Purification and characterization of the goldfish regeneration-associated proteinsgp68/70.

Abstract

Successful regeneration of nerves in the central nervous system in cold-blooded vertebrates is an intriguing phenomenon, in that warm-blooded vertebrates show only a limited capacity for CNS regeneration. By studying regeneration in the goldfish retinotectal projection, strategies may be developed to stimulate regeneration in species that are presently incapable of CNS regeneration. Two acidic proteins (gp68/70) previously shown to be associated with regeneration were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu$\sp{2+}$-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5.2 S. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high resolution DEAE column resolved the protein doublet into three dimeric species of gp68, gp68/70, and gp70. These results indicate that the proteins are highly elongated and associate as either homodimers or as a heterodimer. Sequence analysis of gp68/70 identified homologies to three known proteins. gp68/70 was found to possess 2$\sp\prime$,3$\sp\prime$-cyclic nucleotide 3$\sp\prime$-phosphodiesterase activity. Subcellular localization and membrane extraction experiments indicated gp68/70 to be a component of the plasma membrane associated primarily through hydrophobic interactions. Carbohydrate analysis of gp68/70 indicated it to contain 26% carbohydrate by weight, with fucose, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose and sialic acid present in a 1/0.4/0.9/1.0/0.7/0.3/0.1 ratio. Modification of the purification procedure enabled similar proteins to be purified from the eggs of both goldfish and carp. The results presented in this thesis, together with the results of Wilmot et al (1993), support a dynamic role for gp68/70 in neurite extension and in non-neuronal membrane function.