay of aggregation shown in Fig. 5A (curves 2-6): the more A-particles are present the more soluble A- complexes can be formed and the longer aggregation of is delayed. However, at a certain moment the A-particles become saturated with unfolding -chains, and further interaction with more unfolding -chains leads to insolubilization. At this stage, also precipitation of -chains will occur, because there are no unsaturated A- complexes left to prevent their aggregation. Therefore, oversaturated A- complexes as well as insoluble -aggregates are probably responsible for the aggregation profiles shown in Fig. 5A. When the A to mass ratio is increased to 86% (Fig. 5A, curve 6), the level of oversaturation is not reached, which makes A to remain almost totally in the soluble fraction (Fig. 6B). At this ratio there is apparently an excess of A-particles for which the amount of unfolding -chains is insufficient to form insoluble oversaturated particles. The above postulated mechanism of oversaturation for our recombinant A, largely corresponds with that recently proposed for bovine -crystallin by Wang and Spector (48) .

Unlike for A, increase of the A to mass ratio enlarges the final level of aggregation (Fig. 5B) and does not promote a shift of A to the soluble fraction (Fig. 6D). Apparently, an excess of A, necessary to prevent aggregation, is not reached under our conditions. On the contrary, addition of A contributes to the formation of insoluble particles, because even high concentrations of A precipitate as a result of oversaturation. Therefore, it seems that A has a lower capacity to bind unfolding -chains than A. Titration of A and A with increasing amounts of -crystallin (Fig. 7) indeed confirms this conclusion because A precipitates at a significantly lower -concentration than A.

Our results support the idea that -crystallin complexes have a more or less defined number of binding sites for denaturing proteins. As long as these sites are available, the complex can prevent aggregation by acting as a scavenger for hydrophobic surfaces presented by unfolding proteins. Applying these concepts to native-like A-multimers, it appears that the increased complex size of A, with a concomitant relative decrease of accessible surface area, may already account to some extent for its reduced substrate binding capacity. In addition it can be envisaged that the insert peptides interfere with the accessibility of at least some of the potential binding sites. Such an intervention might be caused by steric hindrance or by an actual interaction of hydrophobic insert residues with the binding sites. Alternatively, the insert peptides might induce a subtle conformational change, which is unfavorable for the substrate binding capacity but not for multimerization and stability.

Alternative splicing allows formation of various related protein isoforms from a single gene (for a review see (54) ). From an evolutionary point of view, alternative splicing might be an important mechanism to generate possibly advantageous variants of essential gene products in the presence of the normally functioning original gene products. It has been shown that A is present in various unrelated mammalian groups, suggesting that it has independently disappeared in most mammalian orders after radiation from a common A-expressing ancestor (30, 34) . The question then is why A has been silenced again in most groups whereas in others, like the rodents, it has been retained for at least 70 million years. It appears that A, in spite of its insertion, still is a viable structural lens protein because it is very thermostable and it can form native-like multimers. Furthermore, it should be realized that A is only a minor component (less than 20%) of native -crystallin complexes in all investigated species where it occurs. This might be expected to result in a proportionally small decrease in the chaperone-like capacity of the complex. We observed indeed that heteromultimeric complexes, reconstituted from equal amounts of A- and A-subunits, display an intermediate chaperoning behavior as compared with the respective homomultimers (data not shown). The fact that A has not become the sole or major A-like subunit in any species may suggest that high levels of expression of A are indeed selectively disadvantageous. However, as long as the proportion of A is small, there is apparently a sufficiently great excess of chaperoning capacity in the lens so that a small decrease can be tolerated. Low levels of expression of A are thus likely to be a selectively neutral character, which may disappear without adverse effects by fortuitous mutations, like in human (55) , but can also be maintained for tens of millions of years, as has been demonstrated for other redundant gene products (56) .

Although the chaperone-like activity of -crystallin and small hsps currently is a major issue in the field of protein folding and interaction (57) , insight into the mode of interaction with denaturing substrate proteins, is still very limited. This is a direct consequence of our poor knowledge of the arrangement of subunits in the multimeric complex of these proteins, making it difficult to design straightforward ``interaction experiments.'' Therefore, studying the structural localization and functional influence of the large insertion in the unique A mutant might provide some general clues about the chaperone-like mechanism displayed by all members of the small heat shock protein and -crystallin family.

  
Table: Size parameters of A and A with and without prior heating as determined by gel permeation chromatography and electron microscopy

Determinations were made with and without prior heating of the proteins at 58 °C. Molecular mass determinations were performed at least in duplicate using high molecular mass standards as reference (see Fig. 2). Diameters were determined by measuring at least 25 particles (see Fig. 4) using the PC Image software package. Volume calculations are based on the assumption that the particles are perfectly spherical.



FOOTNOTES

*
This investigation was carried out under the auspices of the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO) and National Institutes of Health Grant EY09683. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Tel.: 31-80-616848/614254; Fax: 31-80-540525.

The abbreviations used are: HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. N. H. Lubsen for providing the rat lens cDNA library. We also would like to thank Dr. M. A. M. van Boekel and P. S. G. Overkamp for advice and technical assistance.


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