S and N–Crystallin: Function and Evolution in the –Crystallin Family.

Abstract

Abstract: : Purpose: γ–Crystallins are major proteins of the lens and are also expressed in other parts of the eye. Here we describe functional studies on γS, a widespread and ancient member of the family, and the newly discovered γN. Methods: The mouse crygs gene was knocked out by homologous recombination. Opj cataract and crygs –/– mice were compared by microscopic and biochemical techniques. γN was identified by EST analyses and informatics. Expression was examined by 2D–gel/mass spectrometry, northern blot, PCR and western blot using specific peptide antibodies. Results: γS is expressed in lens epithelia, cornea and retina as well as lens fibers. In wt lens, γS is present in cell nuclei in the elongating fiber cells. Mutation of γS in the Opj cataract has multiple serious effects. In Opj/Opj γS is present in dense cytoplasmic plaques, often adjacent to nuclei and the actin cytoskeleton seems to be absent. In γS –/– mice there is no defined cataract but the optical properties of the lens are degraded. Cortical fiber cells are poorly organized and dense nuclear bodies persist deep into the lens. F–actin content is reduced and the cytoskeleton appears to be disorganized. γN is a novel member of the γ–crystallin family with some structural similarity to γS. Surprisingly the exon/intron structure of the γN gene resembles a hybrid of ß– and γ–crystallin genes. In mouse, γN is eye–specific, expressed in lens, retina and RPE. A probable homolog of γN was found in Iguana lens, showing that the protein is ancient and well conserved in evolution. However γN has undergone changes in primates. The stop codon in the human (and chimp) sequence has mutated and a larger sized protein is detectable in human lens. Variant human transcripts were also detected in RPE and testis. Conclusions: The evolutionary conservation of γS and γN suggest they may have ancient functions in the vertebrate eye. While the most severe effects in Opj derive from unfolded γS protein, loss of function in –/– mice indicates a role involving lens cytoskeleton and normal processing of nuclei. The hybrid gene structure of γN suggests it may illustrate an intermediate stage in the evolution of the ßγ superfamily.

Keywords: crystallins • gene/expression • protein structure/function