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.
Highlights
Regeneration of the axotomized optic nerve in cold blooded vertebrates has served as a model system for the characterization of cellular and molecular events underlying regeneration in the central nervous system (CNS)1 [1, 2]
Cloning of a Second Isoform of RICH, gRICH70 —The isolation of a cDNA encoding RICH, based on peptide sequences derived from purified p68/70 has been previously reported [28]
A goldfish genomic library was screened to obtain the sequences corresponding to the 5Ј-end of the new mRNA that were missing in the g-RICH-1 cDNA insert
Summary
Regeneration of the axotomized optic nerve in cold blooded vertebrates has served as a model system for the characterization of cellular and molecular events underlying regeneration in the central nervous system (CNS)1 [1, 2]. The retinal ganglion cells undergo morphological and biochemical changes (8 –10) and regrow their axons to reform connections within the tectum with a high degree of spatial specificity [11, 12], resulting in recovery of visual function [13]. P68/70 is a doublet of acidic proteins that is markedly induced in goldfish retinal ganglion cells and transported into the optic nerve during optic nerve regeneration [15]. The doublet was purified from goldfish tissues, and a polyclonal antibody was generated and used to demonstrate a marked increase of expression of p68/70 in the retinal ganglion cells during regeneration [26]. The RICH mRNA was found to be induced during regeneration as well [28]
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