Cloning and characterization of goldfish and zebrafish RICH proteins.

Abstract

The mammalian central nervous system (CNS) shows a very limited capacity for axonal regeneration, a phenomenon with important clinical consequences for humans. The optic nerve of teleost fish is a vertebrate model system of successful axonal regeneration in the CNS. Biochemical studies of goldfish optic nerve regeneration have led to the identification of several proteins induced during the process of axonal growth. These proteins are of great interest since they provide insight into axonal plasticity mechanisms during regeneration and development. p68/70 is a doublet of acidic proteins highly induced in the retinal ganglion cell neurons during regeneration of the goldfish optic nerve. Using peptide sequence information derived from purified p68/70 proteins, cDNAs encoding both proteins have been cloned and characterized. The predicted proteins were highly homologous with each other, and also showed significant homology to the catalytic domain of mammalian CNPase $(2\sp\prime,3\sp\prime$-cyclic-nucleotide 3$\sp\prime$-phosphodiesterase), a marker enzyme of the myelin sheath. The function of CNPase is not known, although it has been proposed to play a role in membrane-cytoskeletal organization. Based on this information the proteins were designated gRICH68 and gRICH70 (goldfish Regeneration Induced CNPase Homologs of 68 and 70 kDa). Analysis of mRNA abundance by Northern blots or RNase protection assays showed that gRICH68 and gRICH70 mRNAs are strongly induced in the retina during optic nerve regeneration. The recombinant gRICH proteins showed $2\sp\prime,3\sp\prime$-cyclic-nucleotide 3$\sp\prime$-phosphodiesterase activity. A highly homologous protein designated zRICH has been cloned from zebrafish. Characterization of zRICH indicated very similar properties to the goldfish proteins. Additionally both zRICH and site-directed mutants in highly conserved motifs have been characterized and critical residues for catalysis have been identified. The zebrafish may be a highly useful system for future molecular genetic studies with the RICH proteins. The results presented in this dissertation demonstrate a new relationship between CNPases and nerve regeneration and set the stage for future studies regarding the role of RICH proteins in axonal regeneration.