EXPRESSION OF STABLE HUMAN O-GLYCAN CORE 2 BETA -1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE IN SF9 INSECT CELLS

UDP-GlcNAc:Galβ1-3GalNAc-R (GlcNAc to GalNAc) β-1,6- N -acetylglucosaminyltransferase (C2GnT) catalyses the formation of O -glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/ Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per μ l; approx. 0.2 mg/l) and was stable at 4 °C either in solution or after lyophilization. Endoglycosidase H and N -glycanase F treatment of the radiolabelled C2GnT indicated the presence of N -glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.