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.

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... 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. In recent years, great interest has focused on the elucidation of the biological function of glycosphingolipids, in particular gangliosides, which are characteristic constituents at the surface of mammalian cells and abundant in neuronal membranes (1, 2). In the nervous system, gangliosides have been observed to undergo metabolic alterations during development and have been implicated in various biological phenomena, including neuritogenesis (3, 4), synaptic function (5) , neuronal death (6) , neural repair (7) , cell-cell recognition, and cell adhesion (8). Ganglioside accretion is largest during dendritic arborization and synaptogenesis and is accompanied by marked changes in the ganglioside composition, which then persist during adult life (9, 10). Neuroblastoma cell lines can be triggered to undergo neuronal differentiation with a marked alteration of ganglioside patterns by several pharmacological agents (11-13). On the other hand, a number of exogenous gangliosides are known to cause neuritogenesis, including ganglioside GM1 (14) , enhances the effects of neurotrophic factors, such as nerve growth factor (15, 16), in several cell lines. Tsuji et al. (17) reported that very low amounts of exogenous GQ1b specifically stimulated neurite outgrowth in a human neuroblastoma cell line, GOTO cells. Although the general mechanism by which gangliosides promote neuritogenesis has not been elucidated, their desialylation by endogenous as well as exogenous sialidases has been suggested as a critical event for neuronal differentiation (18 -21), myelination (22), synaptogenesis, and synaptic function (23). A potent ganglioside sialidase activity, in fact, is located in the neuronal membranes (24), with a relative enrichment in the synaptosomal fraction as compared with the neuronal perikarya (23, 25) . In this context, it is necessary to clarify the expression mechanisms of endogenous sialidase responsible for ganglioside degradation in the nervous system. In the present study, we obtained new insights into the role of the ganglioside sialidase during neuronal differentiation by studies with a newly cloned cDNA. Mammalian sialidases, catalyzing the first step in glycoconjugate degradation, have been classified based on their subcellular distribution into at least four types: cytosolic, lysosomal matrix, lysosomal membrane, and plasma membrane (26 -28). They differ from each other not only in subcellular location but also catalytic and immunological properties. Membrane-associated sialidases hydrolyze gangliosides preferentially, and those in plasma membrane are distinct from lysosomal membrane sialidase in acting specifically on gangliosides residing in the same membrane (28, 29).We previously cloned a cytosolic sialidase from rat skeletal muscle (30), and recently membrane ganglioside sialidases of bovine (31) and human (32) brains. Lysosomal sialidases of human (33-35) and mouse (36 -38) were also cloned by another three groups. We have now isolated a cDNA encoding mouse ganglioside sialidase capitalizing on the high degree of conservation in the primary structures of bovine plasma membrane (31), rat cytosolic (30), and human lysosomal (33-35) sialidases. The spatial and temporal expression of the sialidase mRNA was investigated in adult mouse brain and during differentiation of Neuro2a cells induced by 5-bromodeoxyuridine (BrdUrd). 1 The results revealed the gan-* This work was supported in part by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s)AB026842 and AB008184 for mouse membrane-associated ganglioside sialidase and mouse cytosolic sialidase, respectively.