Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal␤4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc␤4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding ␤4GalTs responsible for LN synthesis have been reported, the ␤4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc␤-R ␤1,4-N-acetylgalactosaminyltransferase (Ce␤4GalNAcT) that synthesizes the LDN structure. Ce␤4GalNAcT is a member of the ␤4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce␤4GalNAcT expressed in CHOLec8 cells was active using UDP-GalNAc, but not UDPGal, as a donor toward a variety of acceptor substrates containing terminal ␤-linked GlcNAc in both N-and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and 1 H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce␤4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic ␤4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.

mentioning

confidence: 83%

Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-Acetylgalactosaminyltransferase fromCaenorhabditis elegans

Kawar

Die

Cummings

2002

“…The resulting 1.37-kb fragment was cloned into the BglII/XbaI sites of pVL1393 (PharMingen) and pVL1393-MYC. Baculovirus expression constructs, pVL-egghead-full and pVL-egghead-Myc-full, were co-transfected with Baculo-Gold TM DNA (PharMingen) in Sf9 cells as described (12 trol constructs included pVL-GalNAc-T4-full (13) and pVL-brainiac-full (8 (14). Briefly, cells were lysed in lysis buffer (25 mM Tris-HCl (pH 7.4), 250 mM sucrose); after incubation 30 min on ice cells were homogenized and lysate centrifuged at 1,000 ϫ g. Glycerol was added to 20%, and membrane pellets were obtained by 100,000 ϫ g. Pellets were used at 10 mg/ml (protein concentration determined by BCA, Pierce).…”

Section: Methodsmentioning

confidence: 99%

Drosophila egghead Encodes a β1,4-Mannosyltransferase Predicted to Form the Immediate Precursor Glycosphingolipid Substrate for brainiac

Wandall

Pedersen

Park

et al. 2003

The neurogenic Drosophila genes brainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis of egghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:␤Man␤1,3-N-acetylglucosaminyltransferase (␤3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc␤1-3Man␤1-4Glc␤1-1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:␤Glc ␤1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man␤1-4Glc␤1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids.

“…Preparation of Soluble Enzymes-Expression vectors or an empty pcDNA3.1-HSH were transfected individually into Lec20 cells (26), kindly provided by Dr. Pamela Stanley, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. The cells were then cultured for 24 h with ␣-minimum essential medium (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum, and the medium was replaced with Opti-MEM medium (Invitrogen) supplemented with 40 g/ml of L-proline and cultured cells for 24 h at 37°C.…”

Section: Construction Of Expression Vectors Encoding Soluble Forms Ofmentioning

confidence: 99%

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Kitayama

Hayashida

Nishida

et al. 2007

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, ␤1,3-N-acetylglucosaminyltansferase-7 (␤3GnT7) and ␤1,4-galactosyltransferase-4 (␤4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-Nacetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of ␤3GnT7/ ␤4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of ␤3GnT7 or ␤4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.