Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.

Abstract

A cDNA encoding UDP-GkcNAc:Galjll- P-1,6-N-acetylglucsaminyltansferase, the enzyme responsible for the formation of Gall-3(GlcNAcP31-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other 1-6GlcNAc transferases.Most 0-glycosidic oligosaccharides in mammalian glycoproteins are linked via GalNAc to the hydroxyl groups of serine or threonine. Although less well studied than N-glycans, recent studies suggest that O-glycans also have important biological roles. In particular, the appearance of the O-linked

Expression Cloning of a Human Sulfotransferase That Directs the Synthesis of the HNK-1 Glycan on the Neural Cell Adhesion Molecule and Glycolipids

“…In theory, N-CAM cDNA could be also cotransfected transiently since its vector contains only an ampicillin resistant marker. This procedure represents an improved method over previous expression cloning strategy where a vector encoding the polyoma large T antigen was stably expressed (25,39). By avoiding the preparation of stable transfectants, which are necessary for the expression of the acceptor carbohydrates and polyoma large T antigen, the time necessary for cloning has been shortened dramatically.…”

“…Epitope-Lec2-NCAM cells were cotransfected with a human fetal brain cDNA library in pcDNAI, pcDNA3-GlcAT-P, and pPSVE1-PyE (25). The transfected cells were incubated with anti-HNK-1 antibody followed by fluorescein isothiocyanate-conjugated secondary antibody, then subjected to cell sorting.…”

“…Lec2-NCAM cells were thus cotransfected with 18 g of a human fetal brain cDNA library in pcDNAI (19), 6 g of pcDNA3-GlcAT-P, and 6 g of pPSVE1-PyE harboring the polyoma large T cDNA (25), using LipofectAMINE TM (Life Technologies, Inc.) as described previously (19). After 62 h, the transfected cells were dissociated into monodispersed cells using the enzyme-free cell dissociation solution (Hanks' based, purchased from Cell and Molecular Technologies, Lavellette, NJ), followed by fluorescence-activated cell sorting of the HNK-1-positive cells using anti-HNK-1 monoclonal antibody (Becton Dickinson).…”

The human UDP‐N‐Acetylglucosamine:α‐6‐d‐Mannoside‐β‐1,2‐N‐Acetylglucosaminyltransferase II Gene (MGAT2)

“…In addition to GlcNAc-T 11, there are three other cloned GlcNAc-transferases which act on N-glycans: GlcNAc-T I [27 -341, GlcNAc-T I11 [59, 771 and GlcNAc-T V [17,603. Two GlcNAc-transferases, one involved in 0-glycan and the other in branched poly-(N-acetyllactosamine) synthesis, have also been cloned [61,621. Although there are regions of close sequence similarity between the latter two enzymes, it is remarkable that there is no such detectable similarity between the sequences of GlcNAc-T I, 11, I11 and V which all initiate synthesis of N-glycan antennae.…”

Expression of human Wiskott–Aldrich syndrome protein in patients’ cells leads to partial correction of a phenotypic abnormality of cell surface glycoproteins

“…29 Briefly, 1 × 10 7 cells were lysed in 0.4% TritonX-100, 0.9% NaCl and clarified by centrifugation at 5000 g. The lysate at a protein concentration of 10 mg/ml was used for this assay. A reaction mixture containing 50 mm MES (pH 7.0), 100 mm GlcNAc (Sigma), 1 mm UDPGlcNAc (Sigma), 1 Ci UDP-3H-GlcNAc (NEN, Boston, MA, USA), 1 mm p-nitrophenyl-Galb(beta)1-3GalNAc (Sigma) and 25 l of cell lysate was in a total volume of 50 l and such reaction mixture was then incubated for 1 h at 37°C.…”