UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase

Molecular and Cellular Biochemistry 101: 125-143, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

UDP-GIcNAc: Gal 3GalNAc-Mucin: (GIcNAc > GalNAc) [36-N-Acetylglucosaminyltransferase and UDP-GIcNAc: GaI[$3(GIcNAc[ 6) GalNAc-Mucin (GlcNAc-- Gal)[$3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Sreedhara Sangadala, Subramanian Sivakami and Joseph Mendicino Department of Biochemistry, University of Georgia, Athens, Georgia 30602, USA

Received 5 February 1990; accepted 30 July 1990

Key words: GlcNAc transferases, trachea epithelium, mucin glycoprotein

Summary

Two specific ~-N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a ~1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal~3GalNAc-Mucin to yield Ga1133(GlcNAc~6)GalNAc-Mucin and a [33-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal~3(GlcNAc~6)GalNAc-mucin to yield GlcNAc~3Gal~3 (GlcNAc136)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The [31,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Ga1131,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal[31,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 136-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal~I,3GalNAc chains was 0.53/zM; for UDP-N-acetylglucosamine, 12/xM; and for Gall3 1,3GalNAca NO20, 4 mM. The activity of the 136-glucosaminyltransferase was dependent on the extent of glycosylation of the GaI133GalNAc chains in Cowper's gland mucin glycoprotein.

The best substrate for the partially purified 133-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Ga1131,3(GlcNAcI36)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or GaI131,3GalNAc side chains.

The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the ~6- and 133-glucosaminyltransferases were shown to be Gal[~3(GlcNAc~6) GalNAc and GlcNAc133 Ga1133(GlcNAc136)GalNAc respectively.

Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N- acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 136- glucosaminyltransferase converts Ga1133GalNAc chains in mucin glycoproteins to GaI133(GlcNAc136)Gal- NAc chains. This product is the substrate for a second 133-glucosaminyltransferase which converts the

126

Gal~3(GlcNAc[36)GalNAc chains to GlcNAc~3Gal(GIcNAc136)GalNAc chains in the glycoprotein. The ~3-glucosaminyltransferase did not utilize Gal[33GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.

Abbreviat ions: NeuNAc - N-acetylneuraminic acid, GalNAcol - N-acetylgalactosaminitol, CGMG - Cow- per's gland mucin glycoprotein, GalNAc-CGMG - Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine, Gal[53GalNAc-CGMC - Cowper's gland mucin glycoprotein containing Gal[33GalNAc side chains, MES - 2-(N-morpholino) Ethane Sulfonic acid, PBS - Phosphate Buffered Saline

Introduction

The mammalian respiratory tract is protected by mucus secretions which are synthesized in the surface epithelium and submucosal glands in the trachea. The surface epithelium of the tracheobron- chial airway is coated with mucus which is constant- ly removed by the mucociliary transport system. The viscoelastic properties of mucus secretions, the clearance of particulate matter, the maintenance of water balance and the rate of removal of mucus are dependent on the amount and composition of mucin glycoproteins in mucus. The rate of synthesis and secretion of mucin glycoproteins on to the epithelial surface, and the transport of mucus from the surface play a fundamental role in the maintenance of the sterility and stability of the trachea mucus membrane. The carbohydrate chains in mucin glycoproteins play an important role in these processes.

The viscoelastic and rheological properties of mucus is dependent on the amounts and composition of mucin glycoproteins in these secretions. The properties of these glycoproteins are, in turn, dependent on the amounts and structures of numerous oligosaccharide side chains present in these macromolecules. Regulation of the expression of individual oligosaccharide chains is controlled by the specificity and activity of a variety of glycosyltransferases. The biosynthesis of oligosaccharide chains in mucin glycoproteins proceeds via a stepwise addition of monosaccharides from sugar nucleotides to growing oligosaccharide chains. The addition of each monosaccharide requires the action of a specific glycosyltransferase. Elucidation

of the properties and specificities of various glycosyltransferases present in respiratory tissue is es- sential in order to understand the alterations in the viscoelastic properties of mucus secreted by this tissue.

In early studies, a number of glycosyltransferases with specificities for mucin glycoproteins were detected in membrane preparations from canine trachea [1]. These enzymes are found principally in the endoplasmic reticulum and Golgi apparatus. Glycosyltransferases involved in the synthesis of mucin glycoproteins were studied with microsome preparations and some of the enzymes have been solubilized and purified. Glycosyltransferases which add sialic acid, fucose and GalNAc to the non-reducing ends of oligosaccharide chains in mucin glycoproteins have been extensively purified and characterized [2]. GalNAc peptidyltransfe- rase, which adds GalNAc to the polypeptide chain was purified by Sugiura et al. [3]. A ~3-galactosyltransferase which adds Gal to this GalNAc residue to form GalI33GalNAc chains was purified and characterized by Mendicino et al. [4]. The properties of a 136-GIcNAc transferase which adds a GlcNAc residue to the GalNAc moiety in Gal~ 3GalNAc units in glycoproteins has also been examined with microsome preparations from canine submaxillary gland [5, 6] and rabbit small intestine [7].

In the present communication we report the purification and specificity of the [36-and ~3-GlcNAc transferases present in the microsomal fraction of swine trachea epithelium. These enzymes convert Gal~3GlcNAc-O-Ser(Thr) chains present in mucin glycoproteins to Gal~3(GlcNAc~6)GalNAc-O-Ser

(Thr) and GlcNAc~3GaI[33(GlcNAc[36)GalNAc- O-Ser(Thr) chains, respectively. These reactions initiate the branching and elongation of oligosaccharide chains which have been shown to be present in swine trachea respiratory mucin glycoproteins [8, 9].

Experimental procedures

Assay for 136- and ~3-GlcNAc transferase activity

The activity of [36GlcNAc transferase is determined by measuring the rate of transfer of [1- 14C]GlcNAc from UDP-[1-14C]GIcNAc to Gall 3GalNAc-CGMG. Reaction mixtures used in rou- tine assays are incubated at 37 ° for 30min and contain 0.1 M MES, pH 7.0; 10 mM MnC12, 0,2 mM UDP-[1-14C]GlcNAc (22cpm/pmol), 0.25M sucrose, 0.2 mg Gal[33GlcNAc-CGMG and transferase in a total volume of 0.3 ml. Triton X-100 at a final concentration of 0.1% and 1/xmol of NAD + were included in assays of crude extracts or membrane bound preparations.

The activity of ~3-GlcNAc transferase is determined under identical conditions with Gal[33(Glc NAc[56)GalNAc-CGMG as the substrate. Reac- tions are terminated by the addition of 3 ml of 2.8 N perchloric acid containing 2% phosphotungstic acid and the precipitated protein is collected by centrifugation, dissolved in i ml of 1 N NaOH and then reprecipitated with 3 ml of the perchloric acid- phosphotungstic acid mixture. This procedure is repeated once more and the final pellet is dissolved in 0.6 ml of 0.5 N NaOH and counted as described previously [10, 11]. One unit of activity is defined as the amount of enzyme required to transfer 1 t~mol of GIcNAc to Gal[33GalNAc-CGMG per min. The purified transferases are stored at - 80 ° in 0.2 mg/ ml albumin, 0.05mg/ml Gal[33GalNAc-CGMG and 0.1% Triton X-100. The enzyme is diluted 50-fold in 0.05 M MES, pH 6.5 before assay.

The transfer of GIcNAc to reduced oligosaccharides, galactose, pNO2O-[3-D-galactoside, pNO2¢- a-D-galactoside, pNO2¢i-[3-D-GlcNAc and other low molecular weight glycosyl acceptors is measured by an ion-exchange procedure described in

127

earlier studies [10, 11]. These substrates are incubated under the standard assay conditions with 0.1M MES, pH6.5, 10mM MnCla, 0.2mM UDP- [1-14C]GlcNAc (22 cpm/pmol) in a total volume of 0.1 ml. After incubation at 37 ° for 30 min the reaction mixture is diluted to 0.5 ml with 0.001 N HC1 and applied to a Dowex l-C1 column (0.5 x 4 cm) which was previously equilibrated with 0.001N HC1. The column is washed three times with 0.5 ml of 0.001 N HCI and the filtrates are collected in a scintillation vial and counted. Controls which were incubated in the absence of glycosyl acceptors contained little or no radioactivity. The rates obtained with this assay are proportional to the time of incubation and enzyme concentration up to 20% con- version of the substrate. In the presence of 0.5 mM GaI[33GalNAca0 and 0.2 mM UDP-GlcNAc, 5/zg, 10/xg and 15/~g of purified enzyme catalyzed the transfer of 0.30, 0.52 and 0.81 nmole of GlcNAc in 15 min, respectively. After 30 min of incubation under the same conditions, 5 ~g, 10/zg and 15 txg of enzyme transferred, 0.61, 8.7 and 1.5nmole of GlcNAc to the glycosyl acceptor. One unit of activity is defined as the amount of enzyme required to transfer 1/xmol of GIcNAc to Gal[33GalNAca¢ per min.

Assay for other glycosyltransferases

The activity of UDP-GlcNAc: a-mannoside: [32- GlcNAc transferase I and II are assayed in the standard reaction mixture with UDP-[1-14C]Glc NAc and i mg of ovalbumin, ovomucoid or al-acid glycoprotein which were previously treated with neuraminidase, [3-galactosidase and [3-N-acetylglu- cosaminidase. The preparation of these glycosyl acceptors are described in details in our previous reports [10, 11]. These substrates also serve as glycosyl acceptors for other GlcNAc transferases which act on N-asparagine oligosaccharides, but they are devoid of GaI~3GalNAc units. The activity of UDP-Gal : GlcNAc : [M-galactosyltransferase was measured in the standard assay with 1 mg of ovomucoid or ovalbumin devoid of sialic acid and Gal as described in our previous report [12].

The activity of [36- and [33-GlcNAc transferases

128

which transfer GlcNAc to neolactotetraosylcera- mides, Gal~4Glc Ceramide or Gal~4GlcNAc~ 3Gal~4Glc Ceramide were assayed as described by Basu and Basu [13].

Sialyl transferases are assayed as described by Sadler et al. [14] with asialo fetuin, asialo IgG, asialo CGMG and asialo al-acid glycoprotein as g!ycosyl acceptors. The activity is measured in a reaction mixture which is incubated at 37 ° for various times and contained in 0.1ml; 0.1M MES pH 6.9, 0.154 M NaC1, 10 mM MnC12, 0.1% Triton X-100, 0.5 mM CMP-[4-14C]-sialic acid (30 cpm/ pmol) and 0.2rag of glycosyl acceptor. Fucosyl- transferase activity was measured under the same conditions, except that 0.5 mM GDP-[U-14C]-L-fu - cose (30 cpm/pmol) was added in place of CMP- sialic acid and asialo transferrin was used as the glycosyl acceptor. The rate of reaction was measured as described previously. One unit of activity, in each case, is defined as the amount of enzyme required to transfer i pmole of sugar to the modified glycosyl acceptor per min.

Preparation of subcellular fractions

The isolation of subcellular fractions from homogenates of swine trachea membrane is carried out according to procedures described in detail in our previous studies [4]. The purity of the various fractions is monitored by electron microscopy and by determination of the activity of specific marker enzymes [12]. Na+/K+ATPase and 5'-nucleotidase are used for plasma membrane, succinate 2-(p- indophenyl)-3-(p-nitro-phenyl)-5-phenyl tetrazoli- um reductase for mitochondria, NADH-cytochrome c reductase and NADPH-cytochrome c reductase for endoplasmic membranes, and ~2-N- acetylglucosaminyltransferase and [M-galactosyl transferase for Golgi membranes.

Purification of CGMG and preparation of GalNAc-CGMG

CGMG is a constituent of boar semen and it was purified directly from Cowper's glands or from gel

present in ejaculates as described in detail in previous reports [4, 8]. The translucent gel, squeezed from the gland or separated from semen by filtration through cheesecloth, was solubilized in 1% SDS, reduced with 30 mM dithiothreitol and alky- lated with 60 mM iodoacetamide. The mucin glycoprotein was then purified by repeated chromatography on Sepharose CL-6B columns (100 × 5.5 cm) in 0.1% SDS and finally in 3M quanidine- HCI, pH7.5. The glycoprotein which eluted near the void volume from these columns was collected, concentrated and dialyzed against 0.05M Tris- HC1, pH 7.5. The purified preparations employed in the current studies were completely soluble in 0.05 M Tris-HC1, pH 7.5 and they were stored frozen at 10 mg/ml.

The purified glycoprotein contained 30% sialic acid on a dry weight basis, and more than 90% of the oligosaccharide chains were released when preparations were treated with 2M NaBH4 at 45 ° for 36 h [8]. The structure of the oligosaccharide chains in CGMG have been determined by methylation analysis and sequential hydrolysis with specific glycosidases [8]. This glycoprotein contains two short oligosaccharide chains. The principal component, 80%, is NeuNAc~2,6GalNAc, and about 20% of a disialylated oligosaccharide, Neua 2,3Gal[33[NeuNAca2,6]GalNAc is also present. Based on the loss of seryl and threonyl residues and the formation of alanine and 2-aminobutanoic acid as well as the decrease in GalNAc and the recovery of GalNAcol after [3-elimination with alkaline borohydride, CGMG contains about 160 oligosaccharide chains per 200,000 g of glycoprotein [8].

GalNAc-CGMG was prepared by treating asialo CGMG with [3-galactosidase for 24 h at 37 ° to remove terminal Gal residues from the Gal[53Gal- NAc chains as described in an earlier report [4]. More than 90% of the Gal133GalNAc chains in asialo CGMG were converted to terminal GalNAc residues by this treatment.

Preparation of Galfi3GalNAc-CGMG from CGMG with purified fl3-Galactosyltransferase

Sialic acid was completely removed from CGMG

by incubation with viral neuraminidase which was free of protease activity. Bacterial neuraminidase from commercial sources contained appreciable protease activity. CGMG, 500mg, was incubated with 5 units of viral neuraminidase in 0.05 M sodium acetate, pH 5.0 at 37 ° for 24 h under toluene. Afterwards, 2 units of enzyme was added and the mixture was incubated for another 24h. Asialo CGMG was reisolated by chromatography on a Sepharose CL-6B column (6 × 100 cm) in 3 M gua- nidine-HC1 as described previously [8]. The purified asialo CGMG was soluble (10mg/ml) in 0.05 M Tris-HC1, pH 7.5 and did not contain sialic acid. Methylation analysis showed that the glycoprotein now contained about 80% GalNAc and 20% GaI~3GalNAc side chains.

The GalNAc residues in asialo CGMG were converted to disaccharide units, GaI~3GalNAc, with homogeneous ~3-galactosyltransferase [4]. Asialo CGMG was incubated with UDP-Gal:GalNAc- Mucin:[53-galactosyltransferase in an incubation mixture which contained 0.1M MES, pH6.5, 10 mM MnC12, 10 mM UDP-Gal, 0.25 M Sucrose, 0.05% Triton X-100, 5mM KCI, 5mM MgCI2, 10 mM P-enolpyruvate, 10 units of pyruvate kinase, 100 mg asialo CGMG, and 2 units of ~3- galactosyltransferase in a total volume of 35 ml. After incubation for 24h at 37 °, the mucin glycoprotein was reisolated by gel filtration on a Se- pharose CL-6B column, and the high molecular weight fraction was collected, concentrated by ultrafiltration and dialyzed against 0.05 M Tris-HC1, pH7.5. Carbohydrate and methylation analysis showed that more than 95% of the GalNAc residues in asialo CGMG had been converted to Gal[~ 3GalNAc units by this treatment.

129

sodium borotritide (10mCi/ml) in 12mM sodium borate, pH 10.0, was added and the mixture was incubated at 5 ° for 24 h. Afterwards, 20 mg of sodium borohydrate was added and the sample was incubated for an additional 2 h. The sample was adjusted to pH4.0 by careful addition of acetic acid, dialyzed extensively against 0.05 M Tris HCI, pH 7.0 and concentrated by ultrafiltration. The labeled glycoprotein contained 1.1 x 106cpm/mg. When an aliquot was treated with ~-galactosidase more than 90% of the radioactivity was released as free galactose. About 1% of the total counts was present in residual GalNAc residues. The Gal[~ GalNAcol released by treatment with 0.1 N NaOH in the presence of 2M NaBH4 at 45 ° for 36 h was labeled exclusively in the Gal residue.

Preparation of Galt33 (GlcNAcl36) GaINAc-CGMG from Galfl3GalNAc-CGMG with purified fl6-GlcNAc transferase

Purified ~6-GlcNAc transferase was incubated for 24 h at 37 ° in a reaction mixture which contained 0.1 M MES, pH 7.0, 10 mM MnCI2, 0.25 M sucrose, 0.1% Triton X-100, 5 mM KC1, 5 mM MgC12, 50 mg of GaI~3GalNAc-CGMG, 10mM UDP-Gal 10 mM P-enolpyruvate, 10 units of pyruvate kinase in a total volume of 35 ml. The mucin glycoprotein was reisolated by gel filtration on a Sepharose CL-6B column and the high molecular weight fraction was collected, concentrated and dialyzed against 0.05 M Tris-HC1, pH7.5. Carbohydrate composition and methylation analysis showed that more than 90% of the chains in this acceptor had the structure, Gal[33(GlcNAc~6)GalNAc.

Preparation of [3H]GalI33GalNAc-CGMG

The carbohydrate chains in Gal[~3GalNAc-CGMG were labeled by treatment with galactose oxidase and reduction with N a B 3 H 4 . The glycoprotein, 20mg, was incubated with 20 units of galactose oxidase and 80 units of horse radish peroxidase in 3 ml of 0.5 M potassium phosphate, pH 7.2, for 24 h at room temperature under toluene. Then 0.2 ml of

Preparation of affinity columns

DEAE-cellulose containing bound Gal~3GalNAc- CGMG was prepared by stirring 50 mg of the glycoprotein with 20 ml of DEAE-cellulose in 100 ml of a buffer solution containing 0.05 M Tris HC1, pH7.5, 0.25 M Sucrose and 0.1% Triton X-100 for 30min at 3 °. The suspension was poured into a column and washed with three bed volumes of the

130

same buffer. The column contained 2.3mg of Gal[33GalNAc-CGMG per ml of DEAE-cellulose.

DEAE-cellulose containing bound GalNAc- CGMG was prepared in a similar manner. Asialo CGMG was treated with 5 units of seminal ~-galactosidase to remove terminal galactosyl residues [4]. Carbohydrate analysis showed that more than 95% of the chains in this preparation contained only GalNAc. The reisolated GalNAc-CGMG, 1 g, was stirred with 100 ml of DEAE-cellulose in 500 ml of a solution containing 0.05M Tris-HCl, pH7.5, 0.25 M sucrose and 0.1% Triton X-100 for 15 min at 3 °. The suspension was poured into a column and washed with three bed volumes of the same buffer. The column contained 8 mg of GalNAc-CGMG per ml of DEAE-cellulose. The amount of mucin glycoprotein attached to DEAE-cellulose was determined by measuring GalNAc after acid hydrolysis and equating this value to the amount present in purified CGMG.

GalI33GalNAc-CGMG was covalently attached to Sepharose CL-4B by the procedure of Bethell et al. [15] using 1-1'-carbonyldiimidazole.

Affinity columns containing thio-[3-D-galactose and GlcNAc substituents were prepared by reac- ting activated Sepharose 4B with the p-aminophenyl derivatives as described previously [12]. The derivatized Sepharose 4B columns used in the current studies contained about 3.5 t~mol of p-aminophenyl-amino sugar of 5/zmol of p-aminophenyl-thio-~-D-galactose per ml.

Carbohydrate composition and methylation analysis

Oligosacccharides were hydrolyzed in 2N HC1 for 4 h at 100 °. The composition was determined by gas chromatography of alditol acetates as described in previous reports [8, 9]. Analysis of acetylated samples was carried out with a column (0.2 x 200cm) containing 0.75% HiEFF-1BP, 0.25% EGSS-X and 0.1% 144-B (phenyldiethanolamine) on 60/80 mesh Gas Chromosorb Q with inositol or xylitol as internal standards. Permethylation analysis was performed by procedures described in detail in our previous studies [16]. The partially methylated al-

ditol and hexosaminitol acetates were examined by gas chromatography on a column (0.3 × 200cm) containing 3% SE-30 on 100/120 Gas Chrom D (0.3 × 400 cm). Retention times were determined relative to 2,3,4,6-tetramethylglucitol acetate. Peaks were identified by comparison with the retention times of authentic partially methylated alditol and hexosaminitol acetates [16].

Methods and materials

Viral and bacterial neuraminidases obtained from Boehringer Mannheim were purified by affinity chromatography on Sepharose CL-6B columns containing covalently bound N-(~-aminophenyl) oxamic acid (Sigma). Beta-galactosidase and [~-N- acetylgluco-saminidase were purified from Jack bean and seminal fluid [9, 16]. These enzymes were highly purified by affinity chromatography on Se- pharose 4B columns containing immobilized syn- thetic substrates [9, 16]. The purity of each enzyme was examined with 9-nitrophenyl derivatives of GlcNAc, galactose and other sugars. The preparations were flee of other glycosidase activities. Incubation conditions for hydrolysis of oligosaccharides have been described in detail in our previous reports [13, 16].

The concentration of protein was measured col- orimetrically with the Bradford reagent. Triton X-100 was removed from dried samples by extrac- tion with acetone before protein determination. Albumin which was used to stabilize the transferase was removed by passing the sample through a small DEAE-cellulose column (2.2 x 2 cm) before protein determination. Electrophoresis was performed with a BioRad Protein II slab cell with a current of 18 mA per gel for 4 hours. Protein bands were detected with silver stain (BioRad).

The anthrone and phenol-sulfuric acid methods were used to detect carbohydrate. Sialic acid was determined with the resorcinol reagent. Paper chromatography was performed according to procedures described in a previous report [16]. All of the oligosaccharides examined in these studies were purified to homogeneity by repeated gel filtration on Bio-Gel P-6 (400 mesh) columns (2.2 ×

200 cm) with 0.1 M pyridinium acetate, pH 5.5. The recoveries in each case were greater than 95% [8, 9, 16]. Gal~4GlcNAc~3Gal[34Glu-Cer and GlcNAc~ 3GalIMGlcNAc~3Gal~4Glu-Cer were prepared from the NeuGc derivatives of these ceramides which were isolated from bovine erythrocytes as described by Chien et al. [17]. UDP-[1 -14C] GlcNAc and UDP-[3H]GlcNAc were prepared as described in our previous report [18].

Results

Subcellular distribution of fl6-GlcNAc transferase and fl3-GlcNAc transferase in swine trachea epithelium

The relative distribution of transferase activity in subcellular fractions isolated from homogenates of trachea epithelium is shown in Table 1. The activity of ~6-GlcNAc transferase was measured with an excess of Gal[33GalNAc-CGMG and 1/zmol of NAD + was added to the reaction mixture to pro- vide an alternate substrate for hydrolases present in membrane preparations. Under these conditions linear initial rates were obtained with all of the fractions. The addition of Triton X-100, 0.1%, to

Table 1. Subcellular distribution of 136-N-Acetylglucosaminyl- transferase in swine trachea epithelium. The subcellular fractions were isolated from a 20% homogenate prepared from 50 g of trachea as described in the text. Each of the particulate fractions was washed twice with 10 volumes of 0.02M Tris, pH7.0-0.25 M sucrose-2 mM MgC12. The final pellet was suspended in this buffer and assayed for [3-N-acetyl glucosaminyltransferase activity by the standard procedure. Values are taken from the average of four preparations

Fraction Total activity Specific activity (nmol/min/g (nmol/min/mg tissue) protein)

Homogenate 9.6 + 1.8 0.18 Supernatant fraction 0.7 + 0.2 0.02 Plasma membranes 0.4 + 0.1 0.16 Golgi membranes 0.7 + 0.3 4.24 Microsomes 6.9 + 0.8 1.02 Rough microsomes 0.8 + 0.2 0.46 Smooth microsomes 0.9 + 0.3 1.11

131

the reaction mixture and disruption of the membrane preparations by sonication increased the observed transferase activity.

The microsome membrane fractions contained about 70% of the ~-N-acetylglucosaminyltransferase activity. Some activity found in the supernatant fraction obtained by centrifugation at 100,000 × g for 1 h, was probably due to the presence of very small membrane fragments which did not sediment even at high speeds. Subfractionation of the microsomes showed that the transferase was present in both the rough and smooth fractions, however, the smooth ,membrane fraction had a higher specific activity (1.11 compared to 0.46 nmol/min/mg). The specific activity of the purified Golgi membrane fraction was also much higher than that of the homogenate (4.24 compared to 0.18 nmol/min/mg). These results clearly show that the enzyme is associated with membranes in the endoplasmic reticulum and the Golgi apparatus. The specific activity of NADPH-cytochrome c reductase in the microsome fraction was also increased about 10-fold over that in the homogenate (51 nmol/min/mg compared to 4.5 nmol/ min/mg).

As reported earlier [4] the activity of marker enzymes which are reported to be localized in the plasma membrane and mitochondrial fractions were very low in the microsome and Golgi membrane fractions, indicating that the microsome and Golgi membrane fractions could not contain more than 10% contamination from these sources. The activity of 133-GlcNAc transferase was measured under the same conditions with Gal~3(GlcNAc~6) GalNAc-CGMG as the substrate. This enzyme showed a very similar subcellular distribution in trachea epithelium (data not shown). These results strongly suggest that both of the GIcNAc transferases are present in the endoplasmic reticulum and Golgi membranes.

Solubilization and purification of fl6- and fl3-GlcNAc transferase from swine trachea epithelium

Trachea were removed from animals immediately

132

after slaughter and they were trimmed and cooled to 3 ° . The tissue was used directly or stored frozen at - 20 ° without loss in the recovery or final specific activity of the purified enzymes. Only trachea epithelium which showed blood group A + H + specificity in hemagglutination inhibition assays were used for the isolation of these enzymes [8]. About 40 trachea, 2000 g, were sliced longitudinal- ly and the epithelial layer was removed and washed with PBS containing 5mM MgCI2 and 10mM CaC12. All subsequent procedures were carried out at 4 °. The tissue, 130 g, was cut into small pieces and homogenized in a Waring Blender at maxi- mum speed with 5 volumes of 0.05M Tris-HC1, pH 7.5-0.25 M sucrose. The suspension was further dispersed in a Ultra-Turrax T45N homogenizer (Janke & Kunkel GMBH & Co, West Germany) and it was then centrifuged at 27,000 × g for 10 min (Fraction 1, Table 2). Microsomes were isolated from the supernatant solution by centrifugation at 100,000 × g for 2 h and they were suspended in 25 ml of 0.1 M Tris-HCl, pH 7.5-0.25 M sucrose (Fraction 2). The microsomes could be stored frozen for at least six months at - 80 ° without loss of transferase activity. About 75-90% of the activity present in the homogenate was consistently reco- vered in the microsome fraction.

Nearly all of the glycosyltransferases involved in the synthesis of the oligosaccharide chains in mucin glycoproteins are associated with membranes and they can be solubilized by treatment with 1% Tri-

ton X-100 or 1% N0nidet P40 [4]. The microsome suspension was adjusted to 0.1 M NaC1, 1% Triton X-100 and 1% Nonidet P40 and it was stirred for 15 rain and passed into a DEAE-cellulose column (5 × 10 era) which was previously equilibrated with 0.05M Tris-HC1, pH7.5-0.25M sucrose. After- wards the column was washed with 200 ml of a solution containing 0.05M Tris-HCl, pH7.5- 0.25 M sucrose - 0 . 1 M NaC1-0.1% Triton X-100. This procedure yields a soluble enzyme with an overall recovery of 59% (Fraction 3). Much of the RNA and insoluble membrane fragments remain bound to the DEAE-column. Since the transferase do not bind to the DEAE-column under these conditions, they are continuously and efficiently extracted by the eluting buffer.

The filtrate and wash were combined (Fraction 3), 0.2 mg/ml of albumin was added and the solution was adjusted to 0.7 saturation with ammonium sulfate (47.2 g per 100 ml). The suspension was centrifuged at 34,000 × g for 10 rain and the precipitate which collected at the top of the centrifuge tube was separated by carefully pouring off the clear solution underneath. The precipitate was dissolved in 30 ml of 0.05 M Tris-HC1, pH 7.5-0.25 M sucrose-0.1% Triton X-100 and dialyzed twice against one liter of the same buffer (Fraction 4).

The solution was centrifuged at 27,000 × g for 15 min and the supernatant was adjusted to 10 mM MgCI2, 1 mM UMP and 100 mM NaC1 and passed through a Sepharose 4B column (2.2 × 10 cm) p-

Table 2. Purification of UDP-GlcNAc:136-N-acetylglucosaminyltransferase and UDP-GlcNAc:133-N-acetylglucosaminyltransferase from swine trachea epithelium

Fraction Volume Protein Total Specific Yield Purification (ml) (mg) activity activity (%) (fold)

(/~mol/min) (/zmol/min/mg)

1. Crude extract 740 6200 0.70 0.00011 100 1 2. Microsome 25 400 0.52 0.0013 74 12 3. Chromatography on DEAE-ceUulose 220 53 0.41 0.0077 59 70 4. Precipitation with Ammonium Sulfate 32 28 0.37 0.013 53 118 5. Eluent from first affinity chromatography 14 0.8 0.17 0.21 24 1,936

on GaI[53GalNAc-CGMG 6. Second affinity chromatography on 0.8 0.05 0.12 2.40 17 21,800

GalI33GalNAc-CGMG 7. Filtrate from step 5 assayed with 40 26 0.04 0.005 23 53

Gall53 (GlcNAc136) GalNAc-COMG

133

,.ol E _= E

O E 0.5,

>.... !--

>

I-- 0 <:[

0 0 40 80 12__0 160

V O L U M E (ml) Fig. 1. Affinity chromatography of [36- and [33-N-acetylglucosaminyl-transferases on GaI[33GalNAc-CGMG bound to DEAE-cellulose. Curve (O--O) was obtained by applying 4 ml of enzyme from Fraction 4, Table 2 to a DEAE-cellulose column (2.2 × 10 cm). Curve (O--O) was obtained by applying 4 ml of the same solution to a DEAE-cellulose column (2.2 × 10 cm) containing bound GalNAc- CGMG. Curve ( A - - A ) was obtained by applying 4ml of the same solution to a DEAE-cellulose column (2.2 × 10cm) containing bound GaI[33GalNAc-CGMG. Curve ( A - - A ) was obtained by applying 8 ml of the same solution to the DEAE-cellulose column. The application solution contained 0.05 M Tris-HC1, pH7.0, 0.25 M sucrose, 0.1% Triton X-100, 10 mM MgC12 and 1 mM UMP and the columns were equilibrated with this solution. After applying the enzyme, the columns were washed with 30 ml of this buffer containing 0.2 mg/ml of albumin. At the point indicated by the arrow elution was initiated with a solution containing 0.05 M Tris-HC1, pH 7.5, 50 mM EDTA, 0.1% Triton X-100, 0.25 M sucrose and 0.5 mg/ml albumin.

aminophenyl-GlcNAc and then through a Sepha- rose 4B column (2.2 × 10 cm) containing covalently bound p-aminophenyl-thio-~-D-galactoside. The columns were washed with three bed volumes of 0.05 M Tris-HC1, pH 7.5, containing 0.1%. Tri- ton X-100, 0.25 M sucrose, 10 mM MgC12 and I mM UMP. These columns remove 131,4 galactosyltransferase and GlcNAc transferases acting on N-asparagine linked oligosaccharides as described in our previous reports [4, 10-12]. The filtrate and washes were combined, concentrated by ultrafiltration and dialyzed against 0.05 M Tris-HC1, pH7.5 containing 0.25 M sucrose, and 0.1% Triton X-100.

The dialyzed solution was adjusted to 10m MgC12, 1 mM UMP and 100 mM NaC1 and passed

through a DEAE-cellulose column (2.2 × 15 cm) containing bound GalNAc-CGMG as shown in Fig. 1 bound. The column was washed with 50 ml of the same buffer containing 0.1 mg per ml of bovine serum albumin. This column binds [53-galactosyltransferase [4] and other proteins which bind tightly to mucin glycoproteins, but it did not bind the GlcNAc transferases.

The solutions were combined and concentrated as described previously and adjusted to 10mM MgC12, 1 mM UMP and 100 mM NaC1. This solution was applied to a DEAE-cellulose column (2.2 × 15cm) containing bound Gal~3GlcNAc- CGMG (Fig. 1). The column was previously equilibrated with a solution containing 10mM MgC12-

134

.5

E

e--

g - i.o

"-6 E ,C-

v >..- t-.- O.5

0 0 5 I0 15

D I S T A N C E ( c m )

Fig. 2. Polyacrylamide gel electrophoresis of purified 136- GlcNAc transferase. Twenty/zg of the purified enzyme was applied to the top of two wells of a 12% polyacrylamide gel which was previously run for 3 hours to remove persulfate. Electrophoresis was carried out for 4 hours in Tris-glycine buffer at pH8.3. Afterwards one of the lanes was cut out and developed with silver reagent to reveal protein bands. This gel is shown at the top of the figure. The other lane was cut out and sliced into 5 mm sections and the enzyme was extracted with a solution containing 0.1 M MES, pH 7.0, 0.1% Triton X-100 and 0.25 M sucrose. Aliquots were assay for 136-GlcNAc transferase activity as described in the text.

l m M UMP-0.05M Tris-HC1, pH7.5-0.25M sucrose-0.1% Triton X-100. The column was washed with 30 ml of the same buffer containing 0.1 mg per ml of bovine albumin. 136-GIcNAc transferase was then eluted with 60 ml of 0.05 M Tris-HC1, pH 7.5 containing 50mM EDTA, 0.1% Triton X-100, 0.1 mg/ml albumin and 0.25M sucrose. The fractions containing transferase activity were collected, concentrated to 20ml by ultrafiltration with a PM-10 Amicon membrane and dialyzed 3 times against 250 ml of 0.05 M Tris-HC1, pH 7.5-0.25 M sucrose-0.1% Triton X-100 (Fraction 5).

Finally, the solution was adjusted to 10mM MgC12, 1 mM UMP and 100 mM NaC1 and applied to a Sepharose CL-4B column (2.2 x 15 cm) containing covalently bound GaI133GalNAc-CGMG. The column was washed with 75 ml of solution containing 0.05M Tris-HC1, pH7.5, 0.25M sucrose, 0.1% Triton X-100, 15raM MgC12, 1.5mM UMP and 1 mM NaC1. Afterwards the enzyme was

eluted with 30 ml of 0.05 M Tris-HC1, pH 7.5 containing 50 mM EDTA, 0.1% Triton X-100,100 mM NaC1 and 0.25 M sucrose. The fractions with transferase activity were collected, concentrated and dialyzed as described previously. A highly purified preparation of 136-GIcNAc transferase with a yield of about 17% was obtained by the procedure out- lined in Table 2. The final specific activity was 2.4tzmol/min/mg with GalI33GalNAc-CGMG as substrate.

A 53-fold purification of 133-GlcNAc transferase was also obtained. This enzyme was present in the filtrate from the first affinity chromatography step (Table 2). The specific activity of this transferase was about 0.005/xmol/min/mg (Fraction 7).

Properties of purified fl6-GlcNAc transferase

The purified 136-GlcNAc transferase was stable for at least 6 months when stored at 3 ° in the presence of the standard buffer containing 20/zg/ml Gall3 3GalNAc-CGMG. The enzyme showed a broad pH optimum between pH6.5 and 7.5 with a maxi- mum at 7.2. The molecular weight was estimated to be 60Kd by gel filtration on a Sepharose $300 column (1.8 × 100cm) which was previously cali- brated with reference proteins as described in a previous report [4]. A single faint band which cor- responded to a molecular size of about 60,000 was observed with SDS-PAGE. However, no enzymat- ic activity was observed in fractions eluted from these gels. Electrophoresis for 4 h in Tris-glycine buffer at pH 8.3 in the absence of SDS yielded a single protein band as seen in Fig. 2. This band contained nearly all of the 136-GlcNAc activity present in the gel.

The effect of increasing concentrations of UDP- GIcNAc on the initial rates of the purified transferases was measured as a function of the concentration of GaI133GalNAc-CGMG or GaI133(GlcNAc 136)GalNAc-CGMG under the standard assay conditions. Lineweaver-Burk plots of data obtained with UDP-GlcNAc concentrations from 50/xM to 200/zM were linear and intersected on the 1/S axis. An apparent Km of 12/~M for UDP-GlcNAc was obtained with Ga1133GalNAc-CGMG and 136-

GlcNAc transferase and an apparent Km of 17/xM for UDP-GlcNAc was obtained with GaI[33(GIcNAc[36)GalNAc-CGMG and ~3-Glc NAc transferase. Similar data obtained with concentrations of GaI~3GalNAc-CGMG from 0.05/zM to 10/xM in the presence of 0.1 mM UDP- GlcNAc yielded an apparent Km of 0.53~M for this substrate. An apparent Km of 0.45/xM was obtained for Gal~3(GlcNAc[36)GalNAc-CGMG under identicial conditions with ~3-GIcNAc transferase. These values are based on a molecular weight of 200,000 for CGMG.

Acceptor specificity of fl6-GlcNAc transferase

The acceptor substrate specificity of purified [36- GlcNAc transferase was examined with modified glycoproteins, glycolipids and well characterized oligosaccharides. The best glycosyl acceptors were glycoproteins containing terminal O-linked Gal~3 GalNAc units. Little or no activity was observed with glycoproteins containing N-asparagine linked oligosaccharide chains. Ovomucoid, ovalbumin and al-acid glycoprotein devoid of terminal sialic acid, galactose and GIcNAc did not serve on substrates for either the ~6- or ~3GlcNAc transferase. Furthermore, various glycopeptide substrates including Mana6(Mana3)Man~4GlcNAc[34Glc NAc-Asn, Mana6(GlcNAc~2Mana3)ManlMGlc NAcI34GlcNAc-Asn and GlcNAc~2Mana6(Glc NAcl3-Mana3)Man~4GlcNAc~4GlcNAc-Asn which are the preferred substrates for a number of GlcNAc transferases acting on N-asparagine linked oligosaccharides were inactive with the trachea GlcNAc transferase. These observations were further confirmed by directly assaying for a large number of glycosyltransferases which were present in the solubilized extracts. The purified ~6-GlcNAc transferase was completely free of other glycosyltransferases including ~4- and ~3-galactosyltransferases [16, 3], GlcNAc: Mannoside: ~2-GIcNAc transferases [10, 11], fucosyltransferases, sialyl- transferases, exoglycosidases and hydrolases acting on nucleoside diphosphate sugars. These re- suits clearly showed that glycoproteins containing N-asparagine linked oligosaccharide chains with a

135

variety of terminal galactosyl residues did not serve as substrates for the purified enzymes.

The purified trachea GIcNAc transferases showed little or no activity with glycolipid acceptors. No incorporation of [3H]GlcNAc into Gal- ceramide, Galfil4Glc-ceramide or Gal~4Glc NAc~3Gal~4Glc-ceramide was observed when the enzymes were assayed in the standard reaction mixture. The formation of radioactive glycolipids was followed by thin layer chromatography in three different solvent systems [13].

The acceptor substrate specificity of ~6GlcNAc transferase was also examined using mucin glycoproteins and a variety of oligosaccharides with well characterized oligosaccharide sequences which were prepared from swine trachea mucin glycoproteins [8, 9].

The best substrates for the purified ~6-GlcNAc transferase wer asialo CGMG, antifreeze glycoprotein and asialo-afuco procine submaxillary mucin which contain mainly unsubstituted Gal~3Gal- NAc side chains. Intact CGMG, GalNAc-CGMG and Gal[33(GlcNAcI36)GalNAc-CGMG showed very low or nearly undectable activity under the same conditions.

The purified enzyme was active with a number of low molecular weight glycosyl acceptors including: Gal133GalNAcapNO2¢, Km, 4mM; Gal[33Gal NAcotCH3, Km 5.6mM and Gal~3GalNAc, Km 0.9 mM. However, it showed little or no activity with free galactose, pNO2¢-[~-D-galactose, pNO20- ~-D-galactose, pNO2¢-a-D-galactose, pNO20-~- D-GlcNAc, GalNAcol, Gal~3GalNAcol or Gal ~3GlcNAc. The purified enzyme also showed little or no activity with many of the oligosaccharide alditols isolated from swine trachea mucin glycoproteins. The oligosaccharides tested included; Gal~3 (GlcNAc[36) GalNAcol, Gal~3 (Gal~4GlcNAc~6) GalNAcol, Gal~3GlcNAc~3Gal[33(Gal[MGlcNAc~6)GalNA- col, GaI~4GlcNAc~6(GIcNAc[33Gal~3) GalNAcol or Gal~4GIcNAc~6(GlcNAc[~3Gal~3GlcNAc[33 Gal[33)GalNAcol. These results suggest that the 136-GlcNAc transferase does not catalyze the synthesis of the outer branched structures in oligosaccharide chains found in swine trachea mucin glycoproteins [8, 9]. Instead, its action appears to

136

E c~J d E Q_ 0

V

I '0 I 0

x

>- I - >

I--

0 n <~ r r

6 ~

4 - -

2 -

0 -- 4 0 0

A B C

5 0 0 6 0 0

VOLUME (m I)

700

Fig. 3. Elution profiles of radioactive oligosaccharides isolated after incubation of UDP[1-14C]GIcNAc and Ga1133GalNAc-CGMG or Gal~3(GIcNAc136)GalNAc-CGMG with purified 136- or ~3-GlcNAc transferase, respectively. The enzymes were incubated under the standard assay conditions in a total volume of 35 ml with 50 mg of the mucin glycoprotein and UDP[1-14C]GlcNAc(5 x 105 cpm) for 24 h at 37 °. Afterwards the labeled mucin glycoproteins were isolated by gel filtration on Sepharose CL-4B columns and oligosaccharides were released and separated by gel filtration on BioGel P6 columns (2.2 x 200 cm). Fractions of 20 ml were collected and 0.2 ml aliquots were assayed for radioactivity. The elution prifile of radioactive oligosaccharides formed after incubating purified ~6-GlcNAc transferase with Ga1133GalNAc-CGMG is shown in Curve (O--O). The elution pattern of radioactive oligosaccharides formed after incubating 133-GIcNAc transferase with GalI33(GIcNAc136)GalNAc CGMG is shown in Curve (O---Q). The position of elution of GlcNAc133Ga1133(GlcNAc136)GalNAcol (Arrow A), Ga1133(GlcNAc136)GalNAcol (Arrow B) and Gal133GalNAcol (Arrow C) are indicated in the figure.

be localized to the addition of GlcNAc to GalNAc

units attached to Ser or Thr residues in this gly-

coprotein.

Characterization of the products formed from Galfl3 GalNA c-C G M G

The specificity of the purified 136-GlcNAc transfer-

ase was determined by characterizing the products formed in large scale reaction mixtures. Standard incubation conditions with reactants increased 50- fold and the time extended to 24 h were used to

prepare radioactive products. Pyruvate kinase was

added to these reaction mixtures to remove UDP

which inhibits the reaction at high concentrations.

Two different reaction mixtures were used in these studies. One contained UDP-[1J4C]GIcNAc and

Gal[33GalNAc-CGMG and the other contained [3H]Gal[33GalNAc-CGMG and UDP-GlcNAc.

The radioactive mucin glycoprotein products were isolated by gel filtration on Sepharose CL-6B columns and treated with alkaline borohydride to release oligosaccharide chains. More than 90% of the radioactivity was released by this treatment. The labeled reduced oligosaccharide chains were

137

A B C 5

3 x

or"

0400 500 600 700

VOLUME (ml)

Fig. 4. Elution profile of radioactive oligosaccharides isolated after incubation of [3H]Gal[33GalNAc-CGMG with purified [36-N- acetylglncosaminyltransferase. The enzyme was incubated under the standard assay conditions with 10rag of [3H]Gal[33GalNAc- CGMG (1.1 x 106 cpm) and UDPGlcNAc. Afterwards, the mucin glycoprotein was isolated by chromatography on a Sepharose 4B column. Oligosaccharide chains were released by [3-elimination and separated by gel filtration on BioGel P6 columns (2.2 x 200 cm). Arrow A, indicates the position of GIcNAc[~3Gal133(GlcNAc136)GalNAcoI: Arrow B, Gal~33(GlcNAc136)GalNAcol; Arrow C, Gal133GalNAcol. Curve (C)--~) was obtained before incubation with [36-GIcNAc transferase and Curve (0-----0) was obtained after incubation with 136-GlcNAc transferase.

then separated by gel filtration on Bio-Gel P-6 columns (2.2 x 200 cm) as shown in Fig. 3 and Fig. 4. The same radioactive oligosaccharide, Peak B, GaI~3(GIcNAc~6)GalNAcol was formed when la- bel was present in either UDP-[1-14C]GlcNAc or the mucin glycoprotein, [3H]Gal~3GalNAc- CGMG. The trisaccharide formed with UDP[1- 14C]GlcNAc was labeled exclusively in the GlcNAc residue, whereas the same trisaccharide formed from [3H]Gal~3GalNAc-CGMG was labeled in the Gal residue. When ~6-GlcNAc transferase was incubated with UDP [1-14C]GlcNAc and Gal[~3(Glc NAc~6)GalNAc-CGMG under the same conditions no radioactive peaks were found.

Incubation of UDP[1J4C]GlcNAc and Gall33 (GlcNAcf36)GalNAc-CGMG with [33-GlcNAc

transferase resulted in the formation of a tetrasaccharide, Peak A, Fig. 3. A small amount of the trisaccharide, Peak B, was also formed under these conditions. When the oligosaccharides in peak A and peak B were examined by paper chromatography in n-butanol-pyridine-water (6 : 4 : 3), each migrated as a single spot with Rf0.31 and 0.52, respectively, compared to peak C or standard Gal[31,3GalNAcol (Rf 1.00).

The anomeric configuration and linkage of each sugar residue in the isolated oligosaccharides was determined by sequential hydrolysis with glycosidases and methylation analysis. Data obtained in these experiments with oligosaccharides from peak A and peak B is summarized in Table 3.

Oligosaccharide A yielded 2,4,6-Me-Gal, 3;4,6-

138

Me-GIcN(Me)Ac and 1,4,5-Me-GalN(Me)Ac in a ratio of 0.9 : 1.9 : 1.0 following methylation analysis. No hydrolysis was observed when this reduced oligosacchariade was treated with ~-galactosidase. However, extensive hydrolysis with epididimus [3- N-acetylglucosiminidase released two equivalents of radioactive GlcNAc and yielded an unlabeled dissacharide which contained equal amounts of Gal and GalNAcol. Methylation analysis of this product yielded 2,3,4,6-Me-Gal and 1,4,5,6-Me GaIN (Me)Ac in a ratio of 1.0 : 0.9. When oligosaccharide A was treated with Jack bean [~-N-acetylgluco- saminidase only one equivalent of radioactive GlcNAc was released and a radioactive trisaccharide was isolated from the incubation mixture. Me- thylation analysis of this product yielded 2,4,6-Me- Gal, 3,4,6-Me-GIcN(Me)Ac and 1,3,4,5-Me-GaIN (Me)Ac in a ratio of 1 : 0.9 : 1.1. We have previously shown that Jack bean ~-N-acetylglucosamini- dase hydrolyzes ~6-1inked GlcNAc rapidly and [33- linked GlcNAc residues very slowly [4]. Taken collectively these data shown that oligosaccharide A has the structure GlcNac~3Gal~3(GlcNAc~6)Gal- NAcol.

Oligosaccharide B yielded 2,3,4,6-Me-Gal, 2,3,4,6-Me-GIcN(Me)Ac and 1,4,5-Me-GaIN(Me) Ac in a ratio of 0.9 : 1.1 : 1.0 when examined by methylation analysis (Table 3). Treatment with Jack bean [3-galactosidase released one equivalent of Gal and a radioactive disaccharide. Methylation analysis of the disaccharide yielded 3,4,6-Me-GlcN (Me)Ac and 1,3,4,5-Me-GalN(Me)Ac in a ratio of

1.0 : 0.9. Both Jack bean and epididimus B-N-ace- tylglucosaminidase released one equivalent of radioactive GlcNAc from oligosaccharide B (Table 3). Methylation analysis of the resulting reduced disaccharide showed the presence of 2,3,4,6-Me- Gal and 1,4,5,6-Me-GalN(Me)Ac in a ratio of 1.0 : 0.9. These data show that oligosaccharide B has the structure GaI~3(GlcNAc136)GalNAcol. The re- suits indicate that the purified ~6- and ~3-GIcNAc transferases catalyze the formation of a ~6-1inkage between GlcNAc and GalNAc and a [33-linkage between GlcNAc and Gal in a GalI33GalNAc- CGMG to form Gal~3(GlcNAc[36)GalNAc- CGMG and GlcNAc~3GaI~3(GlcNAc~6)Gal NAc-CGMG, respectively.

The activity of fl6-GlcNAc transferase with GalNAc-CGMG as substrate is dependent on the activity of fl3-Galactosyltransferase

The activity of ~6-GIcNAc transferase was measured with GalI33GalNAc-CGMG as substrate. Little or no activity was observed with GalNAc- CGMG or with sialylated native CGMG which contains NeuAca6GalNAc and NeuAca3Gal[33 (NeuAcct6)GalNAc side chains. Experiments with equal activities of ~3-galactosyltransferase and ~6- GlcNAc transferase (0.1 units) in the standard assay system with a saturating concentration of Gal- NAc-CGMG, 4mg, showed that the activity of ~6-GlcNAc transferase was completely dependent

Table 3. M e t h y l a t i o n ana lys i s o f r e d u c e d o l i g o s a c c h a r i d e s A and B

Par t i a l ly m e t h y l a t e d

a ldi tol a ce t a t e

I s o l a t e d o l i g o s a c c h a r i d e A f t e r [3-galactosidase A f t e r [5-N-acetyl g l u c o s a m i n i d a s e

A a B a A a B b A c B 0

2 , 3 , 4 , 6 - M e - G a l - 0 .9 - - 1.0 1.0

2 , 4 , 6 - M e - G a l 0 .9 - 1.1 - - -

3 , 4 , 6 - M e - G l e N ( M e ) A c 1.9 1.1 1.8 1.0 - -

1 , 4 , 5 - M e - G a l N ( M e ) A c 1.0 1.0 1.0 - - -

1 , 3 , 4 , 5 - M e G a l N ( M e ) A c - - - 0.9 - -

1 , 4 , 5 , 6 - M e G a l N ( M e ) A e . . . . 0.8 0.9

a M o l a r r a t io ca l cu l a t ed r e l a t i ve to the a m o u n t o f 1 , 4 , 5 - M e - G a l N A c ( M e ) A c .

b M o l a r r a t io ca lcu la ted r e l a t ive to the a m o u n t o f 3 , 4 , 6 - M e - G l e N ( M e ) A c .

c M o l a r ra t io ca lcu la ted r e l a t ive to the a m o u n t o f 2 , 3 , 4 , 6 - M e - G a l .

on the activity of ~3-galactosyltransferase. One reaction mixture contained 10~mol UDP-gal and 10 ~mol UDP-[3H]GlcNAc and another contained the same concentration, of UDP-[14C]Gal and UDP-GlcNAc. Aliquots were removed from the reaction mixture at various times, i h, 3 h, 6 h, 9 h, 12h and 24h, and assayed for incorporation of [3H]GlcNAc or [14C]Gal into GalNAc-CGMG. The incorporation of [14C]Gal was 8%, 23%, 45%, 61%, 75% and 87% at each time period, respectively. The rate of incorporation of [3H]GIcNAc under identical conditions was 3%, 9.8%, 21%, 29%, 53% and 81%, respectively. These data show that the rate of incorporation of GlcNAc by [~6- GIcNAc transferase was nearly directly proportional to the amount of Gal~3GlaNAc formed by ~3-galactosyltransferase. Since, 133-glactosyltrans- ferase forms the oligosaccharide which acts as a substrate for ~6-GlcNAc transferase it is clear that these enzymes act in a strictly ordered sequence.

Influence of the extent of glycosylation on the activity of fi6-GlcNAc transferase

Samples were removed at various times during the synthesis of Gal~3(GlcNAc~6)GalNAc-CGMG from Gal~3GalNAc-CGMG with [36-GlcNAc transferase. The Gal(GlcNAc136)GalNAc chains in these samples constituted 3%, 5.8%, 21%, 29%, 53% and 81% of the total oligosaccharide chains present. The remainder of the chains in these preparations were Gal~3GalNAc. The initial rate of [56-GlcNAc transferase was then measured as a function of the extent of glycosylation of its mucin glycoprotein substrate with saturating and equal concentrations of each of these samples based on the amount of Gal[33GalNAc chains remaining. Thus, the initial rate of reaction obtained with 5.4mg of CGMG containing 81% Gal~3(Glc NAc~6)GalNAc chains and 19% Gal~3GalNAc chains was compared with the rate observed with 1.25mg of CGMG containing 21% Gal133(Glc NAcI36)GalNAc chains and 79% Gal[33GalNAc chains. In the standard reaction mixture containing 5 ~g of [36-GlcNAc transferase samples of CGMG containing 3%, 5.8%, 21%, 29%, 53% and 81%

139

(GaI~3(GlcNAcI36)GalNAc chains but equal amounts of Gal[53GalNAc chains showed initial activities of 0.52, 0.49, 0.37, 0.32, 0.23 and 0.07 nmol of GIcNAc transfered in 15 min, respectively. These results suggest that the activity of ~6-GlcNAc transferase is influenced by the extent of glycosylation of the glycoprotein substrate. The enzyme shows a significantly decreased activity to- wards oligosaccharide chains remaining as the extent of glycosylation increases. Clustering of oligosaccharide chains, steric hinderance or even the conformation of the polypeptide chain may decrease the affinity or accessibility of some of the oligosaccharide chains. The increased concentration of chains which are a product of the reaction may also promote the formation of dead-end complexes with the enzyme and thereby decrease the amount of enzyme available for the formation of effective active enzyme substrate complexes. In this way as oligosaccharide chains which are a product of the ~6-GlcNAc transferase reaction accumu- late the overall rate of transfer of GlcNAc would decrease. The inability of glycosyltransferases to completely glycosylate mucin glycoproteins which contain numerous oligosaccharide chains, about 200 chains per mole, may contribute to the large variety of chains found in these glycoproteins [8, 9].

Discussion

In an earlier study we used the desialylated deriv- ative of CGMG for the purification of a specific UDP-Gal : GalNAc: ~3-galactosyltransferase [4]. This macromolecular glycosyl acceptor proved to be very useful as a substrate for characterizing the enzyme, as well as for purifying the enzyme by affinity chromatography. CGMG, which is synthesized in the Cowper's gland of the boar, contains numerous short but highly sialylated oligosaccharide chains, 80% NeuAcc~2,6GalNAc and 20% NeuAcc~2,3Gal~3 (NeuAcc~2,6)GalNAcol. Treat- ment with neuraminidase removes all of the sialic acid and subsequent incubation with the purified ~3-galactosyltransferase yields GaI~3GalNAc- CGMG as indicated in the following reaction.

140

GalNAc-CGMG + UDP-Gal--+ Gal[33GalNAc-CGMG + UDP (1)

More than 90% of the chains in asialo CGMG were converted to oligosaccharides with the structure Gal[33GalNAc after long periods of incubation with UDP-Gal and ~3-galactosyltransferase.

In the current studies this substrate was used to purify a ~6-GlcNAc transferase which catalyzes the synthesis of the first branch point in mucin glycoproteins as indicated in the following reaction.

Gall53 GalNAc-CGMG + UDP-GlcNAc-+

GlcNAc[56 ~ GalNAc-CGMG + UDP (2) Gall33 I -

The enzyme was extensively purified from solubilized microsome preparations of swine trachea epithelium by affinity chromatography on DEAE- cellulose columns containing bound Gal~3Gal- NAc-CGMG. The 136-GlcNAc transferase bound to its immobilized marcomolecular substrate in the presence of UMP and Mn ÷÷ and could be specifically released by removing these compounds from the eluting buffer. The purified enzyme did not transfer GlcNAc to glycoproteins containing N- asparagine linked chains nor to the terminal Gal- NAc residue in Type A blood group glycoproteins.

Many tissues including trachea epithelium [10] contain numerous GlcNAc transferases which catalyze the specific incorporation of GlcNAc into N-asparagine linked oligosaccharide chains [19, 20]. Most of these GlcNAc transferases may be separated from those acting on O-Ser (Thr) linked chains in mucin glycoproteins by affinity chromatography on columns containing their immobilized macromolecular substrates. We have previously shown that ~2-GlcNAc transferases [10, 11], which act on N-asparagine linked oligosaccharide chains, could be completely separated from [53-galactosyltransferase [4], which acts on O-Ser (Thr) linked chains, by affinity chromatography with their respective macromolecular substrates. Using the same procedure, Sheares and Carlson [21] showed that the [53-galactosyltransferase isolated by affinity chromatography on a mucin glycoprotein sub- state was completely separated from a fi4-galacto-

syltransferase which acts of N-asparagine linked oligosaccharide chains. In studies with chick em- bryo liver, Furukawa and Roth [22] showed that a ~3-galactosyltransferase acting on asialo ovine submaxillary mucin was completely separated from ~4-galactosyltransferase acting on asialo-agalacto- al-acid glycoprotein by affinity chromatography on columns containing their respective immobilized macromolecular substrates. The two enzymes be- haved identically in most other purification procedures, including electrophoresis, gel filtration and affinity chromatography on UDP-hexanolamine- Sepharose 4B and a-lactalbumin-Sepharose 4B columns. Taken collectively, these observations suggest that macromolecular substrates containing specific oligosaccharide chains which act as substrates for specific glycosyltransferases can serve as effective specific adsorbents in the purification of these enzymes. The polypeptide chain may also contribute to the specificity of these interactions when glycosyltransferases acting near the site of attach- ment of the oligosaccharide chains are being purified.

The characteristics of the affinity columns used in the purification of [56-GlcNAc transferase were examined in the current studies. The enzyme does not bind to DEAE-columns nor to DEAE-columns containing bound GalNAc-CGMG. How- ever, the enzyme did bind to DEAE-cellulose columns containing bound Gal~3GalNAc-CGMG. The enzyme solution was passed through the DEAE column containing bound GalNAc-CGMG first, because this column removes proteins which bind very tightly and non-specifically to mucin glycoproteins. In this way, these proteins were elim- inated before absorbing [56-GlcNAc transferase to the DEAE cellulose column containing bound Gal~3GalNAc-CGMG. CGMG and its derivatives have a very high affinity for DEAE-cellulose and 1 M NaC1 or 0.3 M Na2CO2 are required to elute them from the column. CGMG is not eluted under the conditions which are used to elute the transferase.

136-GIcNAc transferase exhibited a very high specificity for the GalNAc unit in the Gal~3Gal- NAc chain in CGMG. It did not transfer GlcNAc to free Gal or GalNAc, but it did utilize the disaccharide, Gal~3GalNAc and derivatives of this disaccharide. Modifications in the structure of the

disaccharide resulted in a loss of activity. Thus, Gal~4GlcNAc, Gal[33GIcNAc, and Gal~3GalNA- col were all inactive. Furthermore, the purified enzyme was only active with macromolecular substrates containing the disaccharide, Gal~3GalNAc and showed little or no activity with CGMG containing only GalNAc or Gal~3(GlcNAc[36)Gal- NAc chains. Intact CGMG, which contains NeuA- ca6GalNAc and NeuAcc~2Gal(NeuAcc~6)GalNAc chains, was also inactive. The specificity of this enzyme was further evaluated with derivatives of oligosaccharide chains isolated from swine trachea mucin glycoproteins [8, 9]. The purified enzyme showed no activity with oligosaccharide chains hav- ing the following structures.

Gal[MGlcNAc[36 ~ GalNAcol Gal~3

Gal[34GlcNAc[36 ~ GalNAcol Gal~3GlcNAc[33Gal~3

Gal[34GIcNAc[36 ~ GalNAcol GlcNAc~3Gal~3

Gal[34GlcNAc~36 GlcNAc[~3Gal~3GlcNA@3Gal[33 1 GalNAcol

Taken, collectively, these results show that the ~6- GlcNAc transferase purified in the current studies in specific for the synthesis of the first branch point in oligosaccharide chains in respiratory mucin glycoproteins and further suggest that it is not involved in the synthesis of outer branches in these chains. The structures of two of the more complete chains present in swine trachea mucin glycoproteins [8, 9] are shown below:

Fucc~2Gal[34GlcNAcf36 ~ GalNAcol Fuca2Gal134GlcNAc[36 ~ ~ / "

Fuca2Gal134GlcNAc[33 J tJal[33

GalNAc Fucct2Gal133(4)GlcNAc136 /a3 ~ " G a l N A c o l

Fucct2Gal[33 (4) GlcNAc136 . ~ / . . . . . . . . . . . . . . . I t Jail33(4) GlcNAc[33Gal133 r u c q z ~ a l l ~ J O ) u l c r ~ c p ~

,leo GalNAc

Based on the proposition that one transferase may be required for the synthesis of each linkage in an

141

oligosaccharide chain, at least three and as many as six different GlcNAc transferases may be involved in the synthesis of the oligosaccharide chains present in swine trachea mucin glycoproteins [8, 9]. In a recent study Koenderman et al. [23] have shown that Novikoff ascites tumor cells contain at least 2 different ~6-GlcNAc transferase activities. The re- quirements of the [36-GIcNAc transferase isolated in the current studies restrict its action to the first branch point in the synthesis of these oligosaccharide chains. It should further be noted that nearly all of the major oligosaccharide chains present in swine trachea mucin glycoproteins contain the in- ner branched core structure, Gal[33(GlcNAc~6) GalNAc [8, 9].

A ~3-GlcNAc transferase which utilizes the product of the [56-GlcNAc transferase reaction was also isolated in the current studies. This enzyme catalyzes the following reaction.

GlcNAc~--GalNAc-CGMG + UDP-GlcNAc--~ Gal~3

GlcNAc[36 ~ GalNAc-CGMG + UDP (3) GlcNAc[33Gal[33 J

The separation of the [36- and ~3-GlcNAc transferases during affinity chromatography on DEAE- cellulose columns containing bound Gal~3Gal- NAc-CGMG may be due to differences in the re- quirements of these enzymes for binding to this substrate. The formation of dead-end complexes between ~6-GlcNAc transferase, a nucleotide product such as UMP or UDP and Gal[33GalNAc- CGMG requires the participation of Mg ++. These dead-end complexes tend to bind together rather tightly. Gal~3GalNAc-CGMG does not serve as a substrate for the [33-GlcNAc transferase and this enzyme passes through the affinity column.

Evidence obtained in the present studies further suggest that the branched trisaccharide formed by ~6-GlcNAc transferase may serve as the preferred substrate for ~3-GlcNAc transferase, which initi- ates the elongation of oligosaccharide chains in swine trachea epithelium. Thus, the ~6-GlcNAc transferase converts Gal~3GalNAc chains in CGMG to Gal[33(GlcNAc~6)GalNAc chains, which then serve as a substrate for ~3-GlcNAc

142

transferase to form GlcNAc[33Gal[33(GIcNAc~6) GalNAc chains. The partially purified 133-GlcNAc transferase does not transfer GlcNAc to GalNAc or Gal~3GalNAc chains. This leads to an ordered sequence of addition of GlcNAc residues to Gal~3 GalNAc. The branched oligosaccharide, Gal~3 (GlcNAc~6)GalNAc, formed by ~6-GlcNAc transferase serves as an intermediate in the formation of the tetrasaccharide GIcNAc[33Gal(GlcNAc [36)GalNAc by ~3-GlcNAc transferase. Brockhau- sen et al. [24] have described a ~3-GlcNAc transferase in porcine gastric mucosa which shows similar properties.

A completely distinct ~3-GIcNAc transferase which transfers GlcNAc from UDPGlcNAc to terminal GalIMGlcNAc units in glycolipids, proteo- glycans and glycoproteins has been detected in human serum by Piller and Cartron [25]. Both N- asparagine and O-Ser(Thr) chains present in cq- acid glycoprotein and sheep gastric mucin glycoprotein were equally good glycosyl acceptors for the enzyme when Gal~4GIcNAc units were ex- posed by removing terminal sialic acid from these glycoproteins.

In studies on the biosynthesis of O-glycosidically linked chains Bergh et al. [26] have shown that sialic acid is also added in an ordered sequence to the disaccharide chains, Gal~3GalNAc, in fetuin. In this case a ~-galactosidea2--~ 3 sialyltransferase attaches sialic acid to the Gal residue of the disaccharide to form NeuAcc~2---~ 3Gal~3GalNAc. This linear trisaccharide is a substrate for a specific ~2--~6 sialyltransferase which forms NeuAca2--~ 3Ga1133 (NeuAc~x2--~ 6) GalNAc by transfer of sialic acid to the GalNAc residue. Since the (~2--~ 6 sialyltransferase does not sialylate GalNAc or GaI~3GalNAc chains an ordered sialylation sequence is dictated by the specificity of these enzymes. It should be noted that the sequence of addition of sialic acid units by the sialyl transferases is opposite to that of the GlcNAc transferases. GlcNAc is added to the GalNAc unit in Gal[53Gal NAc before GlcNAc is added to the Gal unit.

Acknowledgements

This investigation was supported by Grants HL 26858 and HL 41107 from the Division of Lung Diseases, National Heart, Lung and Blood Insti- tute, National Institutes of Health.

References

1. Baker AP, Sawyer JL, Munroe JR, Weiner GP, Hillegrass LM: Glycosyltransferases in canine trachea. J Biol Chem 247: 5173--5179, 1972

2. Beyer TA, Rearick JI, Paulson JC, Prieels J, Sadler JE, Hill RL: Biosynthesis of mammalian glycoproteins. J Biol Chem 254: 12531-12541, 1979

3. Sugiura M, Kawasaki T, Yamashina I: Purification and characterization of UDP-GalNAc: Polypeptide GalNAc transferase from an Ascites Hepatoma, AH66. J Biol Chem 257: 9501-9507, 1982

4. Mendicino J, Sivakami S, Davila M, Chandrasekaran EV: Purification and Properties of UDP-Gal: N-acetylgalac-to- saminide mucin: [31,3-galactosyltransferase from Swine Trachea Mucosa. J Biol Chem 257: 3987-3994, 1982

5. Williams D, SchachterH: Detection in canine submaxillary glands of an N-acetylglucosaminyltransferase which acts on mucin substrates. J Biol Chem 255: 11247-11252, 1980

6. Williams D, Tongmore G, Matta KL, Schachter H: Sub- strate specificity and product identification studies on canine submaxillary gland UDP-GIcNAc: GaI[31-3GalNAc (GIcNAe---~ GalNAc) [36-N-acetylglucosaminyltransferase. J Biol Chem 255: 11253-11261, 1980

7. Wingert WE, Cheng P: Mucin biosynthesis: Character- ization of rabbit small intestinal UDP-N-Acetylglucosa- mine: Galactose [33-N-acetylgalactosamide (N-acetylglucosamine--* N-acetylgalactosamine) [36-N-acetylglucosaminyltransferase. Biochemistry 23: 690-697, 1984

8. Rana SS, Chandrasekaran EV, Kennedy J, Mendicino J: Purification and Structures of oligosaccharide chains in swine trachea and Cowper's gland muein glycoproteins. J Biol Chem 259: 12899-12907, 1984

9. Chandrasekaran EV, Rana SS, Davila M, Mendicino J: Structures of the oligosaccharide chains in swine trachea mucin glycoproteins. J Biol Chem 259: 12908-12914, 1984

10. Mendicino J, Chandrasekaran EV, Anumula KR, Davila M: Isolation and properties of a-D-Mannose: [3-1,2-N-Ace- tylglucosaminyltransferase from trachea mucosa. Biochem- istry 20: 96%976, 1981

11. Chandrasekaran EV, Davila M, Nixon D, Mendicino J: Purification and properties of c~-D-mannose [3-1,2GlcNAc transferase and a-mannosidases from adenocarcinoma. Canc Res 44: 4059-4068, 1984

12. Rao AK, Garver F, Mendicino J: Purification and proper-

ties of galactosyltransferases froms swine mesentary lymph nodes. Biochemistry 15: 5001-5009, 1976

13. Basu M, Basu S: Biosynthesis in vitro of li core glycosphin- golipids from neolactotetraosylceramide by 131-3- and [31-6- N-Acetylglucosaminyltransferases from mouse T-lympho- ma. J Biol Chem 259: 1255%12562, 1984

14. Sadler JE, Rearick JL, Paulson JC, Hill RL: Purification to homogeneity of a [3-galactoside ct2,3 sialyltransferase and partial purification of an ct-N-acetyl galactosaminide ct2,6 sialyl transferase from porcine submaxillary glands. J Biol Chem 254: 4434-4443, 1979

15. Bethell GS, Ayers JS, Hancock WS, Hearn MTW: A novel method of activation of cross-linked agaroses with 1-1'-carbonyldiimidazole which gives a matrix for affinity chromatography devoid of additional charged groups. J Biol Chem 254: 2572-2574, 1979

16. Chandrasekaran EV, Davila M, Nixon DV, Goldfarb M, Mendicino J: Isolation and structures of the oligosaccharide units of carcinoembryonic antigen. J Biol Chem 258: 7213- 7222, 1983

17. Chien J-L, Li S-C, Laine RA, Li Y-T: Characterization of Gangliosides from Bovine Erythrocyte Membranes. J Biol Chem 253: 4031-4035, 1978

18. Rao AK, Mendicino J: Synthesis of UDP-N-(1-14C)acetyl- D-glucosamine and UDP-N-(1-14C)acetyl-D-galactosamine from (1-14C)acetate. Anal Biochem 91: 490-495, 1978

19. Gleeson PA, Schachter: Control of Glycoprotein Synthe- sis: UDP-GIcNAc: GnGn(GlcNAc to Mancd-3)lM-N-acetylglucosaminyltransferase IV. An enzyme in hen oviduct which adds GlcNAc in 131-4 linkage to the ctl-3 linked man residue of the trimannosyl core of N-glycosyl-oligosaccharides to form a triantennary structure. J Biol Chem 258: 6162-6173

143

20. Schachter H, Narasimhan S, Gleenson P, Vella G: Control of branching during the biosynthesis of asparagine-linked oligosaccharides. Can J Biochem Cell Biol 61: 104%1066, 1983

21. Sheares BT, Carlson: Characterization of UDP GalNAc 3[3-Galactosyltransferase from Pig Trachea. J Biol Chem 258: 9893-9898, 1983

22. Furukawa K, Roth S: Co-purification of galactosyltransferases from chickembryo liver. Biochem J 227: 573-582, 1985

23. Koenderman AHL, Koppen PL, Van den Eijnden DH: Biosynthesis of polylactosaminoglycans: Novikoff ascites tumor cells contain two UDP-GIcNAc : [3-galactoside [31 6-N-acetylglucosaminyltransferase activities. J Biol Chem 263: 12461-12471, 1988

24. Brockhausen I, Williams D, Matta KL, Orr J, Schachter H: Mucin Synthesis III UDP-GlcNAc : GaI[31-3(GlcNAc[31-6) GalNAc-R(GlcNAc to Gal)[33-N-acetyl glucosaminyltransferase, an enzyme in porcine gastric mucosa involved in the elongation of mucin-type oligosaccharides. Can J Biochem Cell Biol 61: 1322-1333, 1983

25. Piller F, Carton JP: UDP-GlcNAc : Gal[31-4GIc(NAc)I31-3 N-acetylglucosaminyltransferase: Identification and characterization in human serum. J Biol Chem 258: 12293- 12299, 1983

26. Bergh MLE, Hooghwinkel GJM, Van den Eijnden: Bio- synthesis of the O-glycosidically linked oligosaccharide chains of fetuin. J Biol Chem 258: 7430-7436, 1983

Address for offprints: J. Mendicino, Department of Biochem- istry, University of Georgia, Athens, GA 30602, USA

Documents

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase