PROCESSING OF ASPARAGINE-LINKED OLIGOSACCHARIDES IN INSECT CELLS - N-ACETYLGLUCOSAMINYLTRANSFERASE I AND II ACTIVITIES IN CULTURED LEPIDOPTERAN CELLS

The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I an d II activities in cultured cells from Bombyx mori (Bm-N), Mamestra br assicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alph a 1-6)-Man beta 1-O(CH2)(8)COOH3 and H-3-labelled UDP-GlcNAc as substr ates, GlcNAc-T I activity was measured with a nonradioactive HPLC meth od using pyridylaminated Man(3)-GlcNAc(2) and Man(5)GlcNAc(2) as accep tor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosid ase digestion and methylation analysis that the product obtained with Man(3)GlcNAc(2) contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor. Compared to the enzyme from the hu man hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man(5) substrate. The GlcNAc-T I from Mb-050 3 cells had apparent K-m and V-max values for pyridylaminated Man(3)- and Man(5)GlcNAc(2) of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man(5)GlcNAc(2) was used as the a cceptor substrate, the levels of GlcNAc-T I activity in the four insec t cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, an d thus were comparable to that of HepG2 cells. Evidence is presented f or the dependence of lepidopteran fucosyltransferase on the presence o f terminal N-acetylglucosamine. GlcNAc-T II activity could be demonstr ated by HPLC using GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc-pyridylamine as the acceptor in the presence of 6-acetamido-6-deoxycastanospermine as an inhibitor of beta-N-acety lglucosaminidase. However, the insect cells exhibited specific activit ies of GlcNAc-T II of only 0.02-0.11 nmol/h/mg of cell protein, much l ess than HepG2 cells.