Molecular cloning of mouse ganglioside sialidase and its increased expression in Neuro2a cell differentiation.
Ganglioside sialidases have been implicated in neuronal differentiation processes, including neurite outgrowth. To understand further the roles and regulation mechanisms of the sialidase in neuronal systems, we have cloned mouse ganglioside sialidase cDNA and observed its expression in Neuro2a cell differentiation. A 3339-base pair cDNA, cloned based on the sequence information of previously cloned enzymes, encodes 418 amino acids containing three Asp boxes characteristic of sialidases. Northern blot analysis revealed a 3.4-kilobase transcript expressed highly in heart but also in several other tissues including brain. In situ hybridization of mouse brain demonstrated the mRNA to be present in the cerebral cortex, as well as in the granule cell layer, Purkinje cells, and deep cerebellar nucleus of the cerebellum. Transient expression of the cDNA in COS-1 cells resulted in over 300-fold increase in sialidase activity toward gangliosides compared with the control level, with a preference for ganglioside substrate. During 5-bromodeoxyuridine-induced Neuro2a cell differentiation, the expression of the sialidase was increased as assessed by activity assays and quantitative reverse transcription-polymerase chain reaction analyses. Stable transfection of the sialidase in Neuro2a cells resulted in accelerated neurite arborization following 5-bromodeoxyuridine treatment, indicating the direct participation of this ganglioside sialidase in neuronal cell differentiation.
Hasegawa
T
T
Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, 47-1 Nodayama, Medeshima-shiode, Natori, Miyagi 981-1293, Japan.
Yamaguchi
K
K
Wada
T
T
Takeda
A
A
Itoyama
Y
Y
Miyagi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000201:enzymology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D019556:COS Cells; D002454:Cell Differentiation; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C036052:GM3 ganglioside sialidase; C511796:Neu3 protein, mouse; D009439:Neuraminidase; D001973:Bromodeoxyuridine