Cloning of human liver cytosolic sialidase from genomic DNA using splicing by overlap extension and its characterization

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-a-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH and temperature of the recombinant Neu2 for 4MU-NeuAc was 6.0 and 37.5°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 GDlb, α2-3 sialyllactose, and sialylated glycoproteins such as fetuin, transferrin, and orsomucoid, but had hardly any activities on a2-6 sialyllactose and ganglioside GM1 and GM2. We concluded that the recombinant Neu2 has a sialidase activity toward glycoproteins as well as gangliosides.