Keith Wyatt

γN‐crystallin and the evolution of the βγ‐crystallin superfamily in vertebrates

The β and γ crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates.

The β and γ crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.

Solution structure of γS‐crystallin by molecular fragment replacement NMR

The solution structure of murine γS‐crystallin (γS) has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of dipolar couplings, recorded in different alignment media, and supplemented by a small number of NOE distance restraints. γS consists of two topologically similar domains, arranged with an approximate twofold symmetry, and each domain shows close structural homology to closely related (∼50% sequence identity) domains found in other members of the γ‐crystallin family. Each domain consists of two four‐strand “Greek key” β‐sheets. Although the domains are tightly anchored to one another by the hydrophobic surfaces of the two inner Greek key motifs, the N‐arm, the interdomain linker and several turn regions show unexpected flexibility and disorder in solution. This may contribute entropic stabilization to the protein in solution, but may also indicate nucleation sites for unfolding or other structural transitions. The method used for solving the γS structure relies on the recently introduced molecular fragment replacement method, which capitalizes on the large database of protein structures previously solved by X‐ray crystallography and NMR.

Gene duplication and separation of functions in αB-crystallin from zebrafish (Danio rerio)

We previously reported that zebrafish αB‐crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone‐like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone‐like activity of a second zebrafish αB‐crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel α‐crystallin transcript designated cryab2 and the resulting protein αB2‐crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human αB‐crystallin and zebrafish αB1‐crystallin, respectively. RT‐PCR showed that αB2‐crystallin is expressed predominantly in lens but, reminiscent of mammalian αB‐crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone‐like activity of purified recombinant αB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of α‐lactalbumin and lysozyme. At 25 °C and 30 °C, zebrafish αB2 showed greater chaperone‐like activity than human αB‐crystallin, and at 35 °C and 40 °C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that αB2‐crystallin makes up ≈ 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different αB‐crystallins. Differences in primary structure, expression and chaperone‐like activity suggest that the two zebrafish αB‐crystallins perform divergent physiological roles. After gene duplication, zebrafish αB2 maintained the widespread protective role also found in mammalian αB‐crystallin, while zebrafish αB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure–function relationships and the regulation of tissue‐specific expression patterns.

A Single Destabilizing Mutation (F9S) Promotes Concerted Unfolding of an Entire Globular Domain in γS-Crystallin

Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. gammaS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse gammaS-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected beta-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at approximately 1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils.

A Role for Lengsin, a Recruited Enzyme, in Terminal Differentiation in the Vertebrate Lens

Lengsin is an eye lens-specific member of the glutamine synthetase (GS) superfamily. Lengsin has no GS activity, suggesting that it has a structural rather than catalytic role in lens. In situ hybridization and immunofluorescence showed that lengsin is expressed in terminally differentiating secondary lens fiber cells. Yeast two-hybrid (Y2H) and recombinant protein experiments showed that full-length lengsin can bind the 2B filament region of vimentin. In affinity chromatography, lengsin also bound the equivalent region of CP49 (BFSP2; phakinin), a related intermediate filament protein specific to the lens. Both the vimentin and CP49 2B fragments bound lengsin in surface plasmon resonance spectroscopy with fast association and slow dissociation kinetics. Lengsin expression correlates with a transition zone in maturing lens fiber cells in which cytoskeleton is reorganized. Lengsin and lens intermediate filament proteins co-localize at the plasma membrane in maturing fiber cells. This suggests that lengsin may act as a component of the cytoskeleton itself or as a chaperone for the reorganization of intermediate filament proteins during terminal differentiation in the lens.

γN-crystallin and the evolution of the βγ-crystallin superfamily in vertebrates: γN-crystallin

The β and γ crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of γ‐crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of γN‐crystallin, an evolutionary bridge between the β and γ families. In all species examined, γN‐crystallins have a hybrid gene structure, half β and half γ, and thus appear to be the ‘missing link’ between the β and γ crystallin lineages. Overall, there are four major classes of γ‐crystallin: the terrestrial group (including mammalian γA–F); the aquatic group (the fish γM‐crystallins); the γS group; and the novel γN group. Like the evolutionarily ancient β‐crystallins (but unlike the terrestrial γA–F and aquatic γM groups), both the γS and γN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, γN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the γ‐crystallins, also in the retina. Although well conserved throughout vertebrate evolution, γN in primates has apparently undergone major changes and possible loss of functional expression.