gRICH68 and gRICH70 are 2',3'-cyclic-nucleotide 3'-phosphodiesterases induced during goldfish optic nerve regeneration

Abstract

Biochemical characterization of changes in gene expression that accompany optic nerve regeneration has led to the identification of proteins that may play key roles in the regeneration process. In this report, a cDNA encoding gRICH70, a novel isoform of the regeneration-induced gRICH68 protein, has been identified and characterized in goldfish. Both gRICH68 and gRICH70 show significant homology (34-36%) to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases), hence the name goldfish regeneration-induced CNPase homolog (gRICH). The predicted 431-amino acid gRICH70 protein is 88% homologous to gRICH68, and the retinal mRNA for gRICH70 is coordinately induced with gRICH68 mRNA during optic nerve regeneration. Enzymatic analysis of recombinant proteins confirms that both gRICH proteins possess CNPase activity. Despite the relatively limited sequence homology, the kinetic constants obtained suggest that both gRICH proteins are at least as efficient as recombinant mouse CNP1 in catalyzing the hydrolysis of 2',3'-cAMP. Immunoprecipitation studies indicate that gRICH proteins are responsible for the majority of the CNPase activity detected in regenerating goldfish retinas. The evidence presented demonstrates that gRICH68 and gRICH70 correspond to a previously described doublet of acidic proteins that are selectively induced in the goldfish retina during optic nerve regeneration. Thus, CNPase enzyme activity is implicated for the first time in the process of nerve regeneration.