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Cloning of Human Liver Cytosolic Sialidase from Genomic DNA Using Splicing by Overlap Extension and Its Characterization

HA KI-TAE    (National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University   ); CHO SEUNG-HAK    (Division of Intestinal Bacteriology, National Institute of Health   ); KANG SUNG-KOO    (National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University   ); KIM YEON-KYE    (National Fisheries Research Institute   ); KIM JUNE-KI    (Division of Intestinal Bacteriology, National Institute of Health   ); KIM CHEORL-HO    (National Research Laboratory for Glycobiology and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University  );
  • 초록

    Cytosolic sialidase (Neu2), a member of the sialidase family that is responsible for hydrolysis of sialic acid from the terminal position of sialoglycoconjugates, is poorly expressed in skeletal muscle and not detected in any other adult tissues. Thus, we isolated Neu2 cDNA using splicing by overlap extension (SOEing). In order to further characterize this enzyme, a His-tagged derivative was expressed in the bacterial expression system and purified by $Ni^{2+}$ -affinity chromatography. A recombinant product of approximately 42 kDa had sialidase activity toward 4-methyl-umbelliferyl- $\alpha$ -D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH and temperature of the recombinant Neu2 for 4MU-NeuAc was 6.0 and $37.5^{\circ}C$ , respectively. The metal ions, such as $Cu^{2+}\;and\;Cd^{2+}$ , showed strong inhibitory effect on the activity of the enzyme. The enzyme efficiently hydrolyzed the gangliosides GM3 and GD3 and had relatively low activities on ganglioside GD1a and GD1b, $\alpha$ 2-3 sialyllactose, and sialylated glycoproteins such as fetuin, transferrin, and orsomucoid, but had hardly any activities on $\alpha$ 2-6 sialyllactose and ganglioside GM1 and GM2. We concluded that the recombinant Neu2 has a sialidase activity toward glycoproteins as well as gangliosides.

  • 주제어

    Cytosolic sialidase .   Chang liver cell .   splicing by overlap extension .   4MU-NeuAc .   ganglioside .   glycolipid .   glycoprotein.  

  • 참고문헌 (31)

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