γ-Crystallins of the chicken lens: remnants of an ancient vertebrate gene family in birds

Abstract

γ-Crystallins, abundant proteins of vertebrate lenses, were thought to be absent from birds. However, bird genomes contain well-conserved genes for γS- and γN-crystallins. Although expressed sequence tag analysis of chicken eye found no transcripts for these genes, RT-PCR detected spliced transcripts for both genes in chicken lens, with lower levels in cornea and retina/retinal pigment epithelium. The level of mRNA for γS in chicken lens was relatively very low even though the chicken crygs gene promoter had lens-preferred activity similar to that of mouse. Chicken γS was detected by a peptide antibody in lens, but not in other ocular tissues. Low levels of γS and γN proteins were detected in chicken lens by shotgun mass spectroscopy. Water-soluble and water-insoluble lens fractions were analyzed and 1934 proteins (< 1% false discovery rate) were detected, increasing the known chicken lens proteome 30-fold. Although chicken γS is well conserved in protein sequence, it has one notable difference in leucine 16, replacing a surface glutamine conserved in other γ-crystallins, possibly affecting solubility. However, L16 and engineered Q16 versions were both highly soluble and had indistinguishable circular dichroism, tryptophan fluorescence and heat stability (melting temperature Tm ~ 65 °C) profiles. L16 has been present in birds for over 100 million years and may have been adopted for a specific protein interaction in the bird lens. However, evolution has clearly reduced or eliminated expression of ancestral γ-crystallins in bird lenses. The conservation of genes for γS- and γN-crystallins suggests they may have been preserved for reasons unrelated to the bulk properties of the lens.

Keywords: crystallin; evolution; eye; promoter; protein folding; proteomics.

FIGURE 1. Conservation of gene structure and linkage

A: Exon/intron junctions of chicken (above) and mouse (below) genes for CRYGS and CRYGN. B: Comparison of the loci for CRYGS and CRYGN in mouse and chicken genomes. Approximate spacing of genes is indicated. Locations of putative orthologs of WDR86and RHEB in chicken were estimated from spliced EST positions in genome build galGal3.

FIGURE 2. Spliced transcripts and γS proteins in eye tissues;

A). RT-PCR of dissected eye tissues detected spliced transcripts retina/rpe (R), cornea (C), lens (L), no RT blank. B). Predicted amino acid sequences of chicken γS and γN aligned with mammalian orthologs. C). Western blot of chicken eye tissues with peptide antibody to chicken γS. M: markers; L: lens; R: retina; C: cornea; E: eyecup (rest of eye); recombinant chicken γS protein.

FIGURE 3. Conservation of crygs promoter sequence and function

A) Alignment of promoters for chicken crygs and mouse crygs. +1 indicates the position chosen as the likely transcription start site and beginning of the 5′UTR. Conserved bases are highlighted and the positions of canonical maf-response elements (MARE), Sox family binding sites (Sox) and TATA boxes are indicated. B) Promoter activity of chicken γS and mouse γS in NIH 3T3 and αTN4-1 cells. The activity was detected by Firefly signal and normalized by renilla signal in a dual luciferase assay. C1: chicken γS promoter without 5′-UTR, C2: chicken γS promoter with 5′-UTR, M1: mouse γS promoter without 5′ -UTR, M2: mouse γS promoter with 5′-UTR.

FIGURE 4. Relative weight abundances of crystallins in 10 week-old chicken lenses

The summed fragment ion intensities from all MS/MS spectra assigned to each crystallin in large-scale shotgun proteomics for water soluble (white bars) and insoluble (black bars) fractions of chicken lens were used to estimate the relative percent weight abundances of each crystallin. High and low abundance crystallins are shown in separate panels with different scales.

FIGURE 5. Sequence and evolution of avian γS-crystallins

A). Homology model of chicken γS. Model was produced using Rosetta [49] and visualized using Chimera [50]. Main chain tracing of the homology model of chicken γS based on the coordinates of the mouse ortholog. Positions of some non-conserved residues that might have effects on solubility, folding or stability are shown. B). Alignment of γS sequences for birds and selected species predicted from genome sequences. Aligned using Clustal as implemented in MEGA [51]. B). C) Evolutionary tree for γS protein sequences. Q16 is present in reptiles and mammals. L16 is present in birds. Tree produced using neighbor joining in MEGA.

FIGURE 6. Structure and stability of recombinant chicken γS proteins

A) Far-UV CD spectra of mouse, chicken L16 and chicken Q16 γS have very similar spectra and predicted contents of secondary structure. B) Thermal stability of secondary structure in in chicken L16 and chicken Q16 γS measured by magnitude of 220nm minimum characteristic of β-sheet. C) Trp fluorescence of chicken L16 and chicken Q16 γS in folded and chemically denatured (7M GdnHCl) states. Both show similar profiles, indicating quenching in the folded state and a shift in the maximum emission upon unfolding.