Functional, biochemical and molecular analyses of the cold stable eye lens crystallins from the Antarctic toothfish Dissostichus mawsoni

The giant Nototheniid Antarctic toothfish (Dissostichus mawsoni), that inhabits the perennially freezing Southern Ocean seawater (-2 degrees C) has a large transparent eye lens. The low temperature stability of the toothfish lens is in contrast to those of temperate fishes and terrestrial mammals, both of which manifest a cold induced cataract when cooled. To investigate the basis of the toothfish lens cold stability, we first determined its lower limit of clarity as -12 degrees C, and verified that its lens was composed of the three most common vertebrate crystallin proteins (alpha, beta and gamma). Chromatographic separation revealed that the toothfish lens has a much higher proportion of gamma crystallins relative to bovine lens. In vitro functional chaperone-like protection assays of the toothfish alpha crystallin confirmed it as a small heat shock protein. However, in a cross-species chaperone-like assay using cow alpha and toothfish gamma, we found that there was virtually no protection of the toothfish gamma crystallin. Mammalian gamma crystallins have previously been identified as the principal agent for cold-cataracts, thus the lack of protection offered by cow alpha crystallin to toothfish gamma indicates the biochemical difference of the latter.; Isolation, cloning and sequencing of the cDNAs encoding for the toothfish crystallins revealed a total of 21 isoforms (2 alphas, 6 betas, and 13 gammas). Phylogenetic analyses suggest that toothfish alpha and beta crystallins are orthologues of mammalian counterparts, while gammas do not. Further separation, partial purification (ion-exchange chromatography) and analyses (isoelectric focussing and 2D SDS-PAGE) of the gamma crystallins has generated eleven sub-fractions, although they are not homogeneous for a single gamma isoform. Cross-species chaperone-like assays with these eleven toothfish gamma fractions show that those containing acidic gammas are protected by cow alpha crystallin, while those which are largely composed of basic isoforms cannot. Solubility of toothfish lens alpha, beta and gamma crystallins are similar to which has been previously reported for cow and humans. Thus, the long-lived, cold-adapted Antarctic toothfish lens is an attractive model system for further investigations into lens crystallin stability.