x-Crystallin/tx-Enolase: One Gene Encodes Both an Enzyme and a Lens Structural Protein

T-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey x-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck x-crystallin was obtained and sequenced, confirming the close relationship with ~t-enolase. Southern blot analysis showed that the duck genome contains a single ct-enolase gene, while Northern blot analysis showed that the message for x-crystallin/ct-enolase is present in embryonic duck lens at 25 times the abundance found in liver, x-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. x-Crystallin/ct-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene. T HE lenses of vertebrates consist of concentric layers of terminally differentiated fiber cells (8). The major macromolecular components of these cells, comprising most of their refractive structure are highly abundant, soluble proteins, the crystallins. These proteins exist at high concentration, exposed to light and other insults essentially without turnover often for many years while maintaining the optical properties of the lens. It might have been expected that these proteins would be highly specialized, adapted for the unusual lens environment. However, it has recently been discovered that some crystallins appear to be common enzymes expressed at remarkably high levels in the lens (3, 10, 29, 33, 34, 35). Some of these enzyme-crystallins may be recently acquired components of the lens, but one, T-crystatlin, is a major protein of vertebrate lenses in lampreys, some fish, reptiles, and birds (24, 31, 32), suggesting an ancient origin in vertebrate evolution. Previously, limited sequence data for purified turtle x-crystallin has suggested a close relationship with the glycolytic enzyme enolase (33). Here the nature of this relationship is elucidated, showing that x-crystallin and A portion of this work was presented by J. Piatigorsky at the First Hans BIoemendal Lecture in June, 1988 in Nijmegen, The Netherlands. Leah A. Williams' present address is Department of Biology, University of West Virginia, Morgantown, West Virginia 26506. a-enolase are the products of the same gene and that a wellcharacterized enzyme has another distinct function as a lens structural protein. Materials and Methods