Characterization of cytosolic sialidase from Chinese hamster ovary cells: part II. Substrate specificity for gangliosides.
Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli. The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Müthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies. The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc). A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer. The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase. The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.
Müthing
J
J
Institute of Cell Culture Technology, Faculty of Technology, University of Bielefeld, Germany. jm@zellkult.techfak.uni-bielefeld.de
Burg
M
M
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D005732:Gangliosides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D005732:Gangliosides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D009439:Neuraminidase