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Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus
Authors:Petros Kingstone Chigwechokha  Masaharu Komatsu  Takao Itakura  Kazuhiro Shiozaki
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
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.
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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