Gain-of-function Chinese Hamster Ovary Mutants LEC18 and LEC14 Each Express a Novel N-Acetylglucosaminyltransferase Activity*

T. Shantha RajuDagger § and Pamela StanleyDagger

From the Dagger  Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461 and § Genentech Inc., South San Francisco, California 94080

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

LEC18 and LEC14 cells are gain-of-function glycosylation mutants isolated from Chinese hamster ovary cells for resistance to pea lectin. Structural studies have shown that LEC18 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-6 position of the core GlcNAc (Raju, T. S., Ray, M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-2 position of the core beta -linked Man residue (Raju, T. S., and Stanley, P. (1996) J. Biol. Chem. 271, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucosaminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to GlcNAcbeta 1-O-pNP and to a GlcNAc-terminating, biantennary, complex N-glycan, with or without a core fucose. By contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have significant activity with simple acceptors, and transfers GlcNAc preferentially to a GlcNAc-terminating biantennary glycopeptide that contains a core fucose residue. The acceptor specificities and other biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previously characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of transferases.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Glycosylation mutants of mammalian cells allow the identification of new molecules involved in complex glycan synthesis by revealing the nature of acceptor substrates, glycosyltransferases, co-factors, and regulatory molecules through mutations that alter, in each case, only one glycosyl transfer reaction. Both loss-of-function and gain-of-function mutants have revealed new aspects of glycosylation in mammals (1, 2).

Gain-of-function glycosylation mutants express an activity that is not detectable in the parental cell. Each of several Chinese hamster ovary (CHO)1 cell gain-of-function mutants express a glycosyltransferase activity that is lacking in parental CHO, and that synthesizes N-glycans with a sugar modification absent from the N-glycans of parent cell glycoproteins. Thus, the LEC10 CHO mutant (3, 4) expresses GlcNAc-TIII (5), and the N-glycans of glycoproteins made in LEC10 cells include a proportion that contain the bisecting GlcNAc (3). LEC11, LEC12, LEC29, and LEC30 CHO mutants each express an alpha (1,3)fucosyltransferase activity that is not detectable in parent CHO cells, and they synthesize N-glycans with fucose in O-3 linkage to the GlcNAc of lactosamine units (6-8).

LEC14 and LEC18 CHO mutants are gain-of-function mutants that were obtained, after mutagenesis, by selection for resistance to pea lectin (9). They have unique and distinct lectin resistance properties and behave dominantly in somatic cell hybrids formed with parent CHO cells (9). Structural analyses of complex N-glycans from LEC14 and LEC18 revealed that both add a specific sugar residue to N-glycans that is not present on N-glycans from CHO cell glycoproteins (10, 11). In LEC18 cells, a proportion of the complex, polylactosamine-containing N-glycans contain an additional GlcNAc in the core region at the O-6 position of the beta (1,4)-GlcNAc adjacent to the beta -linked core Man residue (Ref. 10; Fig. 1). This O-6-linked GlcNAc residue is absent from similar N-glycans of parental CHO cells and has not been observed on glycoproteins from other sources. In LEC14 cells, a proportion of the complex, polylactosamine-containing N-glycans contain an additional GlcNAc linked beta (1,2) to the beta (1,4)-Man residue of the core (Ref. 11; Fig. 1). CHO cells lack this modification, which has to date been observed only in N-glycans from LEC14 cells.

The novel N-glycan cores, which are synthesized by LEC14 and LEC18 mutants, suggested that each mutant expresses a GlcNAc-T activity that is silent in parental CHO cells, in a manner analogous to the previously described gain-of-function CHO mutants (2). In this paper we show that this is indeed the case. These novel activities, termed GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18), generate in vitro, the N-glycan core characteristic of LEC14 and LEC18 glycoproteins, respectively.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

UDP-6-[3H]GlcNAc (6.1 Ci/mmol), concanavalin A (ConA)-Sepharose, and Sephadex G-25 were from Amersham Pharmacia Biotech. Pea lectin (PSA)-agarose was from Vector Laboratories. Bio-Gel P-2 (45-90 mesh), the Bradford protein reagent, and AG1-X4 resin (200-400 mesh, Cl- form) were from Bio-Rad. N-Acetyl-beta -D-glucosaminidases (Diplococcus pneumoniae and bovine kidney), Pronase (Streptomyces griseus), and protease inhibitor mixture tablets were from Boehringer Mannheim or Prozyme, and jack bean N-acetyl-beta -D-glucosaminidase was from Oxford GlycoSciences. D-(+)-Glc, D-(+)-Gal, D-(+)-Man, D-(+)-Fuc, D-(+)-GlcN, D-(+)-GalN, methyl alpha -D-mannoside (MM), methyl alpha -D-glucoside, GlcNAcbeta 1-O-pNP, Galbeta 1-O-pNP, Glcbeta 1-O-pNP, Manbeta 1-O-pNP, GalNAcbeta 1-O-pNP, GlcNAcbeta 1,4GlcNAcbeta 1-O-pNP, GlcNAcbeta 1-S-pNP, GlcNAcbeta 1-O-benzyl, UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc, ATP, Triton X-100, Nonidet P-40, human IgG, human fibrinogen, chicken ovalbumin, chitin, chloramine T, potassium metabisulfite, and bovine serum albumin were from Sigma. Sodium hydroxide (50%, w/v) and other reagent grade chemicals were from Fisher.

Purification of Glycopeptides from Standard Glycoproteins

Oligomannosyl glycopeptides were prepared from chicken egg ovalbumin as described (12); biantennary N-linked glycopeptides (GnGn) with no fucose and terminating with GlcNAc were isolated from human fibrinogen as described (13) and the corresponding glycopeptides with fucose (GnGn(Fuc)) were prepared from human IgG. N,N'-Diacetylchitobiose and N,N',N"-triacetylchitotriose were isolated from chitin essentially as described by Rupley et al. (14). Glycopeptides with the structures shown in Fig. 1 were obtained from glycoproteins of LEC14 and LEC18 cells as described (10, 11).

Cell Lines and Cell Culture

CHO cells were grown in suspension at 37 °C in complete alpha  medium (Life Technologies, Inc.) containing 10% bovine calf serum. Parent CHO (Pro-5) and Pro-LEC18.21B, Pro-LEC14.4A, and Pro-LEC10.3C mutant CHO cells were described previously (3, 9).

Preparation of Cell Extracts

Postnuclear supernatant from LEC18, LEC14, and parent CHO cells was prepared essentially as described (15). Briefly, cells (~6 × 106) were washed two times with saline, followed by one wash with homogenizing buffer (10 mM Tris-HCl, pH 7.4, with 250 mM sucrose), and incubated in 1 ml of homogenizing buffer on ice. After 20 min, the swollen cells were homogenized using a Balch Homogenizer (Industrial Tectonics Inc., Dexter, Michigan; basic diameter = 0.2489 inches, tungsten carbide, part p592492) at 4 °C. The lysate was centrifuged at 3000 rpm for 30 min at 4 °C. Glycerol was added to the supernatant to a final concentration of 20%, before storage at -70 °C. For preparation of microsomal membranes, postnuclear supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. For LEC10 cells, extraction after cell washing was in 1.5% Triton X-100, to which glycerol was added to 20% final volume before storage at -70 °C.

N-Acetylglucosaminyltransferase Assays

Enzyme assays with cell extracts were carried out in 1-ml Eppendorf tubes in a 50-µl reaction volume containing 12.5 nmol of UDP-[3H]GlcNAc (~5500 cpm/nmol), 1.5 µmol of MnCl2, 0.5 µmol of ATP, 0.5% Triton X-100, 0.5 µmol of sodium cacodylate buffer, pH 7.0, 5-10 µl of extract (~50-100 µg of protein), and one of the following as acceptor molecule: 50 nmol of GlcNAc, GlcNAcbeta 1-O-pNP, Galbeta 1-O-pNP, Glcbeta 1-O-pNP, Manbeta 1-O-pNP, GlcNAcbeta 1-S-pNP, GlcNAcbeta 1-O-benzyl, or ~74 nmol of GnGn or GnGn(Fuc) for 90 min at 37 °C. To assay GlcNAc-TIII activity, ~50-100 µg of LEC10 extract was incubated in a final volume of 40 µl with ~40-180 nmol of GnGn or GnGn(Fuc), in the presence of 50 mM PIPES buffer, pH 7.0, protease inhibitors (Boehringer Mannheim tablet) according to manufacturer's instructions, 0.5% Triton X-100, 10 mM MnCl2, 0.2 M GlcNAc, and 24 nmol of UDP-GlcNAc (~25,000 cpm/nmol) for 2 h at 37 °C. Assays lacking acceptor were used to determine incorporation into endogenous acceptors and degradation of donor sugar. After incubation at 37 °C for 15-120 min, the reaction was stopped by adding 950 µl of cold water. Reactions containing simple sugars or the glycopeptides, GnGn or GnGn(Fuc) were passed through a 1-ml column of AG1-X4 (Cl- form) that was subsequently washed with 3 ml of water. Eluate and washings were combined, mixed with 17 ml of Ecolume, and counted by liquid scintillation spectroscopy. Reactions with GlcNAcbeta 1-O-pNP, GlcNAcbeta 1-S-pNP, and GlcNAcbeta 1-O-benzyl acceptors were passed through a Sep-Pak C18 cartridge (Waters), and radiolabeled product was eluted with 50% aqueous methanol as described (16). Radioactivity was measured by liquid scintillation spectroscopy.

Product Characterization

Size-exclusion Chromatography-- To characterize GlcNAc-T reaction products, terminated reactions were pooled, concentrated to 2 ml, and desalted on a Bio-Gel P-2 column (1.5 cm × 70 cm). The column was eluted under pressure with glass distilled water at a flow rate of 15-20 ml/h, and fractions of 2 ml were collected. Under these conditions, charged molecules may interact with the acrylamide and elute anomalously rather than according to molecular weight. A portion of each fraction (0.5 ml) was mixed with 5 ml of Ecolume, and radioactivity was counted by liquid scintillation spectroscopy.

Lectin Affinity Chromatography-- Assay tubes, in which GnGn or GnGn(Fuc) were acceptors, were adjusted to 1 ml with cold water and passed through a 1-ml column of AG1-X4 resin (Cl- form) that was subsequently washed with 3 ml of water. Combined eluate and washings were concentrated to ~250 µl, mixed with 2× ConA buffer (0.2 M sodium acetate, 0.02 M MgCl2, 0.02 M CaCl2, 0.02 M MnCl2, 0.04% sodium azide, pH 7.3), and applied to a ConA-Sepharose column (0.5 cm × 20 cm). The column was washed with at least 10 column volumes of ConA buffer before bound glycopeptides were eluted with at least 4 column volumes of ConA buffer containing 10 mM methyl-alpha -D-glucoside, followed by ConA buffer containing 10 mM MM, and finally 200 mM MM in ConA buffer. Reaction products were also fractionated on PSA-agarose (0.5 cm × 20 cm) and eluted with ConA buffer followed by ConA buffer containing 200 mM MM. Fractions of 1 ml were collected, and a portion was counted by scintillation spectroscopy. Pooled products from lectin columns were desalted on Bio-Gel P-2 (1.5 cm × 70 cm) and characterized by glycopeptide mapping using a Dionex HPAEC-PAD.

Glycopeptide Mapping by Dionex HPAEC-PAD-- Product analysis by HPAEC-PAD was performed using a Dionex Bio-LC gradient pump and a pulsed amperometric model PAD-2 detector (Dionex Corp., Sunnyvale, CA) as described (17). Briefly, GlcNAc-T reactions containing GlcNAcbeta 1-O-pNP acceptor were passed through a Sep-Pak C18 cartridge, and the 50% aqueous methanol eluant was dried using a Savant Speed Vac, resuspended in 50-100 µl of glass distilled water, passed through a Centrex filter (Schleicher & Schuell), and analyzed by HPAEC-PAD using a CarboPac PA-10 (4 mm × 250 mm) pellicular anion-exchange column equipped with a CarboPac guard column. The column was eluted with 15 mM NaOH generated from 15% eluant 1 (100 mM NaOH) and 85% eluant 2 (water) for 25 min, followed by eluant 3 (500 mM NaOH) for 10 min, at a flow rate of 0.9 ml/min. The following pulse potentials and durations were used: E1 = 0.05 V (t1 = 300 ms); E2 = 0.65 V (t2 = 180 ms); E3 = -0.65 V (t3 = 60 ms). Detection was with 1000 nm full scale. A Dionex Advanced Computer Interface connected to a Gateway 2000, 4SX-33V computer with Dionex AI-450 software (release 3.32.00) was used to collect the data. For radioactivity measurement, fractions of 0.5 min (0.45 ml), were collected, mixed with 5 ml of Ecolume, and counted by liquid scintillation spectrometry.

Transferase reactions containing GnGn or GnGn(Fuc) glycopeptide acceptors were passed through a 1-ml column of AG1-X4 resin (Cl- form) that was subsequently washed with 3 ml of water. The combined eluate and washings were concentrated to ~1 ml, desalted on Bio-Gel P-2 (1.5 × 70 cm), dried, resuspended in 50-100 µl of glass distilled water, passed through a Centrex filter, and analyzed by HPAEC-PAD. A CarboPac PA-1 column (4 mm × 250 mm) was used with 100 mM NaOH (eluant 1) and 100 mM NaOH containing 1 M NaOAc (eluant 4). Elution was isocratic with 2% eluant 4 for 10 min, linearly increasing eluant 4 to 80% over 60 min and, finally, 80% eluant 4 isocratically for another 20 min. For radioactivity determination, fractions of 1 min were collected and counted by scintillation spectroscopy.

Temperature and Chemical Inactivation of GlcNAc-T Activity

The novel GlcNAc-T activity in LEC18 cells transferred GlcNAc to both GlcNAcbeta 1-O-pNP and to a glycopeptide substrate (see "Results"). To investigate whether a single GlcNAc-T performed both reactions, the activities were compared following treatment with increasing temperatures or with the oxidizing agent chloramine T (18). For heat inactivation, LEC18 cell extracts (~500 µg of protein in 50 µl of extraction buffer/tube in duplicate) were placed in a water bath set at the appropriate temperature for 10 min before being returned to 4 °C and then assayed at 37 °C under standard conditions with both GlcNAcbeta 1-O-pNP and glycopeptide acceptor GnGn, and in the absence of acceptor. For chemical inactivation, LEC18 cell extract (~300 µg of protein in 30 µl of extraction buffer/tube in duplicate) were incubated on ice with 15 µl of buffer alone or 15 µl of buffer containing increasing concentrations of chloramine T. After 5 min at 4 °C, 15 µl of potassium metabisulfite at the same concentration as the chloramine T was added to stop the oxidation reaction. Subsequently, treated extracts were assayed under standard conditions with both GlcNAcbeta 1-O-pNP and GnGn acceptors, and in the absence of acceptor.

Product Analysis following N-Acetyl-beta -D-Glucosaminidase Treatment

Transferase reactions containing GnGn or GnGn(Fuc) acceptors were passed through a 1-ml column of AG1-X4 resin (Cl- form), washed with 3 ml of water, and the combined eluate was desalted on Bio-Gel P-2, dried, and redissolved in buffer appropriate for digestion with different N-acetyl-beta -D-glucosaminidases. Jack bean N-acetyl-beta -D-glucosaminidase digestion was performed in 50-100 µl of sodium citrate phosphate buffer (pH 4.5) at 37 °C under toluene for 48 h with a total of 50 milliunits of enzyme. D. pneumoniae or bovine kidney N-acetyl-beta -D-hexosaminidase digestion was performed in 50-100 µl of sodium citrate phosphate buffer (pH 5.0) at 37 °C under toluene for 25-48 h with a total of 50-100 milliunits of enzyme. Digestion was stopped by heating in a boiling water bath for 5 min, and products were desalted on a Bio-Gel P-2 column (1.5 cm × 70.0 cm) before analysis by HPAEC-PAD as described above for GnGn and GnGn(Fuc) product analysis.

To determine if the LEC18 product from GlcNAcbeta 1-O-pNP was susceptible to digestion with N-acetyl-beta -D-glucosaminidase, ~3,500 cpm product was digested for 24 h under the conditions described above with N-acetyl-beta -D-glucosaminidase from D. pneumoniae. As controls, N,N'-diacetylchitobiose, N,N',N"-triacetylchitotriose, and N,N'-diacetylchitobiosebeta 1-O-pNP (100 µg each) were digested under the same conditions with either jack bean or bovine testis or D. pneumoniae N-acetyl-beta -D-glucosaminidase. Chitobiose and chitotriose digests were dried, resuspended in 15 µl of 15% aqueous acetic acid containing 9-aminopyrene 1,3,5-trisulfonate to which ~5 µl of sodium cyanoborohydride in THF was added, and incubated at 65 °C for 2 h. The derivatization reaction was stopped by adding 0.5 ml of cold water. The mixture was analyzed using a capillary electropherograph (P/ACE System 5000, Beckman Instruments, Palo Alto, CA) equipped with an argon ion laser induced fluorescence detector and a coated capillary at 20 kV. The digests of LEC18 product from GlcNAcbeta 1-O-pNP and the chitobiosebeta 1-O-pNP standard were analyzed by Dionex HPAEC-PAD as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

LEC18 Cells Have a GlcNAc-T Activity Undetectable in CHO Cells-- A subset of complex N-glycans in LEC18 glycoproteins carry a GlcNAc linked to the O-6 position of the core GlcNAc residue (Fig. 1). A GlcNAc-T capable of performing this transfer might act on simple monosaccharide acceptors, similar to chitin synthases (19, 20), or to the GlcNAc-T from the snail, Lymnaea stagnalis, that transfers GlcNAc in beta 1,4-linkage to simple GlcNAc derivatives and to terminal GlcNAc residues of N- and O-glycans (21-23). Therefore, extracts from LEC18 cells were tested for their ability to transfer GlcNAc to simple monosaccharide acceptors.


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Fig. 1.   N-Glycan cores of LEC10, LEC14, and LEC18 glycopeptides. The core GlcNAc residue attached to GnGn(Fuc) that is characteristic of LEC10, LEC14, and LEC18 CHO N-glycans is shown in bold. These structures have been previously established by a variety of techniques (3, 10, 11). These and related glycopeptides were used as standards in Figs. 6 and 7.

Initial assays, under conditions similar to those used for the snail GlcNAc-T (21), revealed that, among a variety of simple sugars, GlcNAcbeta 1-O-pNP was the only significant acceptor of GlcNAc with LEC18 cell extract, and that parent CHO cell extract had no comparable activity. Interestingly, LEC18 extract had no detectable activity with GlcNAcbeta 1-S-pNP, whereas this acceptor is optimum for the snail GlcNAc-T (21). The LEC18 GlcNAc-T also did not transfer GlcNAc at significant levels under these assay conditions to free GlcNAc, chitobiose (GlcNAcbeta 1,4GlcNAc), or chitotriose (GlcNAcbeta 1,4Gl cNAcbeta 1,4GlcNAc), showing that it also differs from chitin synthase activities (19, 20). Several other simple oligosaccharides were also not acceptors (Table I), and neither UDP-Glc nor UDP-GalNAc could substitute for UDP-GlcNAc as a donor sugar (data not shown).

                              
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Table I
LEC18 cell extract transfers GlcNAc to GlcNAcbeta 1-O-pNP
Cell extracts were incubated with ~50 nmol of acceptor as described under "Experimental Procedures." Duplicates were performed and the results shown are the average of at least two and up to six independent experiments.

Conditions for optimal solubilization of the LEC18 GlcNAc-T were tested with 14 different detergents. At 0.5% final concentration, maximum activity was obtained with Triton X-100 followed by Surfactant Amps-PX, Lubrol, and Nonidet P-40. A dose-response curve showed that 0.5% was the optimal concentration for Triton X-100. In the absence of detergent, no activity was obtained, and, when microsomal membranes prepared by centrifugation from the postnuclear supernatant were assayed in 0.5% Triton X-100, 98-99% of the LEC18 GlcNAc-T activity was membrane-associated.

A variety of organic buffers at 10 mM and pH 7.0 or 7.5 gave similarly good activity, but there was no activity in phosphate-buffered saline, pH 7.0. In 20 mM sodium cacodylate buffer, LEC18 GlcNAc-T activity gave a relatively sharp optimum at pH 7.0 (Fig. 2A), and had an absolute requirement for Mn2+, with a rather broad optimum between 20 mM and 60 mM (Fig. 2B). By contrast, the snail beta (1,4)GlcNAc-T is strongly inhibited at 40 mM MnCl2 (21). Like the snail GlcNAc-T, the LEC18 GlcNAc-T requires the presence of ATP (Fig. 2C). In its absence no transfer was observed, and high concentrations were also inhibitory. The optimum concentration of ATP was 10 mM (Fig. 2C).


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Fig. 2.   Optimum pH, Mn2+, and ATP concentrations for the LEC18 GlcNAc-T. The acceptor GlcNAcbeta 1-O-pNP (~50 nmol) was incubated with LEC18 extract (~100 µg of protein) in 0.5 µmol of sodium cacodylate of varying pH (A) under the conditions described under "Experimental Procedures." Similar results were obtained in two experiments. GlcNAc-T activity was also determined in LEC18 cell extract under conditions in which MnCl2 (B) or ATP (C) concentration was varied. Similar results were obtained in three separate experiments.

The transfer of GlcNAc to GlcNAcbeta 1-O-pNP by LEC18 cell extract was linear for 20 min and stayed constant from 40 to 100 min under the conditions used in Fig. 2B. To determine apparent Km values, LEC18 extract was incubated with increasing concentrations of either acceptor or donor substrate (Fig. 3, A and B). Maximal activity was obtained at ~0.22 mM UDP-GlcNAc and ~0.8 mM GlcNAcbeta 1-O-pNP. From Lineweaver-Burk plots, the apparent Km for UDP-GlcNAc was calculated as 1.1 mM (Vmax = 10 nmol/mg/h) and for GlcNAcbeta 1-O-pNP as 1.8 mM (Vmax = 20 nmol/mg/h), respectively.


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Fig. 3.   Kinetic analysis of the LEC18 GlcNAc-T. Using optimal assay conditions and 1 mM GlcNAcbeta 1-O-pNP, the UDP-[3H]GlcNAc concentration was varied (A); under the same conditions with 0.25 mM UDP-[3H]GlcNAc, the GlcNAcbeta 1-O-pNP concentration was varied (B).

Under the optimal conditions established for LEC18 extract, parent CHO extract routinely produced a small amount of labeled material (Table I). However, fractionation on Bio-Gel P-2 showed that CHO "product" included a substantial amount of unutilized UDP-GlcNAc, as well as a small amount of material that eluted one fraction beyond the product from LEC18 extract (Fig. 4A). When the latter was analyzed on Dionex HPAEC-PAD, it was clear that CHO extract produced no oligosaccharide product, because all label eluting with product on Bio-Gel P-2 was found to elute at the position of free GlcNAc on Dionex HPAEC-PAD (Fig. 4B). By contrast, LEC18 product from Bio-Gel-P2 eluted on Dionex HPAEC-PAD prior to the substrate GlcNAcbeta 1-O-pNP, and after authentic GlcNAcbeta 1,4GlcNAcbeta 1-O-pNP, consistent with its being the predicted product, GlcNAcalpha /beta 1,6GlcNAcbeta 1-O-pNP, for which no standard was available. Digestion of the LEC18 product with N-acetyl-beta -hexosaminidases from D. pneumoniae failed to release GlcNAc (data not shown). However, this result is not surprising as digestion of chitobiose or chitotriose with N-acetylglucosaminidases from three sources also failed to release GlcNAc, as measured by capillary electrophoresis. It seems that the terminal GlcNAc in such linear oligosaccharides is highly resistant to removal by beta -hexosaminidases.


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Fig. 4.   Product analysis for LEC18 GlcNAc-T by Bio-Gel P2 chromatography. LEC18 and CHO extracts were incubated with GlcNAcbeta 1-O-pNP (~50 nmol) under optimal assay conditions for 2 h. At termination, 2 tubes of LEC18 and 10 tubes of CHO were separately combined, concentrated to 2 ml, and desalted, on a BioGel P-2 column eluted in water. UDP-GlcNAc elutes anomalously under these conditions due to an interaction with BioGel-P-2 (A). Products (fractions 25-30) were pooled as indicated, concentrated, and resuspended in 100 µl of water, and 50 µl was analyzed on Dionex HPAEC-PAD (B) as described under "Experimental Procedures." The elution positions of standard markers, GlcNAcbeta 1-O-pNP, and GlcNAcbeta 1,4-GlcNAcbeta 1-O-pNP are indicated. Under these conditions, free GlcNAc eluted at ~22 min. The parent "product" eluted as free *GlcNAc only.

LEC18 and LEC14 Cells Have Distinct GlcNAc-Ts That Act on the Biantennary N-Glycan GnGn(Fuc)-- Attempts to identify a GlcNAc-T that transferred GlcNAc to simple sugar acceptors in LEC14 extracts failed (data not shown). Therefore, acceptors that could potentially be biosynthetic intermediates were tested. The novel GlcNAc residues found in the core of LEC14 and LEC18 N-glycans (Fig. 1) are somewhat analogous to the bisecting GlcNAc (Fig. 1), in the case of LEC14, and to a fucose that modifies the core GlcNAc (24), in the case of LEC18. GlcNAc-TIII transfers the bisecting GlcNAc to the O-4 position of Man in GnGn (3, 5), and GnGn is also the preferred substrate of core alpha (1,6)Fuc-T (25) and core beta (1,2)xylosyl-T (26). It has previously been established that parent CHO extract does not transfer GlcNAc to GnGn under assay conditions in which GlcNAc-TIII in LEC10 extract transfers the bisecting GlcNAc at a specific activity of 6-10 nmol/mg/h (3, 4). Therefore, the branching transferases, GlcNAc-TIV and GlcNAc-TV, which are certainly active in CHO cells based on structural studies of CHO-derived glycoproteins (for example Ref. 27), and from assays with GnGn and CHO extracts performed under different conditions (15), were not detected under the conditions optimal for the LEC18 GlcNAc-T.

When LEC18 and LEC14 extracts were incubated with GnGn(Fuc) and UDP-[3H]GlcNAc, both extracts gave significantly more transfer than did parent CHO extract (Table II). Mixing experiments showed that there was no inhibitor in parent CHO extracts (Table II). Glycopeptide products of these assays were therefore characterized by lectin affinity chromatography on ConA-Sepharose and PSA-agarose, and by oligomapping on Dionex HPAEC-PAD, both before and after digestion with beta -hexosaminidases.

                              
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Table II
GlcNAc-T activity of LEC18, LEC14, and parent CHO extracts with the biantennary glycopeptide GnGn(Fuc).
Cell extracts containing ~100 µg of protein were incubated with GnGn(Fuc) as described under "Experimental Procedures." For mixed reactions, ~50 µg of protein from each extract was combined. The results are from one experiment and were reproduced once.

Biantennary glycopeptides like GnGn and GnGn(Fuc) bind to ConA-Sepharose even if they contain a bisecting GlcNAc (3, 4). However, GnGn(Fuc) glycopeptides from LEC14 and LEC18 cells that contain an extra core GlcNAc do not bind to ConA-Sepharose (10, 11). When the products of GlcNAc-T assays with GnGn(Fuc) were chromatographed on ConA-Sepharose, >90% of the counts from parent CHO products bound to ConA (data not shown), suggesting that they arose not by the addition of a branching GlcNAc, but by replacement of a GlcNAc in the acceptor due to the action of GlcNAc-TI or GlcNAc-TII (reviewed in Ref. 28). Approximately 20-30% of LEC14 and LEC18 products also bound to ConA (data not shown), presumably for the same reason. However, the majority of the LEC14 and LEC18 products (70-80%) did not bind to ConA, as expected if they contained the additional core GlcNAc.

The biantennary N-glycan glycopeptides from LEC14 and LEC18, which carry an extra core GlcNAc (Fig. 1), were also shown previously not to bind to PSA-agarose (10, 11). Thus authentic products of a novel GlcNAc-T in LEC14 and LEC18 cells should not bind to PSA-agarose. Consistent with this, a majority of the reaction products from LEC18 and LEC14 extracts did not bind to PSA-agarose (Fig. 5). By contrast, almost all the products from CHO extracts bound to this column.


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Fig. 5.   Product analysis by PSA-agarose affinity chromatography. LEC14, LEC18, and CHO extracts were incubated with GnGn(Fuc) (~74 nmol) for 2 h under the conditions described under "Experimental Procedures." Products were partially purified on Bio-Gel P-2 as shown in Fig. 4, and subsequently analyzed on PSA-agarose. Products that bound to PSA-agarose were eluted with 200 mM MM. Shown are the products from 10 assay tubes of CHO extract and 2 assay tubes from each of LEC14 and LEC18 extracts.

To further characterize GlcNAc-T assay products, they were analyzed by oligomapping on a Dionex HPAEC-PAD (17) after partial purification by chromatography on Bio-Gel P-2. Structurally characterized glycopeptides shown in Fig. 1, as well as GnGn and GnGn(Fuc), were used to calibrate the column (Fig. 6). All extracts gave rise to a small amount of product eluting at ~21 min. This may be triantennary glycopeptides arising from the action of GlcNAc-TIV and/or GlcNAc-TV. However, the major product of each cell extract eluted at a unique position. The major CHO product eluted earliest, at the same position as authentic GnGn(Fuc), providing strong evidence that it had acquired a labeled GlcNAc by hydrolysis and re-addition of a GlcNAc residue; the major product from LEC14 extract eluted at the position of authentic LEC14 glycopeptide at 22 min (Ref. 11; Fig. 1); the LEC18 product interacted most tightly with the column, eluting at ~42 min at the position of authentic LEC18 glycopeptide (Ref. 10; Fig. 1); and LEC10 product containing the bisecting GlcNAc (Fig. 1), eluted at 29 min (Fig. 6). These data clearly show that CHO extract has no activity similar to GlcNAc-TIII or LEC14 or LEC18 GlcNAc-Ts. In addition, the major product from LEC10, LEC14 and LEC18 eluted beyond GnGn(Fuc) at the position predicted from the respective glycopeptide standards.


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Fig. 6.   Product analysis by glycopeptide mapping on Dionex HPAEC-PAD. The acceptor GnGn(Fuc) (~74 nmol) was incubated with LEC10, LEC14, LEC18, and CHO cell extract (~100 µg of protein) under optimized conditions for 2 h as described under "Experimental Procedures." Reaction products, partially purified on Bio-Gel P-2 were analyzed by Dionex HPAEC-PAD. Ten tubes from parent CHO extract and 2 tubes from each of LEC10, LEC14 and LEC18 extracts were analyzed. Standard, structurally characterized glycopeptides GnGn(Fuc), LEC18 core N-glycan (10), and LEC14 core N-glycan (11) designated by symbols were shown to elute at the position of the major product in the respective panels (square , GlcNAc; open circle , Man; triangle , Fuc; see Fig. 1).

Further characterization of GlcNAc-T products was obtained by digestion with N-acetyl-beta -D-hexosaminidases from jack bean, bovine kidney, or D. pneumoniae (Fig. 7). CHO product was converted almost completely to free GlcNAc by hexosaminidase treatment, as predicted if the CHO product was labeled GnGn(Fuc). The LEC10 product was also completely digested to give free GlcNAc, as expected because the bisecting GlcNAc is susceptible to beta -hexosaminidases (29, 30). Some of the LEC14 products were hydrolyzed to release GlcNAc, but the majority were resistant to digestion and eluted at the same position as untreated product. This is exactly what was observed in the N-glycans characteristic of LEC14 glycoproteins (11). Despite exhaustive digestion with jack bean and D. pneumoniae hexosaminidases, the major LEC14 N-glycans with a beta 1,2-linked core GlcNAc remained unaffected (11). The combined evidence shows that LEC14 cells express a novel GlcNAc-T activity that is undetectable in CHO cells, LEC10 cells, or LEC18 cells. This new transferase activity will henceforth be called GlcNAc-TVII.


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Fig. 7.   Product analysis by Dionex HPAEC-PAD after digestion with N-acetyl-beta -D-hexosaminidase. Cell extracts were incubated with GnGn(Fuc) (~74 nmol) under optimal conditions for 2 h. Reaction products from 2 tubes of LEC10, LEC14, or LEC18 and 10 tubes parent CHO were separately combined and passed through a 1-ml AG1-X4 (Cl-) column. The flow-through was desalted on Bio-Gel P-2. Products were treated with N-acetyl-beta -D-glucosaminidase (100 milliunits, D. pneumoniae) in 50 µl of citrate-phosphate buffer, pH 4.5, at 37 °C for 24 h. Reaction mixtures were boiled, centrifuged, desalted on Bio-Gel P-2, and analyzed by Dionex HPAEC-PAD. Similar results were obtained after treating GlcNAc-T products with N-acetyl beta -D-glucosaminidase from jack bean or bovine kidney. Elution positions of GlcNAc and authentic glycopeptides are shown as in Fig. 6.

The products of LEC18 extract also behaved as expected (Fig. 7). Hexosaminidase digestion almost completely eliminated the major LEC18 product that eluted at 42 min in Fig. 6. Previous results have shown that LEC18 N-glycans containing the extra core GlcNAc are relatively resistant to hexosaminidase digestion (10), but prolonged digestion removes both arm beta 1,2-linked GlcNAc residues, although it does not remove the extra core GlcNAc residue, which is highly resistant to digestion (10). Thus, the major product of beta -hexosaminidase digestion of LEC18 N-glycans is Man3GlcNAc3(Fuc)Asn (10). This glycopeptide, previously characterized by mass spectrometry and composition analysis (10), was shown to elute from the glycopeptide mapping column at 16 min, the same position as the major digestion product in Fig. 7. The combined results provide strong evidence that the LEC18 GlcNAc-T indeed synthesizes the novel N-glycan core of LEC18 cells. This represents a GlcNAc-T activity not present in CHO, LEC10, or LEC14 cells that will henceforth be known as GlcNAc-TVIII.

GlcNAc-TVIII Is Responsible For Transferring GlcNAc to GlcNAcbeta 1-O-pNP and to the Core of a N-Glycan-- Because LEC18 extract transfers GlcNAc to GlcNAcbeta 1-O-pNP, the question arose as to whether LEC18 extracts possess two novel GlcNAc-Ts: GlcNAc-TVIII that transfers GlcNAc to the core of N-glycans and another GlcNAc-T that transfers GlcNAc to GlcNAcb1-O-pNP. This question was addressed by comparing the heat and chemical inactivation profiles of LEC18 extracts with both acceptors. The data in Fig. 8 (upper panel) show that increasing temperature reduced GlcNAc transfer by LEC18 cell extract to GlcNAcbeta 1-O-pNP and to the N-glycan GnGn to a similar degree. In like manner, when the oxidizing agent chloramine T was used to inactivate enzyme activity (18), the reduction in GlcNAc transfer to GlcNAcbeta 1-O-pNP and to GnGn was essentially identical (Fig. 8, lower panel). Therefore, LEC18 cells contain one activity, GlcNAc-TVIII, that can transfer GlcNAc to both a simple beta -linked GlcNAc and to the predicted physiological acceptor, GnGn.


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Fig. 8.   GlcNAc-TVIII transfers GlcNAc to both GlcNAcb1-O-pNP and to GnGn. To obtain a heat inactivation profile, LEC18 cell extract was treated for 10 min at increasing temperatures before being assayed under standard conditions for transfer of GlcNAc to GlcNAcbeta 1-O-pNP or GnGn as described under "Experimental Procedures." Specific activity for the 100% value was 2.8 nmol/mg/h for GlcNAcbeta 1-O-pNP and 6.1 nmol/mg/h for GnGn. For chemical inactivation, LEC18 cell extract was treated with increasing concentrations of chloramine T for 5 min on ice, potassium metabisulfite was added, and the extract was assayed for transfer of GlcNAc to GlcNAcbeta 1-O-pNP or GnGn as described under "Experimental Procedures." Specific activity at 100% was 2.1 nmol/mg/h for GlcNAcbeta 1-O-pNP and 5.7 nmol/mg/h for GnGn. The results are the average of one or two experiments performed in duplicate. Other experiments gave similar results.

GlcNAc-TVII from LEC14 Cells, GlcNAc-TVIII from LEC18 Cells, and GlcNAc-TIII from LEC10 Cells Exhibit Distinct Acceptor Preferences-- The data in Table II show that GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18) transfer GlcNAc to GnGn(Fuc) to different extents, indicating that the GlcNAc-Ts may not be identical in their ability to act on this acceptor. Therefore, specific activities were determined over a range of GnGn(Fuc) concentrations (Fig. 9, upper panel). Lineweaver-Burk plots revealed apparent Km values of 5 mM and 6.3 mM with a Vmax in each case of ~25 nmol/mg/h for GlcNAc-TVII and GlcNAc-TVIII, respectively. Most interestingly, when the same experiment was performed with GnGn lacking the core Fuc, LEC18 GlcNAc-TVIII had approximately similar activities (Vmax = 29 nmol/mg/h; Km(app) = 11 mM), whereas LEC14 GlcNAc-TVII utilized this acceptor very poorly (Fig. 9, lower panel). It seems that GlcNAc-TVII strongly prefers a core glycopeptide acceptor that contains an alpha (1,6)-linked core fucose, and suggests that this transferase may not act until after the addition of this fucose to N-glycan cores. This is entirely consistent with the fact that LEC14 glycopeptides with the beta 1,2-linked core GlcNAc were core fucosylated (11). Interestingly, glycopeptides containing the core GlcNAc unique to LEC18 glycoproteins were also core fucosylated (10). However, in contrast to GlcNAc-TVII, GlcNAc-TVIII utilized GnGn and GnGn(Fuc) rather equivalently in vitro (Fig. 9).


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Fig. 9.   GnGn and GnGn(Fuc) as acceptors for GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18). Acceptors GnGn or GnGn(Fuc) were varied from ~20 to ~200 nmol/reaction under the optimal assay conditions described under "Experimental Procedures."

Under our assay conditions, neither GlcNAc-TVII nor GlcNAc-TVIII transferred GlcNAc to Man5GlcNAc2Asn, the intermediate acted on by GlcNAc-TI (see Ref. 28), despite the fact that the sugar residues to which both enzymes transfer are present in Man5GlcNAc2Asn (Table I). This N-glycan intermediate will also not serve as an acceptor for GlcNAc-TIII (reviewed in Ref. 31). However, GlcNAc-TVII and GlcNAc-TVIII are clearly distinct transferases from GlcNAc-TIII, as became apparent when GnGn and GnGn(Fuc) were compared as acceptors for GlcNAc-TIII. At saturating levels of glycopeptide acceptor, GlcNAc-TVII transferred GlcNAc preferentially to GnGn(Fuc) compared with GnGn, GlcNAc-TVIII transferred well to GnGn(Fuc) and had good activity with GnGn, whereas GlcNAc-TIII from LEC10 cells transferred the bisecting GlcNAc preferentially to GnGn (Fig. 10). These results suggest that each transferase recognizes a different aspect of the GnGn(Fuc) core acceptor.


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Fig. 10.   Different acceptor specificities of GlcNAc-TIII, GlcNAc-TVII, and GlcNAc-TVIII. Acceptors GnGn or GnGn(Fuc) were assayed at 3 equivalent concentrations under optional conditions (see "Experimental Procedures") for the transferase activities GlcNAc-TIII, GlcNAc-TVII, and GlcNAc-TVIII in LEC10, LEC14, and LEC18 extracts, respectively.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Molecules that participate in glycosylation reactions responsible for the synthesis of complex glycans are identified from knowledge of glycan structures that act as acceptors in assays for glycosyltransferases, by characterizing the products of new glycosyltransferase genes, and by determining the biochemical basis of mutations that affect individual glycosylation reactions. The advantage of the mutant strategy is that it often leads to the identification of an activity for which existence would not be predicted from structural studies because the activity generates an intermediate never found on a mature glycoconjugate, or because glycoconjugates bearing the modification generated by that activity have never been isolated. This latter situation is the case with LEC14 and LEC18 CHO cells, which we have shown in this paper to express two new GlcNAc-T activities termed GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18).

Previous studies of the N-glycans present on LEC14 and LEC18 glycoproteins showed that each of these mutants adds a GlcNAc to the core of N-glycans in a linkage not observed in previously characterized N-glycans (Fig. 1; Refs. 10 and 11). Based on the properties of similar gain-of-function CHO glycosylation mutants (2), the biochemical basis for the LEC14 and LEC18 phenotypes was predicted to be the de novo expression of a glycosyltransferase activity (10, 11). In vitro assays with detergent extracts or microsomal membranes have now shown that GlcNAc-TVII and GlcNAc-TVIII are unique to LEC14 and LEC18 cells, respectively. They appear to be Golgi transferases because of their membrane association and because biantennary glycopeptides that are generated in the medial or trans Golgi are acceptors.

The major product obtained from GnGn(Fuc) by GlcNAc-TVIII behaved exactly as authentic LEC18-derived N-glycans described previously (10); it did not bind to either ConA-Sepharose or PSA-agarose, eluted at the expected position from CarboPac PA-1, and digestion with N-acetyl-beta -glucosaminidases released only the arm GlcNAcs (Fig. 7). GlcNAc-TVIII is a GlcNAc-to-GlcNAc transferase as is the snail beta (1,4)GlcNAc-T (21-23), and, like the snail enzyme, GlcNAc-TVIII utilizes simple GlcNAcbeta 1-O-pNP and glycopeptides as acceptors (Table I). The snail beta (1,4)GlcNAc-T is cloned, and it is therefore quite clear that the same enzyme transfers to both simple and complex acceptors. The heat and chemical inactivation profiles of LEC18 cell extract (Fig. 8) provide strong evidence that GlcNAc-TVIII transfers to GlcNAcbeta 1-O-pNP and to N-glycan acceptors.

GlcNAc-TVII in LEC14 cells does not act on simple acceptors and is similar in this regard to most GlcNAc-Ts (31). Neither parent CHO nor LEC18 cells possess GlcNAc-TVII activity, which is unique to LEC14. The major product obtained from the action of GlcNAc-TVII on GnGn(Fuc) behaved as expected (11); it did not bind to ConA-Sepharose or PSA-agarose, it eluted from CarboPac PA-1 at the same position as the authentic LEC14 glycopeptide shown in Fig. 1, and it was completely resistant to digestion with N-acetyl-beta -glucosaminidases (Fig. 7). GlcNAc-TVII is a GlcNAc-to-Man transferase and catalyzes a reaction most similar to a beta (1,2)xylosyltransferase recently purified from plants (26). However, in contrast to the beta (1,2)xylosyltransferase, which utilizes GnGn and GnGn(Fuc) equivalently as acceptors, GlcNAc-TVII strongly prefers GnGn(Fuc).

Two outcomes of this study are of some interest with respect to the properties of mammalian GlcNAc-Ts. The first is that GlcNAc-TI and GlcNAc-TIII are not active under the in vitro conditions optimal for GlcNAc-TVII and GlcNAc-TVIII (Table I),2 and GlcNAc-TIV and GlcNAc-TV activities are minimal under these conditions (Fig. 6). The second is the clear difference in acceptor preference of the three GlcNAc-Ts that modify the core of N-glycans. When presented with GnGn versus GnGn(Fuc), GlcNAc-TIII clearly prefers GnGn and GlcNAc-TVII clearly prefers GnGn(Fuc), whereas GlcNAc-TVIII uses both acceptors equivalently (Figs. 9 and 10). These results provide additional evidence of the distinct nature of GlcNAc-TIII, GlcNAc-TVII, and GlcNAc-TVIII. In addition, they indicate that the in vivo action of these GlcNAc-Ts may depend on the prior action of other glycosyltransferases, such as alpha (1,6)fucosyltransferase or branching GlcNAc-Ts. In this context, it is of note that monoclonal antibodies that recognize the alpha (1,6)fucose residue in an N-glycan core do not bind well to LEC14 or LEC18 glycoproteins (32).

It will now be important to clone the genes that encode GlcNAc-TVII and GlcNAc-TVIII. This should be possible by expression cloning using a cDNA library from LEC14 or LEC18 to complement CHO cells. We have recently shown that expression of GlcNAc-TIII in LEC10 CHO cells corresponds to transcriptional activation of the Mgat3 gene. Northern blot analyses, with probes from the mouse Mgat3 gene that encodes GlcNAc-TIII (33), revealed the predicted ~4.7-kilobase Mgat3 gene transcript in LEC10 cells and no signal from the silent Mgat3 gene of CHO cells.3 Expression of the alpha (1,3)Fuc-T in LEC11 cells also corresponds to transcriptional activation of a Chinese hamster FUT gene. Three independent LEC11 mutants have been shown to possess an ~1.8-kilobase transcript that hybridizes to a CHO FUT gene probe, whereas this gene is transcriptionally inactive in parent CHO cells (34). It therefore seems likely that GlcNAc-TVII and GlcNAc-TVIII reflect transcriptional activation of new glycosyltransferase genes. Although it is possible that, like the blood group A, B, and O transferases that represent alleles of a single gene (35), GlcNAc-TVII and/or GlcNAc-TVIII might arise from the alteration of a known GlcNAc-T gene, the properties of genes encoding GlcNAc-Ts make this unlikely. To date, any GlcNAc-T that transfers GlcNAc to a unique substrate (e.g. GlcNAc TI versus GlcNAc-TII; see Ref. 28), or in a specific linkage, is encoded by a unique gene. There is no significant sequence homology between the genes encoding GlcNAc-TI, GlcNAc-TII, GlcNAc-TIII, GlcNAc-TV, or core 2 GlcNAc-T (reviewed in Ref. 36). Although there is homology between core 2 GlcNAc-T and the GlcNAc-T that creates the I antigen (37), these genes encode transferases that perform essentially identical reactions. By contrast, GlcNAc-TVII (LEC14) and GlcNAc-TVIII (LEC18) perform unique reactions, distinct from all the other GlcNAc-Ts described to date, and therefore are predicted to be encoded by distinct genes.

Cloning of both genes is an essential step to determining biological functions for the GlcNAc residues they add to N-glycans. The reason N-glycans with these GlcNAc residues have not previously been observed is presumably because they are present on a limited number of tissue specific glycoproteins or serum glycoproteins, or present in small amounts, or present only at particular times of development. Cloning the genes that encode GlcNAc-TVII and GlcNAc-TVIII will allow their spatio-temporal expression patterns to be determined and their expression to be altered or ablated in a complex organism such as the mouse.

    ACKNOWLEDGEMENTS

We thank Subha Sundaram for superb technical assistance and E. Richard Stanley for suggesting the chloramine T experiment.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1 36434 (to P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-3346; Fax: 718-829-7619; E-mail: stanley{at}aecom.yu.edu.

1 The abbreviations used are: CHO, Chinese hamster ovary; ConA, concanavalin A agglutinin; PSA, Pisum sativum (pea) agglutinin; GlcNAc-T, N-acetylglucosaminyltransferase; GlcNAcbeta 1-O-pNP, p-nitrophenyl-N-acetyl-D-glucosamine; MM, methyl-alpha -D-mannoside; GnGn, biantennary N-linked glycopeptide terminating with GlcNAc; GnGn(Fuc), biantennary N-linked glycopeptide (GnGn) containing a core fucose residue linked alpha 1,6 to the Asn-linked GlcNAc; HPAEC-PAD, high performance anion-exchange chromatography with pulsed amperometric detection; PIPES, 1,4-piperazinediethanesulfonic acid.

2 T. S. Raju, unpublished observations.

3 M. Bhaumik, J. Chang, X. Zhang and P. Stanley, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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