Novel Nile tilapia Neu1 sialidases: Molecular cloning and biochemical characterization of the sialidases Neu1a and Neu1b.

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

Regulation of triglyceride metabolism in medaka (Oryzias latipes) hepatocytes by Neu3a sialidase.

Fish store triglycerides (TGs) in the liver, muscle, and adipose tissue and TGs constitute an energy source upon metabolic demand. The liver generally plays important roles in lipid metabolism. Recent studies have suggested the possibility of hepatic lipid metabolic regulation by ganglioside in mammals; however, ganglioside-mediated regulation of lipid metabolism is unclear in fish. This study aimed to clarify the role of ganglioside in fish TG metabolism, with particular reference to Neu3a, a ganglioside-specific sialidase expressed in the fish liver. Under fasting conditions, there was a decrease in hepatic TG contents, and neu3a mRNA level was significantly up-regulated in the medaka liver. To determine the role of Neu3a in hepatic lipid metabolism, Neu3a stable transfectants were generated using fish liver Hepa-T1 cells. After treating Neu3a cells with oleic acid, reduction of TG was detected in comparison with the mock cells. Furthermore, lipase activity was greater in Neu3a cells than in mock cells. To examine which ganglioside regulates these events, alterations of ganglioside composition in Neu3a cells were analyzed. Neu3a cells exhibited increased level of lactosylceramide (LacCer), a Neu3 enzymatic product originating from GM3. In addition, exposure of LacCer toward Hepa-T1 cells resulted in an increase of neutral lipase activity. The present results suggest that Neu3a up-regulation in medaka under fasting condition accelerates hepatic TG degradation for energy production via GM3 desialylation.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness.

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/ß.

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin ß and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin.

Suppression of Neu1 sialidase delays the absorption of yolk sac in medaka (Oryzias latipes) accompanied with the accumulation of α2-3 sialo-glycoproteins.

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Recently, medaka sialidase Neu1 has been cloned and its enzymatic properties were investigated. Although enzymatic properties of this sialidase, such as optimal pH and substrate specificity, exhibits high similarity with human NEU1, Neu1 physiological functions in fish are still unclear. Here, to understand Neu1 significance in medaka embryogenesis, sialidase translation knockdown was carried out with one-cell stage fertilized egg using morpholino oligo injection. Neu1 exhibited desialylation of α2-3 sialic acid linkage in vitro and lysosomal localization in medaka caudal fin primary cells. Chloroquine treatment, inhibitor of lysosomal enzymes, caused an accumulation of α2-3 sialo-glycoproteins in the primary cells. During the embryogenesis neu1 mRNA level was elevated until 3.5 day post fertilization (dpf) while an initial decrease of α2-3 sialo-glycoprotein was observed around the same developmental stage. Neu1 knockdown by morpholino oligo induced some abnormal phenotypes such as delay of yolk sac absorption and small embryos. Sialidase-knockdown embryos also showed increase of heart rate in 5.5 and 6.5 dpf. Furthermore, about 37% decrease of hatching rate was observed in Neu1-MO treated embryos compared with control MO. Embryos showing severe phenotypes stopped embryogenesis at the late stage of development. Alteration of embryonic sialo-glycoproteins induced by morpholino injection was examined by lectin blotting to clarify the mechanism of abnormal development. As a result, degradation of several α2-3 sialo-glycoproteins was suppressed in Neu1-MO embryo, possibly induced by the interruption of lysosomal desialylation toward yolk glycoprotein. Our results suggest that medaka Neu1 could be crucial for embryonic development through the degradation of yolk sac nutrition.

Naringenin suppresses Edwardsiella tarda infection in GAKS cells by NanA sialidase inhibition.

Edwardsiella tarda (E. tarda) is a gram-negative bacterium, which causes Edwardsiellosis in aquaculture. Previous studies indicate that E. tarda NanA sialidase plays crucial roles in infection through the desialylation of glycoproteins in fish cells. On the other hand, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, classic sialidase inhibitor, negatively regulates E. tarda infection of goldfish scale GAKS cells. Here, to development the suppression model of E. tarda infection for aquaculture application, the possibility of NanA inhibitory activities in citrus phytochemicals was evaluated as citrus extracts have widely been used as a supplement in fish diets for the improvement of meat quality. Some flavanones such as naringenin, hesperetin, hesperidin and naringin showed sialidase inhibitory activity toward recombinant NanA in vitro. Among them, naringenin showed the most potent inhibitory activity and its inhibitory pattern was non-competitive. Naringenin significantly suppressed E. tarda infection in GAKS cells at 200 and 400 µM without bactericidal effect on E. tarda. On the other hand, naringin, glycosylation form of naringenin, showed slight suppression of E. tarda infection toward GAKS cells, suggesting the glycosides on flavanone could be important for NanA inhibition. Fluorescence microscopy analysis verified that number of invading E. tarda in GAKS cells was declined by naringenin treatment. The present study exhibited the possibility of naringenin as an effective ingredient in fish diet for the inhibition of E. tarda infection.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth.

Positive regulation of myoblast differentiation by medaka Neu3b sialidase through gangliosides desialylation.

Sialidase Neu3b is an unique enzyme conserved in medaka and tilapia, but not in mammals. Previous study revealed that medaka Neu3b is localized at cytosol and is a ganglioside-specific sialidase. Neu3b functions, however, have not been understood, while Neu3a sialidase, which is widely conserved from human to fish, is known as a regulator of neurite formation. Here, we investigated the biological function of Neu3b for C2C12 myoblast cell differentiation. Bioinformatics analysis using genome browser revealed the presence of neu3b gene in some orders of fish species such as Beloniformes, Perciformes and Cyprinodontiformes. With the treatment of 2% horse serum, Neu3b-overexpression accelerated myoblast cell differentiation to myotubes accompanied with up-regulation of myogenesis biomarkers mRNA, myod and myog. Neu3b altered ganglioside composition in C2C12 cells results showing a decrease in GM2, and the increase of Lac-Cer, while desialylation of glycoproteins were not detected. Contrary to cell differentiation, Neu3b cell proliferation was suppressed in normal growth medium. To understand the mechanism of the alteration of cell differentiation and proliferation, phosphorylation of signal molecules in EGFR/ERK pathway was investigated. Neu3b induced a decline in phosphorylation of ERK and EGFR. Surprisingly, immuno-blot and real-time PCR analysis revealed that down-regulation of egfr gene could be involved in the acceleration of cell differentiation by Neu3b. These results suggested that Neu3b sialidase is a positive regulator for myoblast differentiation, similar with mammalian cytosolic sialidase Neu2.

Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.

Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate α2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection.

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3.

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown. In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.

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

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 1227bp, 1194bp and 1155bp, 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 45.9kDa, 44.4kDa and 43.6kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH4.6. 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.

Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase.

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.