Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus |
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Authors: | Petros Kingstone Chigwechokha Masaharu Komatsu Takao Itakura Kazuhiro Shiozaki |
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Institution: | 1. The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Japan;2. Department of Fisheries, Mzuzu University, Mzuzu, Malawi;3. Faculty of Fisheries, Kagoshima University, Kagoshima, Japan |
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Abstract: | Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish. |
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Keywords: | 4-MU-NANA 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid BSA bovine serum albumin DMEM Dulbecco modified eagle medium GAPDH glyceraldehyde 3-phosphate dehydrogenase GD1a NeuAcα2 &rarr 3Galβ1 &rarr 3GalNAcβ1 &rarr 4[NeuAcα2 &rarr 3]Galβ1 &rarr 4Glcβ1 &rarr Cer GD3 NeuAcα2 &rarr 8NeuAc α2 &rarr 3Gal β1 &rarr 4Glcβ1 &rarr Cer GM1 Gal β1 &rarr 3GalNAcβ1 &rarr 4[NeuAcα2 &rarr 3]Galβ1 &rarr 4Glcβ1 &rarr Cer GM3 NeuAcα2 &rarr 3Galβ1 &rarr 4Glcβ1 &rarr Cer HA hemagglutinin PCR polymerase chain reaction SNP single nucleotide polymorphism |
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