The presence of UDP-N-acetylglucosamine:alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I activity in Spodoptera frugiperda cells (IPLB-SF-21AE) and its enhancement as a result of baculovirus infection.
A Golgi preparation from Spodoptera frugiperda (IPLB-SF-21AE) cells was incubated in the presence of the mannosidase II inhibitor, swainsonine, with the oligosaccharide, M(alpha 1,3)[[M(alpha 1,3)[M(alpha 1,6)]M(alpha 1,6)]] M(beta 1,4)Gn(beta 1,4)Gn (M5Gn2), the preferred substrate for the enzyme, UDP-N-acetylglucosamine:alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (Gn-TI). This resulted in formation of the product, Gn(beta 1,2)M(alpha 1,3)[[M(alpha 1,3)[M(alpha 1,6)]M(alpha 1,6)]]- M(beta 1,4) Gn(beta 1,4)Gn (Gn(I)M5Gn2). A significantly increased (> 4-fold) rate of conversion of M5Gn2 to Gn(I)M5Gn2 occurred with insect cell-derived Golgi preparations that had been infected with a recombinant baculovirus for 66 h, a time at which significant amounts of complex-type oligosaccharides were assembled on a heterologous protein, human plasminogen, expressed in this system. Coupled with previous results (Davidson, D.J., Bretthauer, R.K., and Castellino, F.J. (1991) Biochemistry 30, 9811-9815) that demonstrated the occurrence of virally induced activation of a specific M6-mannosidase in IPLB-SF-21AE cells, it is clear that viral infection of lepidopteran insect cells makes available enzymes that provide and utilize the substrate, M5Gn2-protein. This is a key feature in the explanation of the previous original observations made by this laboratory, that lepidopteran insect cells are capable of assembly of complex-type oligosaccharides on glycoproteins.
Velardo
M A
MA
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Bretthauer
R K
RK
Boutaud
A
A
Reinhold
B
B
Reinhold
V N
VN
Castellino
F J
FJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D004789:Enzyme Activation; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D008361:Mannosidases / Q000037:antagonists & inhibitors; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009036:Moths; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011134:Polysaccharides / Q000096:biosynthesis; D017026:Swainsonine / Q000494:pharmacology
D009844:Oligosaccharides; D011134:Polysaccharides; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D017026:Swainsonine