Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli.
Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.
Ha
Ki-Tae
KT
National Research Laboratory for Glycobiology, Ministry of Science and Technology, Korean Government.
Lee
Young-Choon
YC
Cho
Seung-Hak
SH
Kim
June-Ki
JK
Kim
Cheorl-Ho
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D005732:Gangliosides; D011994:Recombinant Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; C026226:ganglioside, GD3; C465436:Neu3 protein, human; D009439:Neuraminidase