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

• Published in: Journal of microbiology and biotechnology pp. 722 - 727
• Main Authors: HA, KI-TAE, SEUNG-HAK CHO, SUNG-KOO KANG, YEON-KYE KIM, JUNE-KI KIM, CHEORL-HO KIM
• Format: Journal Article
• Language: Korean
• Published: 한국미생물·생명공학회 01.08.2005
• Subjects: 생물학
• ISSN: 1017-7825

Cytosolic sialidase (Neu2), a member of thesialidase family that is responsible for hydrolysis of sialic acidfrom the terminal position of sialoglycoconjugates, is poorlyexpressed in skeletal muscle and not detected in any otheradult tissues. Thus, we isolated Neu2 cDNA using splicing byoverlap extension (SOEing). In order to further characterize thisenzyme, a His-tagged derivative was expressed in the bacterialexpression system and purified by Ni2+-affinity chromatography.A recombinant product of approximately 42 kDa had sialidaseactivity toward 4-methyl-umbelliferyl-α-D-N-acetylneuraminicacid (4MU-NeuAc). The optimal pH and temperature of therecombinant Neu2 for 4MU-NeuAc was 6.0 and 37.5oC,respectively. The metal ions, such as Cu2+ and Cd2+, showedstrong inhibitory effect on the activity of the enzyme. Theenzyme efficiently hydrolyzed the gangliosides GM3 andGD3 and had relatively low activities on ganglioside GD1aand GD1b, α2-3 sialyllactose, and sialylated glycoproteinssuch as fetuin, transferrin, and orsomucoid, but had hardlyany activities on α2-6 sialyllactose and ganglioside GM1 andGM2. We concluded that the recombinant Neu2 has a sialidaseactivity toward glycoproteins as well as gangliosides. KCI Citation Count: 0