Taxon-specific ζ-Crystallin in Japanese Tree Frog (Hyla japonica) Lens

Abstract

The present study demonstrated that the 38-kDa protein, instead of ρ-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from ρ-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was ζ-crystallin. Analysis of cDNA sequence encoding ζ-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with ζ-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. ζ-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC 1.6.99.2, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 μM) using NADPH (Km, 60 μM) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 μM. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as ζ- and ρ-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.