The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins

Abstract

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and βγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the βγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens βγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in βB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even β-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Keywords: cataract; crystallin; cysteine; disulfide; eye lens; methionine; protein aggregation; protein evolution; protein misfolding; refractive index.

Figure 1

Protein sequence similarity among βγ- and γ-crystallins. Crystallin sequences from chordates (tunicate, lancelet, hagfish) cluster with that of the sea lamprey, a representative of the most basal vertebrates (Cluster 1). The next major group of crystallins to split (Cluster 2) include γS-crystallins from many organisms. From there, two more large groups of γ-crystallins split off. Group 3 includes mammalian γD-crystallins (kangaroo, human, cow, mouse). as well as other γ-crystallins found in lobe-finned fish (coelacanth), chondrichthyans (bamboo shark), bony fish (zebrafish), crocodilians (alligator), and amphibians (clawed frog). Finally, group 4 comprises fish-specific γM-crystallins from the zebrafish and bamboo shark.

Figure 2

Phylogenetic tree for βγ- and γ-crystallins. Nucleic acid sequences coding for crystallin proteins from the tunicate and lancelet are highly similar. The labels are color coded according to the protein sequence clusters described in Figure 1.

Figure 3

Experimentally determined structures of representative βγ- and γ-crystallins. The double Greek key fold is common to βγ-crystallins. (A) Tunicate (Ciona intestinalis) βγ-crystallin (PDB ID: 2BV2) [132]. (B) Human γS-crystallin (PDB ID: 2M3T) [58]. (C) Human γD-crystallin (PDB ID: 2KLJ) [133]. (D) Zebrafish γM7-crystallin (PDB ID: 2M3C) [134]. The individual Greek key motifs are colored in pink, green, yellow, and blue from N- to C-terminus.

Figure 4

Cysteine in representative γ-crystallins. (A–C) Human γS-crystallin (PDB ID: 2M3T) [58]. Inset of the cysteine loop, formed by C23, C25, and C27 along with surrounding residues. (D–F) Human γD-crystallin (PDB ID: 2KLJ) [133]. (G–I) Zebrafish γM7-crystallin (PDB ID: 2M3C) [134]. Note that we use the UniProt convention for numbering γ-crystallin residues in this paper, counting Met1 as the first residue even though it is absent from the mature form of the protein. In other papers, we and others have used the PDB numbering scheme of Basak et al. [137] or the traditional scheme based on alignment to human γB-crystallin.

Figure 5

Methionine in representative γ-crystallins. (A) Human γS-crystallin (PDB ID: 2M3T) [58]. (B) Human γD-crystallin (PDB ID: 2KLJ) [133]. (C) Zebrafish γM7-crystallin (PDB ID: 2M3C) [134].

Figure 6

Combined solvent-accessible surface area (SASA), in square Angstroms, of Cys residues by domain in solution NMR structures or D-I-TASSER predicted structures of representative γ-crystallins. Protein nomenclature follows UniProt. Orange = N-terminal cysteines; blue = C-terminal cysteines. Red dashed lines indicate the mean SASA for a control buried Cys that is conserved in almost all of the γ-crystallins, e.g., C83 in human γS (see Figure 4C). Black dashed lines indicate the mean + standard deviation for the control buried SASA.