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 s oluble glutathione S-transferase (GST)-sialidase fusion protein with an app arent molecular weight of 69 kD in Escherichia coli. The enzyme has then be en produced in mg quantities at 25-L bioreactor scale and purified by one-s tep affinity chromatography on glutathione sepharose (Burg, M.; Muthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for d esialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosid es were separated on silica gel precoated TLC plates, incubated with increa sing concentrations of sialidase (50 muU/mL up to 1.6 mU/mL) without deterg ents, and desialylated gangliosides were detected with specific anti-asialo ganglioside antibodies. The enzyme exhibited almost identical hydrolysis ac tivity in degradation of G(M3)(Neu5Ac) and G(M3)(Neu5Gc). A slightly enhanc ed activity, compared with reference Vibrio cholerae sialidase, was detecte d towards terminally alpha (2-3)-sialylated neolacto-series gangliosides IV 3-alpha -Neu5Ac-nLc(4)Cer and VI3-alpha -Neu5Ac-nLc(6)Cer. The ganglio-seri es gangliosides G(D1a), G(D1b), and GT(1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less su itable targets for the CHO cell sialidase. The increasing evidence on coloc alization of gangliosides and sialidase in the cytosol strongly suggests th e involvement of the cytosolic sialidase in ganglioside metabolism on intra cellular level by yet unknown mechanisms. (C) 2001 Elsevier Science Ltd. Al l rights reserved.