Elsevier

Gene

Volume 552, Issue 1, 15 November 2014, Pages 155-164
Gene

Nile Tilapia Neu3 sialidases: Molecular cloning, functional characterization and expression in Oreochromis niloticus

https://doi.org/10.1016/j.gene.2014.09.029Get rights and content

Highlights

  • Five neu3 genes, neu3a, neu3b, neu3c, neu3d and neu3e are present in Nile Tilapia.

  • Neu3a possessed sialidase activity towards gangliosides, same as human NEU3.

  • neu3b and neu3c showed no expression in all tested tissues from adult fish.

  • Neu3d and Neu3e showed no sialidase activity towards all tested substrates.

  • Overexpressed Neu3a and Neu3d were at plasma membrane, while Neu3e was at cytosol.

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.

Introduction

Gangliosides are ubiquitous, membrane-associated, sialic acid containing glycosphingolipids that have been implicated in a broad range of biological functions. They play roles in a wide array of physiological processes (Watanabe et al., 2002, Anderson, 1998, Lloyd and Furukawa, 1998, Rampersaud et al., 1999, Sorice et al., 2004, Hakomori and Igarashi, 1995). Moreover, previous reports suggest that gangliosides are receptors of various pathogens in mammals (Kanda and Watanabe, 2001, Tsai et al., 2003, Low et al., 2006, Markwell et al., 1981, Oda et al., 2013, Kuziemko et al., 1996) and in avian (Ferreira et al., 2004). Despite several reports on the significance of gangliosides as receptors of pathogens in mammals and avian, such information is still obscure in fish. Only recently, Vibrio trachuri has been reported to adhere to GM3 and GM4 in red sea bream (Pagrus major) (Matsunaga et al., 2011, Chisada et al., 2013). Based on these studies, enzymatic modulator of gangliosides could be indispensable in regulating infection and invasion of various pathogens, although the underlying mechanism remains unclear in fish.

In nature, sialidase are among important modulators of gangliosides. Sialidases are glycohydrolytic proteins that catalyze the cleavage of sialic acids from sialoglycoconjugates (Miyagi and Yamaguchi, 2007). They are found widely distributed in nature ranging from viruses, bacteria and higher animals (Saito and Yu, 1995, Rosenberg and Schengrund, 1976) where they modulate and influence various physiological functions by their sialoglycohydrolytic activity (Miyagi, 2008, Valaperta et al., 2006, Yamaguchi et al., 2006). Various groups of mammalian sialidases have been identified and extensively characterized. They are designated as Neu1, Neu2, Neu3 and Neu4 (NEU1, NEU2, NEU3 and NEU4 in humans). Each group of mammalian sialidase has been reported to possess unique properties including subcellular localization and substrate specificity. Among mammalian sialidase, Neu3, a plasma membrane and ganglioside specific sialidase holds a strategic role in glycoconjugate modulation; consequently, affecting cell surface events and cellular physiology such as apoptosis (Valaperta et al., 2006), cell proliferation and myoblast differentiation (Anastasia et al., 2008), neurite formation (Hasegawa et al., 2000, Proshin et al., 2002), regulation and regeneration of neurons (Rodriguez et al., 2001, Da Silva et al., 2005).

Fish Neu3 sialidases have not been extensively characterized. Molecular cloning and characterization of neu3 genes have so far been reported only in zebrafish (Danio rerio) with five neu3 sialidases genes identified (neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5) (Manzoni et al., 2007) and medaka (Oryzias latipes) with neu3a and neu3b (Shiozaki et al., 2013). Medaka and zebrafish Neu3 sialidases possess well conserved YRIP, VGPG and Asp-box motifs. Interestingly, variations that exist in mammalian sialidase properties have also been observed in fish Neu3 sialidases. For instance, medaka Neu3a is localized at plasma membrane (Shiozaki et al., 2013), while in zebrafish, Neu3.1 is present at endoplasmic reticulum (Manzoni et al., 2007). These results suggest the existence of evolutionary diversified sialidase properties among fish species. Therefore, the expansion of sialidase studies to other fish species could reveal more striking property patterns of these enzymes.

Medaka and zebrafish, with significant sialidase exploration, are smaller in size and not aquaculture candidates. This potentially limits other types of investigations such as the application of in vitro biochemical results to a full scale aquaculture operation. For example, an aquaculture species could not only be studied with regard to its economical interest but also as a model for more fundamental research in immunology, physiology, toxicology and others. Hence, a widely cultured fish species would be a suitable model animal for understanding the biological consequences of ganglioside modulation by sialidase Neu3 in fish diseases. Recently, the intensification of fish farming has led to increased outbreaks of infectious disease (FAO, 2012), which have caused huge losses to the industry. Therefore, the exploration of sialidase Neu3 and its target gangliosidic substrates could provide an ideal focal point towards further elucidation of the regulatory mechanisms in cultured fish species. Based on all of these, in the present study we focused on molecular cloning and characterization of neu3 genes in Nile tilapia, Oreochromis niloticus, one of the most important aquaculture species in the world (http://www.fao.org/fishery/culturedspecies/Oreochromis_niloticus/en). The positive attributes of the species in aquaculture such as its wide environmental tolerance, high growth rates and large harvest body weights make it a valuable resource as an aquaculture experimental animal, especially for in situ gene and protein regulation and functionality. Therefore, the present study expanded the understanding of the structure and evolution of neu3 sialidase genes in teleosts and other vertebrates.

Section snippets

Animals and tissue preparation

Wild O. niloticus were used in this study. The fish were collected from Ibusuki River (Ibusuki, Japan). Tilapia tissues were excised from each fish, immediately frozen by dry ice and kept at − 80 °C until use.

Determination of putative tilapia sialidase genes and post-cloning sequence analysis

Putative tilapia sialidase nucleotide sequences were identified from genomic sequence available in central repositories including National Center of Biotechnological Information (NCBI) (http://www.ncbi.nlm.nih.gov/), Ensembl Genome Browser (http://asia.ensembl.org/index.html) and University

Identification of sialidase genes in O. niloticus

In order to clone sialidase genes in tilapia genome, O. niloticus genomic sequence databases were queried by BLAST search for sequences showing similarities to human NEU1, NEU2, NEU3 and NEU4 genes. As a result, eight putative sialidase genes were found in tilapia genome: two neu1-like genes, five neu3-like genes and one neu4-like gene, seven of which were predicted in Ensembl database. One neu3-like sequence, not predicted in Ensembl database was further analyzed in silico using gene

Discussion

Expansion of molecular cloning and biochemical characterization of sialidase genes and proteins to various species continue to reveal their remarkable diversity. Recent efforts have focused on uncovering other roles of sialidases, especially Neu3, which based on mammalian studies is plasma-membrane associated sialidase and is known to have strict substrate specificity towards gangliosides (Miyagi et al., 1999, Monti et al., 2000). Studies of fish Neu3 sialidase have lagged behind and have

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgment

We appreciate the useful discussions with Prof. Taeko Miyagi and the expert technical assistance of Kazuki Takeshita, Naoto Matsushita, Sena Ryuzono and Asami Ikeda. This work was supported by Grant-in-Aid for Young Scientist (B) (Grant No. 24780204) from Japan Society for the Promotion of Science (JSPS).

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