Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration.
We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.
Ballestero
R P
RP
Department of Biological Chemistry and Mental Health Research Institute, University of Michigan, Ann Arbor 48104-1687, USA.
Dybowski
J A
JA
Levy
G
G
Agranoff
B W
BW
Uhler
M D
MD
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000737:chemistry / Q000235:genetics*; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics; D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D029941:Fish Proteins; D015870:Gene Expression / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009416:Nerve Regeneration / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics; D009900:Optic Nerve / Q000166:cytology / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D012165:Retinal Ganglion Cells / Q000737:chemistry / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015027:Zebrafish
D029941:Fish Proteins; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C078182:beta-ketoacyl-acyl carrier protein synthase I; D000102:Acetyl-CoA C-Acyltransferase; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; C105609:gRICH68 protein, Carassius auratus; C105610:gRICH70 protein, Carassius auratus