Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.

Crocodilian tau-crystallin: overexpression, purification, and characterization.

tau-Crystallin is a taxon-restricted crystallin found in eye lenses of reptiles and a few avian species but presumably absent in mammals. The level of tau-crystallin in the lens varies among different species. In the crocodile lens, it is the least abundant crystallin and is present in trace amounts. We present a method for cloning, overexpression, and purification of crocodilian tau-crystallin utilizing a combination of gel filtration and ion-exchange chromatography yielding an extremely purified protein. The protein gets profusely expressed resulting in a fairly high yield and exists as a monomeric entity of 47.5 kDa molecular mass. The recombinant tau-crystallin exists in a properly folded native state as probed by circular dichroism and fluorescence spectroscopy and exhibits enolase activity.