UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II in Caenorhabditis elegans

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) and UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II) are key enzymes in the synthesis of Asn-linked hybrid and complex glycans. We have cloned cDNAs from Caenorhabditis elegans for three genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14) and one gene homologous to mammalian GnT II. All four cDNAs encode proteins which have the domain structure typical of previously cloned Golgi-type glycosyltransferases and show enzymatic activity (GnT I and GnT II, respectively) on expression in transgenic worms. We have isolated worm mutants lacking the three GnT I genes by the method of ultraviolet irradiation in the presence of trimethylpsoralen (TMP); null mutants for GnT II have not yet been obtained. The gly-12 and gly-14 mutants as well as the gly-14;gly-12 double mutant displayed wild-type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that the GLY-13 protein is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behavioral defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Our data indicate that complex and hybrid N-glycans may play critical roles in the morphogenesis of C. elegans, as they have been shown to do in mice and men.     

Purification and characterization of rat kidney UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase.

In order to investigate the molecular mechanism of the specific increase of UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (GlcNAcT-V, EC 2.4.1.155) activity after viral or oncogenic transformation, we have purified the enzyme from a Triton X-100 extract of rat kidney acetone powder. GlcNAcT-V was purified by sequential affinity chromatography using first UDP-hexanolamine-agarose and then a synthetic oligosaccharide inhibitor-agarose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed two major bands at apparent molecular masses of 69 and 75 kDa. The enzyme was recovered in a 26% final yield with a 450,000-fold increase in specific activity to a Vmax of 18.8 mumols/(mg.min). Enzyme activity was stabilized and enhanced by the addition of 20% glycerol, 0.5 mg/ml IgG, and 0.2 M NaCl. The optimal ranges of pH and Triton X-100 concentrations for enzyme activity were 6.5-7.0 and 1.0-1.5%, respectively. The divalent cations, Mn2+, Ca2+, and Mg2+, were each found to have a negligible (less than 10%) effect on activity; moreover, the enzyme was fully active in the presence of 20 mM EDTA. The Km value of the purified enzyme toward a synthetic trisaccharide acceptor was 90 microM, and the Ki value toward a synthetic active site inhibitor was 140 microM.     

Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B

Hep 3B, a human hepatoma cell line was examined for its RNA hybridizable to the hepatitis B virus sequence. Using probes that covered different regions of the hepatitis B virus genome, five species of RNA were observed of sizes 4.0, 3.3, 2.9, 2.6 and 2.2 kilobases. The RNAs covered surface antigen gene, pre-S and X regions. None of them had a core antigen sequence. RNA with a 4.0 kilobase size was the most abundant. Using S1 nuclease analysis, its 5' end of hepatitis B virus sequence was mapped at pre-S region and its 3' end of viral sequence was mapped at DR region.     

Molecular cloning and characterization of the mouse UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I gene.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.     

Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I catalyzes an essential first step in the conversion of high mannose to hybrid and complex N-glycans (Schachter, H. (1986) Biochem. Cell Biol. 64, 163-181; Oppenheimer, C.L., and Hill, R.L. (1981) J. Biol. Chem. 256, 799-804), i.e. the addition of GlcNAc to (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(Man alpha 1-3)Man beta 1-4GlcNAc-OR to form (Man alpha 1-6(Man alpha 1-3)Man alpha 1-6)(GlcNAc beta 1-2Man alpha 1- 3)Man beta 1-4GlcNAc-OR. The enzyme has been purified from Triton X-100 extracts of rabbit liver by chromatography on CM-Sephadex, Affi-Gel blue, UDP-hexanolamine-Sepharose, and a novel adsorbent in which UDP-GlcNAc is linked to thiopropyl-Sepharose at the 5-position of uracil. The enzyme exists in crude liver extracts in two molecular weight forms separable on Sephadex G-200. The low molecular weight form was purified 64,000-fold with a specific activity of 19.8 mumol/min/mg. The pure enzyme was free of N-acetylglucosaminyltransferase II-V activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single major band of Mr 45,000 and two minor bands of Mr 54,000 and 50,000. All three bands showed retarded elution from an affinity column in which the acceptor substrate for the transferase was covalently linked to Sepharose. Kinetic analysis indicated a largely ordered sequential mechanism with UDP-GlcNAc binding to the enzyme first and UDP leaving last. Studies with synthetic analogues of the substrate Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc showed that an unsubstituted equatorial hydroxyl on carbon 4 of the beta-linked Man residue was essential for enzyme activity.     

Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3- D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP- N- acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N- acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N- glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c- Myc epitope tag at the C-terminus elucidates that the N - terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.      

UDP-N-acetylglucosamine:alpha-6-D-mannoside beta1,6 N-acetylglucosaminyltransferase V (Mgat5) deficient mice

Targeted gene mutations in mice that cause deficiencies in protein glycosylation have revealed functions for specific glycans structures in embryogenesis, immune cell regulation, fertility and cancer progression. UDP-N-acetylglucosamine:alpha-6-D-mannoside beta1,6 N-acetylglucosaminyltransferase V (GlcNAc-TV or Mgat5) produces N-glycan intermediates that are elongated with poly N-acetyllactosamine to create ligands for the galectin family of mammalian lectins. We generated Mgat5-deficient mice by gene targeting methods in embryonic stem cells, and observed a complex phenotype in adult mice including susceptibility to autoimmune disease, reduced cancer progression and a behavioral defect. We found that Mgat5-modified N-glycans on the T cell receptor (TCR) complex bind to galectin-3, sequestering TCR within a multivalent galectin-glycoprotein lattice that impedes antigen-dependent receptor clustering and signal transduction. Integrin receptor clustering and cell motility are also sensitive to changes in Mgat5-dependent N-glycosylation. These studies demonstrate that low affinity but high avidity interactions between N-glycans and galectins can regulate the distribution of cell surface receptors and their responsiveness to agonists.     

Mucin biosynthesis: characterization of rabbit small intestinal UDP-N-acetylglucosamine:galactose beta-3-N-acetylgalactosaminide (N-acetylglucosamine----N-acetylgalactosamine) beta-6-N-acetylglucosaminyltransferase

We have characterized a UDP-GlcNAc:Gal beta-3-GalNAc (GlcNAc----GalNAc) beta-6-N-acetylglucosaminyltransferase from rabbit small intestinal epithelium by using freezing point depression glycoprotein as the acceptor. Optimal enzyme activity was obtained at pH 7.0-7.5, at 3 mM MnCl2, and at 0.08% Triton X-100. Ca2+, Mg2+, and Ba2+ also enhanced enzyme activity. The apparent Michaelis constant was 4.80 mM for freezing point depression glycoprotein, 0.59 mM for periodate-treated porcine submaxillary mucin, 0.49 mM for Gal beta 1----3 GalNAc alpha Ph, and 1.03 mM for UDP-GlcNAc. No enzyme activity was observed when asialo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was shown to be a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel filtration, and high-performance liquid chromatography. The structure of the trisaccharide was identified as Gal beta 1----3-(GlcNAc beta 1----6)GalNAc-H2 by (a) identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and 1,4,5-trimethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by gas-liquid chromatography-mass spectrometry and (b) the complete cleavage of the newly formed glycosidic bond by jack bean beta-hexosaminidase. The structure of the trisaccharide was confirmed by 1H nuclear magnetic resonance (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 N HCl hydrolysis, a second NaBH4 reduction, and the identification of threosaminitol on an amino acid analyzer. By acceptor competition studies, the enzyme activity was shown to be a much N-acetylglucosaminyltransferase. We postulate that this glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents – transforming growth factor β₁ (TGF-β₁), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) – on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-β₁; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-β₁, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of core 2 beta-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes.

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.     

25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.     

Pulse-chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2

We have used pulse-chase methodology to study the synthesis of apolipoprotein B in a human hepatoma-derived cell line, the Hep G2 cells. A 2-min pulse with [35S]methionine was followed by a chase period varying from 5-90 min. A protein of large molecular mass (estimated molecular mass: 312 +/- 41 kDa, mean +/- SD, n = 8) could be immunoprecipitated from the cells at all chase periods between 5 min and 60 min with both monoclonal antibodies to a narrow density cut of the low density lipoprotein LDL-2 (density: 1.030-1.055 g/ml) and polyclonal antibodies to the apolipoprotein B apo B 100 or to a narrow density cut of LDL-2 (density: 1.030-1.055 g/ml). In addition to this large molecular mass protein, nascent polypeptides could be precipitated after 5, 10 and 15 min of chase. The apolipoprotein B molecules that had been labelled during the pulse disappeared from the cells after 60-90 min of chase, while they started to appear in the medium after 30-35 min of chase. The results obtained indicate (a) that apolipoprotein B is synthesized as one polypeptide with a large molecular mass, (b) that newly synthesized apolipoprotein B molecules are secreted after a delay of 30-35 min, (c) that no intracellular accumulation of apolipoprotein B occurs, and (d) that apolipoprotein B is recovered in the density fraction less than 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.     

Augmentation of haptoglobin production in Hep3B cell line by a nuclear factor NF-IL6.

The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6-mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin.     

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.     

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.