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Gewählte Publikation:

Altmann, F; Kornfeld, G; Dalik, T; Staudacher, E; Glössl, J.
(1993): Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells.
Glycobiology. 1993; 3(6):619-625 FullText FullText_BOKU

Abstract:
The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)(8)COOH3 and H-3-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a nonradioactive HPLC method using pyridylaminated Man(3)-GlcNAc(2) and Man(5)GlcNAc(2) as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase 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 human 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-0503 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 acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine. GlcNAc-T II activity could be demonstrated 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-acetylglucosaminidase. However, the insect cells exhibited specific activities of GlcNAc-T II of only 0.02-0.11 nmol/h/mg of cell protein, much less than HepG2 cells.
Autor*innen der BOKU Wien:
Altmann Friedrich
Dalik Thomas
Glößl Josef
Staudacher Erika
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Animals -
Asparagine - metabolism
Baculoviridae - physiology
Carbohydrate Sequence - physiology
Cells, Cultured - physiology
Fucosyltransferases - metabolism
Humans - metabolism
Molecular Sequence Data - metabolism
Moths - metabolism
N-Acetylglucosaminyltransferases - metabolism
Oligosaccharides - metabolism
Tumor Cells, Cultured - metabolism

Find related publications in this database (Keywords)
N-ACETYLGLUCOSAMINYLTRANSFERASE
BACULOVIRUS INFECTION
GLYCOPROTEINS
INSECT CELLS
N-LINKED OLIGOSACCHARIDES


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