PMID:23032629

Citation	Shiozaki, K, Takeshita, K, Ikeda, M, Ikeda, A, Harasaki, Y, Komatsu, M, Yamada, S, Yamaguchi, K and Miyagi, T (2013) Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes). Biochimie 95:280-9
Abstract	Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.
Links	PubMed Online version:10.1016/j.biochi.2012.09.026
Keywords	Amino Acid Sequence; Animals; Brain/enzymology; Cell Membrane/enzymology; Cloning, Molecular; Conserved Sequence; Cytosol/enzymology; Evolution, Molecular; Gangliosides/metabolism; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; Molecular Sequence Data; Neuraminidase/genetics; Neuraminidase/metabolism; Oryzias/genetics; Oryzias/metabolism; Phylogeny; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Alignment; Substrate Specificity; Zebrafish/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ORYLA:G3XKV6		GO:0016020: membrane	ECO:0000314:		C		Figure 6 A shows an immunofluorescence image of neu3a localizing to the cell membrane	complete CACAO 10918
ORYLA:G3XKV6		GO:0004308: exo-alpha-sialidase activity	ECO:0000314:		F		Figure 5 A shows the sialidase activity on neu3a on different sialated substrates.	complete CACAO 10919
ORYLA:G3XKV6		GO:0045666: positive regulation of neuron differentiation	ECO:0000315:		P		Figure 8A shows that Neuro2a cells transfected with neu3a differentiated faster after retinoic acid induction than cells that were mock-transfected.	complete CACAO 10920
ORYLA:G3XKV7		GO:0005829: cytosol	ECO:0000314:		C		Figure 6C shows neu3b localizing to the cytosol, along with GAPDH, a cystolic marker.	complete CACAO 10921
ORYLA:G3XKV7		GO:0004308: exo-alpha-sialidase activity	ECO:0000314:		F		Figure 5B shows the sialidase activity of neu3b on several different sialated substrates.	complete CACAO 10922
ORYLA:G3XKV7	part_of	GO:0005829: cytosol	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete

References

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