Here we describe the association of mouse βB1- and human βA3-crystallins. When present alone, each protein associates into homodimers but neither forms homotetramers, similar to results obtained with βB2- and βA3-crystallins. However, in contrast to the previous results obtained from βB2- and βA3-crystallins, mixed βB1- and βA3-crystallins show a dimer-tetramer equilibrium, with a Kd of 1.1 μM. Thus mixed βB1- and βA3-crystallins associate predominantly into heterotetramers in vitro.
Mouse βB1-crystallin was faithfully expressed with high yield in BL21(DE3)pLysS, which is the same system used previously for expressing human βB1-crystallin by Lampi et al.
). The identity of the expressed βB1-crystallin is supported by its correct molecular mass on SDS-PAGE and mass spectrometry. Human βA3-crystallin was previously expressed using the baculovirus system (6
). This study utilized the bacterial system BL21(DE3), which is a more convenient and equally productive method for the expression of relatively stable proteins, such as human βA3-crystallin. The identity of the expressed βA3-crystallin is confirmed by its molecular mass on SDS-PAGE and its strong reactivity with antibodies that specifically target the first Greek-key motif of bovine βA3-crystallin (data not shown). While mouse and human βB1-crystallins are only 80.9% identical (90% similar), mouse and human βA3-crystallins share a 95.3% identity (100% similarity), suggesting that the use of human and mouse crystallins provides a reasonably accurate model of crystallin behavior, especially in the mouse lens. Also, while the conserved PAPA sequence is more evident in mouse βB1-crystallin, human βB1-crystallin is predicted to share similar structural characteristics in this region by both Chou-Fasman and Garnier-Robson algorithms (data not shown).
Size-exclusion chromatography of mouse βB1-crystallin at 1 mg/mL shows a somewhat broad and asymmetrical single peak with an apparent molecular weight that is intermediate between the monomeric and dimeric forms, consistent with a monomer-dimer equilibrium (17
). An intermediate molecular weight is observed as monomers constantly associate and dissociate in a rapidly reversible manner as the protein is chromatographed (17
). The homodimer formation observed in this study agrees with other reports of βB1-crystallin (16
) and most other β-crystallins, including βA3- and B2-crystallins (6
). However, while size exclusion chromatography in these studies has suggested that βB1-crystallin might behave as a dimer as concentrations increase above 10 mg/ml (18
) or at 0.7 mg/ml (16
), light scattering suggests molecular masses consistent with a dimer with a small population in aggregates of 264 kDa (16
) or varying between 48 kDa and 125 kDa depending on the protein concentration (18
). While the nature of the higher aggregates is unclear from the data presented, they do not appear to represent the reversible high affinity association described here. Rather, the data suggest either a stable high molecular weight complex co-existing with a lower dimeric form in some cases and a gradual smooth increase in molecular mass in others.
The stability of βB1-crystallin over time is supported by its molecular weight, which remains relatively constant after 24 hours of incubation at room temperature. Similar results were obtained for βA3-crystallin, which also displays persistent monomer-dimer equilibrium. Despite having a smaller predicted molecular size, βA3-crystallin shows a higher apparent molecular weight on size exclusion chromatography than βB1-crystallin. Delayed elution of βB1-crystallin from size exclusion chromatography has been reported previously(16
) and has been attributed to interaction between βB1-crystallin and the column matrix, as is seen with amino-truncated βA3-crystallin,(8
) . However, it is also consistent with the higher affinity for self-dimerization of βA3-crystallin relative to βB1-crystallin. The increase of the apparent molecular weight with increasing βB1-crystallin concentration on size exclusion chromatography suggests that the delayed elution of βB1-crystallin is due to reversible monomer-dimer equilibrium rather than interactions with the column or unusual effects of an unusual shape of the molecule, or perhaps both might contribute to some degree.
Sedimentation equilibrium of mouse βB1-crystallin at 0.6 mg/mL and 20°C again confirms a monomer-dimer equilibrium. In contradistinction to some previous reports,(18
) no evidence for homotetramer formation was seen with analytical ultracentrifugation or size exclusion chromatography in these studies. The apparent average molecular weight estimated from sedimentation equilibrium at 20°C (47 kDa) is significantly higher than that estimated from size-exclusion chromatography (37 kDa). This discrepancy may be attributed to the fact that the association of crystallins is dependent on protein concentration, but also might reflect a tendency of βB1-crystallin to stick to the gel matrix in a size-exclusion column as discussed above. While the sedimentation study does not involve any elution buffer, size-exclusion chromatography subjects protein samples to dilution by the running buffer. As protein concentration drops, the monomer-dimer equilibrium is expected to shift towards monomer, thus resulting in a lower apparent molecular weight.
When mouse βB1-crystallin and human βA3-crystallin are incubated together at room temperature, heterocomplex formation is readily appreciable within 30 minutes by size-exclusion chromatography, native gel electrophoresis, and isoelectric focusing. In , the higher-molecular-weight species that increases in amount with incubation time represents a dimer-tetramer equilibrium, as it corresponds to an apparent molecular weight that is intermediate between that of a dimer (either homodimer or heterodimer) and that of a tetramer. A number of observations suggest that this peak represents tetramer rather than trimer formation. As is seen with βA3- and βB1-crystallins in isolation, the apparent molecular weight increases towards the predicted tetramer size with increasing protein concentration.
SDS-PAGE of the eluted fractions shows that this peak contains equal amounts of βB1- and βA3-crystallins (). This 1:1 ratio suggests that each tetramer is composed on average of two subunits of βB1-crystallin and two subunits of βA3-crystallin. Conversely, the lower-molecular-weight species corresponds to a monomer-dimer equilibrium that decreases in amount over time. SDS-PAGE of this peak shows an asymmetric distribution of βB1- andβA3-crystallins, suggesting lack of interaction at the beginning of the incubation, and perhaps a tendency of βB1-crystallin to stick to the column matrix. In theory, three types of dimers can be present: βB1-homodimers (56 kDa), βA3-homodimers (50 kDa), and βB1/βA3-heterodimers (53 kDa). It is likely that the proximity in molecular masses of these three species has exceeded the resolution capacity of the Superdex 75 column, resulting in a broad single peak. While the exact mechanism of heterotetramer formation is unknown, it seems likely that it is via the association of two βB1/βA3-heterodimers rather than association of a βB1-homodimer with a βA3-homodimer. Ultimately, one would expect the mixture to reach a steady state of heterodimer-heterotetramer equilibrium. These data are in agreement with those of Bateman et al.(12
). As expected, the final amount of heterotetramer formed is dependent on crystallin concentration. As protein concentration increases, the percentage of protein at the molecular masses representing both monomer-dimer and dimer-tetramer equilibria increase, and vice versa (). This indicates that higher concentrations shift the equilibria towards association.
Native gel electrophoresis, which separates proteins in their native states, provides additional information on the shapes of the complexes. Of interest, the migration of βB1-crystallin by itself is markedly retarded with some protein not entering the gel (). One possible explanation is that the N-terminal extension of βB1-crystallin, being the longest among all β-crystallins, severely hinders the migration of monomeric/dimeric βB1-crystallin through the polyacrylamide gel matrix. The intermediates formed over time, representing heterocomplex formation with βA3-crystallin, migrate further down the gel compared to βB1-crystallin alone. This suggests the interaction between βB1- and βA3-crystallins gives rise to a conformation that is more compact and thus penetrates the matrix more easily. One possibility is that the N-terminal arm of βB1-crystallin may be involved in holding the heterocomplex in place and thus becomes less flexible in the tetramer (19
), protruding less and allowing migration of the complex into the gel. Alternatively, the βB1-crystallin N-terminal arm might interact with the gel matrix less in heterotetramers, which would also allow the heterocomplex to migrate further than the βB1-crystallin dimer.
Heterocomplex formation is also confirmed by isoelectric focusing. As expected, the heterocomplex has a pI that is intermediate between that of βB1-crystallin and of βA3-crystallin (). While this technique does not distinguish between heterodimer and heterotetramer formation, the absence of homodimers or homotetramers, which would have identical isoelectric points, after longer incubation times suggests that most of the intermediate band is composed of heterotetramers. This can be seen in comparison to isoelectric focusing of mixed βB2- and βA3-crystallins, in which the heterodimer and two homodimers coexist in a 2-1-1 ratio (13
). That is, at equilibrium there is twice as much heterodimer as there is of each homodimer, suggesting that none of the three possible dimers is energetically favored over the other.
Finally, analytical ultracentrifugation provides thermodynamic characterization of the heterocomplex equilibrium. The experimental data fit well with the expected gradient of the heterodimer-heterotetramer equilibrium, with a Kd of 1.1 μM. The observed molecular weight (96 kDa) is consistent with the predicted molecular weight of the heterotetramer consisting of two βB1- and two βA3-crystallin subunits (106.2 kDa), but significantly higher than the apparent molecular weight estimated from size-exclusion chromatography (69.6-76.4 kDa). Again, this may be due to the dilution effect during chromatography or adherence of the βB1-crystallin protein to the size exclusion chromatography gel matrix or both.
The formation of heterotetramers by mixed βB1- and βA3-crystallin contrasts with the lack of tetramers seen under similar conditions with mixed βB2- and βA3-crystallin (7
). However, this is consistent with the high βB1-crystallin content observed in βH aggregates synthesized in vivo
). βB2-crystallin, on the other hand, is present in all size-classes and is the primary constituent of βL2-crystallin (20
), suggesting βB2-crystallin might have a low affinity for higher-order association with other β-crystallins. These observations might relate to the structural difference between βB2- and βB1-crystallins. The major factor that distinguishes βB1-crystallin from other β-crystallins, and especially basic β-crystallins, is its extremely long N-terminal extension containing 57 residues including a PAPA sequence (21
). We hypothesize thatβB1-crystallin may promote higher-order complex formation with other β-crystallins in the lens through the action of its long N-terminal extension. While it cannot, by itself, account for the formation of βH-crystallin oligomers, this does agree with the presence of βB1-crystallin preferentially in the βH complexes and truncated βB1-crystallins in βL1 and βL2 complexes previously described (9
It is important to realize that under physiologic conditions, where protein concentration exceeds 300 mg/mL, higher-order association between β-crystallins would be favored. Therefore, βB1-crystallin might have a crucial role in associating with other β-crystallins in higher order complexes. We have not been able to measure the dissociation constant of the putative βB1 βA3-crystallin heterodimer. However, if it has a similar dissociation constant to the βB1 homodimer the fraction of heterotetramer at 300 mg/ml would be greater than 99.99%, and this fraction would still approach 99.9% at 1 mg/ml. Thus, as has been previously suggested, the rapid interchange of crystallins might have more physiological implications than the existence of monomer or even dimer forms of the protein. This being said, the findings in this study have implications in cataractogenesis of the aging lens where N-terminal truncation of βB1-crystallin has been well documented (10
). In this regard, the size distribution of β-crystallins is dependent on the age of the lens, which is itself correlated with crystallin modifications including truncation of the terminal arms (10
). Finally, the lack of homotetramer formation in vitro
indicates that higher-order association between different β-crystallin subtypes is favored over self-association. This supports a previous suggestion that acidic β-crystallins may preferentially associate with basic β-crystallins (23
), although this may well result from heterotetramer/ heterodimer equilibrium with βB1- and βA3-crystallins as opposed to the heterodimer in which βB2- and βA3-crystallins associate into βB2 homodimers:βB2-βA3 heterodimers:βA3 homodimers with a roughly 1:2:1 ratio (13
In summary, this study has demonstrated for the first time reversible spontaneous in vitro formation of heterotetramers by β-crystallins. When present alone under these conditions, βB1- and βA3-crystallin associate into homodimers but neither forms homotetramers. Upon mixing under physiological conditions, heterocomplex formation between these two β-crystallin subtypes was observed by size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to the previous results obtained from βB2- and βA3-crystallins, which did not show tetramer formation, analytical centrifugation shows a dimer-tetramer equilibrium with a Kd of 1.1 μM, suggesting that βB1- and βA3-crystallins associate predominantly into heterotetramers in vitro. Although we suspect that heterotetramers are formed preferentially through the interaction of two heterodimers (βB1/βA3 + βB1/βA3) these studies are unable to discriminate this mechanism from that involving the interaction between two homodimers (βB1/βB1 + βA3/βA3) or mixtures of both hetero- and homodimers. Future studies will address this question, as well as elucidating the molecular mechanisms of βB1-crystallin association into both dimers and heterotetramers.