Expression of stable human O-glycan core 2 beta-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells.
UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-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 microl; approx. 0.2 mg/l) and was stable at 4 degrees 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.
Toki
D
D
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Sarkar
M
M
Yip
B
B
Reck
F
F
Joziasse
D
D
Fukuda
M
M
Schachter
H
H
Brockhausen
I
I
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D004795:Enzyme Stability; D016695:Glycosyltransferases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D011134:Polysaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase