Biosynthesis of Branched Polylactosaminoglycans
EMBRYONAL CARCINOMA CELLS EXPRESS MIDCHAIN beta 1,6-N-ACETYLGLUCOSAMINYLTRANSFERASE ACTIVITY THAT GENERATES BRANCHES TO PREFORMED LINEAR BACKBONES*

Anne LeppänenDagger , Ying ZhuDagger , Hannu MaaheimoDagger , Jari HelinDagger , Eero Lehtonen§, and Ossi RenkonenDagger

From the Dagger  Institute of Biotechnology and Department of Biosciences, University of Helsinki, P. O. Box 56, FIN-00014 Helsinki and § Division of Pathology, Haartman Institute, University of Helsinki, P. O. Box 21, FIN-00014 Helsinki, Finland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Two types of beta 1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta 1-3)Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta 1-4GlcNAcbeta 1-3(GlcNAcbeta 1-6)Galbeta 1-4GlcNAc from the linear tetrasaccharide Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3(GlcNAcbeta 1-6)Galbeta 1-4GlcNAc and Galbeta 1-4GlcNAcbeta 1-3(GlcNAcbeta 1-6)Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc from the linear hexasaccharide Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta 1-3Galbeta 1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Biosynthesis of branched backbones of type 2 polylactosamines involves reactions catalyzed by beta 1,3-N-acetylglucosaminyltransferase (GnT3),1 beta 1,4-galactosyltransferase (GalT4), and beta 1,6-N-acetylglucosaminyltransferases (GnT6). However, the actual pathways leading to in vivo biosynthesis of branched polylactosamine backbones have not been clearly identified. The branch-forming reactions, in particular, are poorly understood. Two candidate branching reactions involving distinct beta 1,6-N-acetylglucosaminyltransferases (IGnT6) have been described in vitro (1). The "distally acting" dIGnT6 transfers a GlcNAc unit in the beta 1,6 linkage to the penultimate galactose residue at the growing end of the linear polylactosamine chain (Scheme 1) (2-8). By contrast, the "centrally acting" cIGnT6 transfers a GlcNAc residue in the beta 1,6 linkage to midchain galactose units of preformed as well as growing linear chains (3, 9, 10). The dIGnT6 reactions proceed only with acceptor chains bearing distal GlcNAc residues, whereas the cIGnT6 works with polylactosamine backbones carrying either a galactose or a GlcNAc residue at the distal position. There is very little overlapping in the acceptor specificities of the two types of enzymes in vitro.


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Scheme 1.   Formation of GlcNAc branches at different positions of a linear polylactosaminoglycan by the two types of IGnT6s (2-10).

In naturally occurring branched backbones, uniformly short LacNAcbeta 1-6 branches are linked to linear primary chains; this is the case e.g. in human embryonal carcinoma (EC) cells (11) and adult erythrocyte band 3 (12). It has been suggested that the dIGnT6 is responsible for the biosynthesis of polylactosamines in these cells (5, 11, 12). However, the suggested role of dIGnT6 in the biosynthesis of molecules containing exclusively short branches is doubtful because the extension enzyme, e.g. the GnT3 of human serum elongates both branches of the hexasaccharide LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAc (where LacNAc is Galbeta 1-4GlcNAc) (13), paving routes to the formation of complex as well as short branches (14).

We have recently suggested that midchain branching enzymes similar to the cIGnT6 activity present in blood serum of mammals may be responsible for the conversion of linear polylactosamine chains into branched backbone arrays in vivo (10). The reasoning was based on data showing that the cIGnT6 activity of rat serum catalyzed the transformation of the linear hexasaccharide LacNAcbeta 1-3LacNAcbeta 1-3LacNAc in two steps into the doubly branched octasaccharide LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAc. The latter was beta 1,4-galactosylated enzymatically into the mature decasaccharide backbone LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAc (10), which strikingly resembles the polylactosamine backbones of human EC cells of line PA1 (11).

Here, we report experiments involving lysates of human PA1 cells, exogenous UDP-GlcNAc, and either the tetrasaccharide LacNAcbeta 1-3'LacNAc or the hexasaccharide LacNAcbeta 1-3'LacNAcbeta 1-3'LacNAc that established the presence of the cIGnT6 activity in the PA1 cells. In contrast, the dIGnT6 activity was not detected in experiments where UDP-GlcNAc and GlcNAcbeta 1-3Galbeta 1-4GlcNAc were incubated with PA1 cell lysates. Lysates of murine EC cells of line PC13, known to carry large amounts of branched polylactosamines (15), also expressed the cIGnT6 activity but not the dIGnT6 activity. The data imply that cIGnT6 rather than dIGnT6 activity is involved in the biosynthesis of branched polylactosaminoglycans in human as well as murine EC cells. Hence, it is suggested that the linear polylactosamine backbones are probably synthesized first and branched afterward in these cells.

    EXPERIMENTAL PROCEDURES
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Results
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References

Cells-- Mouse embryonal carcinoma cells of line PC13 established from the pluripotent OTT6050 teratocarcinoma tumor (16) were obtained from Dr. C. F. Graham (Department of Zoology, University of Oxford, UK). The human PA1 teratocarcinoma-derived cells (17) were obtained from Dr. Jorma Wartiovaara (Institute of Biotechnology, University of Helsinki, Finland). The cells were maintained in Eagle's minimum essential medium supplemented with 10% fetal calf serum as described (18). For the experiments, the cells were detached from the dishes with 0.02% EDTA in NaCl-P buffer (140 mM NaCl, 10 mM sodium phosphate, pH 7.2) and washed twice in Dulbecco's phosphate-buffered saline, pH 7.2-7.4 (with Ca2+ and Mg2+).

Preparation of Cell Lysates-- Washed human (PA1) and mouse (PC13) EC cell pellets (50-150 µl) were lysed with 200 µl of 0.9% NaCl, 1% TX-100, 1 mM phenylmethylsulfonyl fluoride. In some experiments, more concentrated cell lysates were prepared by suspending the cell pellets in 50 µl of 1.8% NaCl, 2% TX-100, 2 mM phenylmethylsulfonyl fluoride with the cells. Small amounts (0.6 µl) of 40 mM phenylmethylsulfonyl fluoride in ethanol were added to the mixture once each h to a final concentration of 1 mM. In some experiments aprotinin and leupeptin were also added to the lysis buffer to a final concentration of 17 µg/ml and 20 µg/ml, respectively. The lysed cells were kept at 0 °C and homogenized by 5 × 3 strokes in a Potter homogenizer. The lysates were used immediately as the enzyme source in glycosyltransferase reactions.

Acceptor Oligosaccharides-- The acceptor oligosaccharides (for the structures see also Table I) were synthesized as described: LacNAcbeta 1-3[14C]Galbeta 1-4GlcNAc (1) (9), unlabeled 1 (19); two isotopomers of hexasaccharide 3 (LacNAcbeta 1-3[3H]Galbeta 1-4GlcNAcbeta 1-3'LacNAc and LacNAcbeta 1-3'LacNAcbeta 1-3[14C]Galbeta 1-4GlcNAc) (10); [14C]GlcNAcbeta 1-3'LacNAc (7) (7). A (1:1)-mixture of [3H]heptasaccharides 4 and 5 was synthesized from [3H]Galbeta 1-4GlcNAcbeta 1-3'LacNAcbeta 1-3'LacNAc (3) as described in Leppänen et al. (10).

Marker Oligosaccharides-- The following radiolabeled marker oligosaccharides were synthesized as described: 7, 8, and GlcNAcbeta 1-6'LacNAc (7); LacNAcbeta 1-3Gal (9); 2 (19); LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAc (13); a mixture of the heptasaccharides [3H]Galbeta 1-4GlcNAcbeta 1-3LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAc (4) + [3H]Galbeta 1-4GlcNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'LacNAc (5) and the octasaccharide LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAc (10). [3H]Galbeta 1-4GlcNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3Gal was obtained by endo-beta -galactosidase cleavage of authentic appropriately radiolabeled heptasaccharide 5 (10). The octasaccharide 6 (RMP = 0.29, RMH = 0.63; solvent A) was synthesized by beta 1,4-galactosylating the hexasaccharide GlcNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'LacNAc (8).

Glycosyltansferase Reactions-- The cIGnT6 reactions were performed by incubating the acceptors (3 pmol-100 nmol) and 3.7 µmol of UDP-GlcNAc with 25 µl of the EC cell lysate for 4 h and in some cases for 21-23 h in a total volume of 25 µl of 50 mM Tris-HCl buffer, pH 7.5, 8 mM NaN3, 20 mM EDTA, 0.5 mM ATP, 20 mM D-galactose, 60 mM gamma -galactonolactone, and 100 mM GlcNAc. EDTA inhibited the serum GnT3 activity (20), D-galactose and gamma -galactonolactone were added to inhibit beta -galactosidase activity, and GlcNAc was used to inhibit beta -N-acetylhexosaminidase activity. The dIGnT6 reactions with the teratocarcinoma cell lysates were carried out essentially as described for hog gastric mucosal microsomes (7), but incubation times of 4 h were used, and the total volume of the reaction mixture was 25 µl. All IGnT6 reaction mixtures were passed through a mixed bed of Dowex AG1 (AcO-) and Dowex AG 50 (H+), and the eluates were lyophilized.

Galactosylation with bovine milk beta 1,4-galactosyltransferase (EC 2.4.1.90) (Sigma) was performed essentially as described in Brew et al. (21).

Chromatographic Methods-- Paper chromatographic runs of desalted radiolabeled saccharides were performed on Whatman III Chr paper with the upper phase of 1-butanol/acetic acid/water (4:1:5 v/v; solvent A) or with 1-butanol/ethanol/water (10:1:2 v/v, solvent B). Radioactivity on the chromatograms was monitored as in Leppänen et al. (10) using Optiscint (Wallac, Turku, Finland) as scintillant. Marker lanes of malto-oligosaccharides on both sides of the sample lanes were stained with silver nitrate.

Gel permeation chromatography on a column of Superdex 75 HR 10/30 or Superdex Peptide HR 10/30 (Amersham Pharmacia Biotech) was performed as in Niemelä et al. (19).

Degradative Experiments-- Digestions with endo-beta -galactosidase from Bacteroides fragilis (EC 3.2.1.103) (Boehringer Mannheim) were performed according to Leppänen et al. (9); parallel control reactions cleaved over 90% of radiolabeled GlcNAcbeta 1-3Galbeta 1-4GlcNAc.

Digestions with jack bean beta -galactosidase (EC 3.2.1.23) were carried out as described in Renkonen et al. (22). Partial digestions with jack bean beta -N-acetylhexosaminidase (EC 3.2.1.30) were performed as in Leppänen et al. (10).

Partial acid hydrolysis was carried out using 0.1 M trifluoroacetic acid at 100 °C essentially as in Seppo et al. (7).

1H NMR Experiments-- The 1H NMR experiments were carried out as in Niemelä et al. (19).

Matrix-assisted Laser Desorption/Ionization Mass Spectrometry-- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed in the positive-ion delayed-extraction mode with a BIFLEXTM mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) using a 337-nm nitrogen laser. 1 µl of sample (10 pmol) and 1.5 µl of 2,5-dihydroxybenzoic acid matrix (10 mg/ml in water) were mixed on the target plate and dried with a gentle stream of air. Dextran standard 5000 from Leuconostoc mesentroides (Fluka Chemica-Biochemica) was used for external calibration.

    RESULTS
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Procedures
Results
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References

The structures of key oligosaccharides of the present experiments are shown in Table I and are identified in the text by using appropriate bold face digits.

                              
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Table I
Structures of the key saccharides

Branching Reactions of Tetrasaccharide 1, Catalyzed by Lysates of PA1 and PC13 Cells, Gave Pentasaccharide 2-- Incubation of tetrasaccharide Galbeta 1-4GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc (1) and UDP-GlcNAc with lysates of human embryonal carcinoma cells (line PA1) gave an oligosaccharide product that chromatographed on paper like authentic pentasaccharide 2 marker (peak 1 in Fig. 1A). Yields of 3-7% were obtained. The identity of the product as glycan 2 was established by enzymatic degradation. First, a treatment of the pentasaccharide with beta -galactosidase gave a product co-chromatographing with authentic GlcNAcbeta 1-3(GlcNAcbeta 1-6)[14C]Galbeta 1-4GlcNAc (8) (Fig. 1B). Next, the putative glycan 8 was identified by a partial treatment with beta -N-acetylhexosaminidase that gave the trisaccharides GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc and GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc as well as the disaccharide [14C]Galbeta 1-4GlcNAc (Fig. 1C). In addition to degradation, the original pentasaccharide product was subjected to MALDI-TOF mass spectrometry that gave a major signal at m/z 974.8 (Fig. 1D), assigned to the sodiated molecular ion of Gal2GlcNAc3 (calculated m/z = 974.9). Finally, the identity of the pentasaccharide product generated by PA1 cell lysates was confirmed by the 1H NMR spectrum (Fig. 1E, Table II); the resonances of the structural reporter groups were practically identical with those of authentic glycan 2 (23). Some of these resonances probably would have been different if the GlcNAc branch had been transferred to C-2 or C-4 of the central galactose unit of glycan 1 (24).


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Fig. 1.   Product analysis of the branching reaction of tetrasaccharide 1 catalyzed by the cIGnT6 activity of PA1 cells. A, paper chromatography (solvent A for 113 h) of the reaction mixture. Peak 1 represents the pentasaccharide product that chromatographed like authentic pentasaccharide 2; peak 2 is the unreacted acceptor 1. Unlabeled markers MT, MTet, MP, (MH), maltotriose, -tetraose, -pentaose (and -heptaose), respectively. The position of the radiolabel in the glycans is shown by an asterisk. B, paper chromatography (solvent A for 96 h) of a beta -galactosidase digest of peak 1 of A. The major product chromatographed like authentic tetrasaccharide 8. C, paper chromatography (solvent B for 305 h) of a partial beta -N-acetylhexosaminidase digest of peak 1 of B. Peak 1 chromatographed like uncleaved 8, peak 2 like GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc, peak 3 like GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc (7), and peak 4 like [14C]Galbeta 1-4GlcNAc. Unlabeled markers are as in A. D, MALDI-TOF mass spectrum of the original pentasaccharide product. The signal at m/z 974.8 was assigned to (M + Na)+ of Gal2GlcNAc3 (calculated m/z 974.9). The signal at m/z 990.8 was assigned to (M + K)+ (calculcated m/z 990.3). E, reporter group signals of a 500-MHz 1H NMR spectrum of the original pentasaccharide product (20.5 nmol) at 23 °C; the vertical scale at 4.1-5.3 ppm is multiplied by 4. The spectrum was very nearly identical with the spectrum of authentic pentasaccharide 2 generated by the cIGnT6 of rat serum (23) (see Table II).

                              
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Table II
1H chemical shifts of tetrasaccharide 1 and pentasaccharide 2 at 23 °C in 2H2O
<AR><R><C></C></R><R><C></C></R><R><C><UP>  D      C</UP></C></R><R><C><UP>Gal&bgr;1–4</UP><UP>GlcNAc&bgr;1 </UP></C></R></AR><AR><R><C><UP>      B      A</UP></C></R><R><C><UP> 3 Gal&bgr;1–4GlcNAc </UP>(<UP><B>1</B></UP>) </C></R><R><C><UP>&cjs0604;</UP></C></R></AR>
<AR><R><C><UP>E     </UP></C></R><R><C><UP>GlcNAc&bgr;1&cjs0605; </UP></C></R><R><C><UP>6</UP></C></R><R><C><UP>D      C    3</UP></C></R><R><C><UP>Gal&bgr;1–4GlcNAc&bgr;1&cjs0604;</UP></C></R></AR><AR><R><C><UP>   B      A</UP></C></R><R><C><UP>  Gal&bgr;1–4GlcNAc </UP>(<UP><B>2</B></UP>)</C></R></AR>

The pentasaccharide 2 was formed from tetrasaccharide 1 in yields of 6-10%, also in reactions catalyzed by lysates of murine EC cells of line PC13 (Fig. 2A). The endo-beta -galactosidase-resistant product was identified as glycan 2 by enzymatic degradation as above. First, beta -galactosidase converted the putative pentasaccharide 2 into a tetrasaccharide that chromatographed like glycan 8 (Fig. 2B). Then the putative glycan 8, upon a partial treatment with beta -N-acetylhexosaminidase gave a mixture of the trisaccharides GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc and GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc as well as the disaccharide [14C]Galbeta 1-4GlcNAc (Fig. 2C). Finally, enzymatic beta 1,4-galactosylation of the putative pentasaccharide 2 gave the hexasaccharide LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAc (Fig. 2D), establishing the presence of a distal GlcNAc in the acceptor.


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Fig. 2.   Product analysis of the branching reaction of tetrasaccharide 1 catalyzed by the cIGnT6 activity of PC13 cells. A, paper chromatography (solvent A for 114 h) of the reaction mixture. Peak 1 represents the pentasaccharide product, and peak 2, the unreacted acceptor 1. The position of the radiolabel is shown by an asterisk. B, paper chromatography (solvent A for 113 h) of beta -galactosidase digest of peak 1 of A. Peak 1 migrated like the marker tetrasaccharide 8. C, paper chromatography (solvent B for 305 h) of partial beta -N-acetylhexosaminidase digest of peak 1 of B. Peak 1 chromatographed like the uncleaved 8, peak 2 like GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc, peak 3 like GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc, and peak 4 like [14C]Galbeta 1-4GlcNAc. D, paper chromatography (solvent A for 141 h) of products from GalT4 reaction of peak 1 of A. Peak 1 chromatographed like authentic hexasaccharide LacNAcbeta 1-3'(LacNAcbeta 1-6')LacNAc.

Branching Reactions of Hexasaccharide 3, Catalyzed by PA1 Cell Lysates Gave Heptasaccharide Isomers 4 and 5-- Two isotopomers of glycan 3 (LacNAcbeta 1-3'LacNAcbeta 1-3[14C]Galbeta 1-4GlcNAc and LacNAcbeta 1-3[3H]Galbeta 1-4GlcNAcbeta 1-3'LacNAc) were synthesized and were separately incubated with PA1 cell lysates and UDP-GlcNAc to establish whether both the galactose 2 (labeled in the [14C]-acceptor) and the galactose 4 (labeled in the [3H]-acceptor) of glycan 3 serve as independent acceptor sites. A heptasaccharide-like product was formed from both acceptors. The product chromatographed as a single peak (peak 1 in Fig. 3A, RMP = 0.49, RMH = 1.04, solvent A), showing the same migration rate as an unresolved mixture of authentic heptasaccharides 4 and 5 (10). The net yield of the heptasaccharide-like fraction varied from 3 to 7% in 4-h incubations in several separate experiments; it was not improved in 22-h incubations. To ensure that peak 1/Fig. 3A represented an authentic product resulting from the transfer of GlcNAc to radiolabeled acceptor 3, an aliquot of the material was treated with UDP-Gal and GalT4. This treatment gave 62% [3H]glycans chromatographing like authentic octasaccharide 6 (Fig. 3B, RMP = 0.32, RMH = 0.68, solvent A), establishing the presence of a distal GlcNAc residue in most of the glycans of peak 1/Fig. 3A. In addition, the data of Fig. 3B suggest that the glycans of peak 1/Fig. 3A also included the [3H]hexasaccharide 3 acceptor itself, which contaminated the heptasaccharide products because of obvious chromatographic "tailing." Other experiments that are described below show that the distal GlcNAc units of the heptasaccharide products of the branching reaction of glycan 3 were beta 1,6-bonded in some molecules to the galactose 2 and in others to the galactose 4 of the acceptor.


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Fig. 3.   Product analysis of the branching reactions of hexasaccharide 3 catalyzed by the cIGnT6 activity of PA1 cells. A, paper chromatography (solvent A for 331 h) of the products from the [3H]hexasaccharide 3. Peak 1 contained the heptasaccharide mixture (4 + 5) and some contaminating 3, whereas peak 2 represented the bulk of the unchanged acceptor 3. The position of the radiolabel in the glycans is shown by an asterisk. B, paper chromatography (solvent A for 264 h) of the products from a GalT4 reaction performed with the concentrate of [3H]heptasaccharides (4 + 5) present in peak 1 of A. Peak 1 chromatographed like the authentic octasaccharide 6, and peak 2 migrated like the hexasaccharide 3. C, paper chromatography (solvent A for 115 h) of products from endo-beta -galactosidase digestion of the [14C]-labeled mixture of crude heptasaccharides 4 + 5. Peak 1 chromatographed like LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3[14C]Gal marker, and peak 2, like authentic tetrasaccharide 8. The position of the [14C]-label is shown by an asterisk. D, paper chromatography (solvent B for 359 h) of a partial beta -N-acetylhexosaminidase digest of peak 2 of C. Peak 1 chromatographed like uncleaved 8, peak 2 like GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc, peak 3 like GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc. E, paper chromatography (solvent B for 359 h) of a partial acid hydrolysate of the hexasaccharide LacNAcbeta 1-3(GlcNAcbeta 1-6)[3H]Galbeta 1-4GlcNAcbeta 1-3Gal isolated from an endo-beta -galactosidase digest of the crude [3H]-labeled heptasaccharides 4 + 5. Peak 1 contained a mixture of [3H]-labeled trisaccharides, peak 2 was GlcNAcbeta 1-6[3H]Galbeta 1-4GlcNAc, and peak 4 was GlcNAcbeta 1-6[3H]Gal. Unlabeled markers are as in Fig. 1C.

The presence of glycan 4 in the heptasaccharide fraction of the branching reaction was established by using endo-beta -galactosidase, which cleaves internal beta -galactosidic linkages of polylactosamines but does not attack these linkages at branch-bearing N-acetyllactosamine units (25). Treatment of the crude [14C]heptasaccharide fraction with this enzyme gave two labeled oligosaccharide products, shown in Fig. 3C. Peak 2, chromatographing like the branched tetrasaccharide 8, was derived from the putative glycan 4. It was isolated and cleaved by partial beta -N-acetylhexosaminidase treatment into GlcNAcbeta 1-6[14C]Galbeta 1-4GlcNAc (peak 2) and GlcNAcbeta 1-3[14C]Galbeta 1-4GlcNAc (peak 3) as shown in Fig. 3D. These data established that during the incubation with UDP-GlcNAc and PA1 cell lysate, a beta 1,6-bonded GlcNAc branch had been transferred to galactose 2 of the [14C]-labeled glycan 3. Peak 1 of Fig. 3C represented the hexasaccharide LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 3[14C]Gal, a cleavage product of glycan 5; it was not studied further because it contained no easily accessible structural information about the branch structure.

The presence of glycan 5 in the crude [3H]heptasaccharide fraction of the branching reaction was also established by using endo-beta -galactosidase, which gave two radiolabeled oligosaccharides, LacNAcbeta 1-3[3H]Gal (from glycan 4) and LacNAcbeta 1-3(GlcNAcbeta 1-6)[3H]Galbeta 1-4GlcNAcbeta 1-3Gal, that were separated by paper chromatography (not shown). The hexasaccharide product, which was derived from the putative glycan 5 of the [3H]heptasaccharide fraction, was then subjected to partial acid hydrolysis. This resulted in formation of several [3H]oligosaccharides shown in Fig. 3E. The important products were GlcNAcbeta 1-6[3H]Galbeta 1-4GlcNAc (peak 2) and GlcNAcbeta 1-6[3H]Gal (peak 4), which proved that the galactose 4 of the [3H]-labeled glycan 3 had accepted a beta 1,6-bonded GlcNAc branch during the incubation with UDP-GlcNAc and a PA1 cell lysate.

The Branching Reaction of the Mixed Heptasaccharides 4 and 5, Catalyzed by PA1 Cell Lysates, Gave an Octasaccharide-- Incubation of an authentic 1:1 mixture of [3H]-labeled heptasaccharides 4 and 5 with UDP-GlcNAc and PA1 cell lysates gave small amounts (2.4-4.4%) of a product that chromatographed on paper like the doubly branched octasaccharide LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAc (not shown; RMP = 0.74, RMH = 1.10). In the MALDI-TOF mass spectrum the product gave a sodium-containing molecular ion at m/z 1543.4 (calculated m/z for Gal3HexNAc5 1543.4). Together with the (M + K)+ signal, this ion represented 70% of the total polylactosamines in the molecular ion range (26). A treatment of the octasaccharide concentrate with GalT4 and UDP-Gal gave a crude decasaccharide. In MALDI-TOF mass spectrum of this product, a major signal at m/z 1867.8 was observed that was assigned to (M + Na)+ of Gal5HexNAc5 (calculated m/z 1867.7). These data suggest that the octasaccharide formed from the mixture of the heptasaccharides 4 and 5 by the PA1 cell lysate was [3H]LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAcbeta 1-3'(GlcNAcbeta 1-6')LacNAc.

Human (PA1) and Mouse (PC13) Embryonal Carcinoma Cells Did Not Reveal the Presence of the dIGnT6 Activity-- When the trisaccharide [14C]GlcNAcbeta 1-3Galbeta 1-4GlcNAc (7) (21 pmol) was incubated with UDP-GlcNAc and human or mouse EC cell lysates, chromatographically detectable tetrasaccharide-like products were formed in less than 0.3% yield (not shown), implying that human and mouse EC cells did not express significant dIGnT6 activities.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present data show that lysates from human embryonal carcinoma cells of line PA1 contained centrally acting beta 1,6-N-acetylglucosaminyltransferase activity, which catalyzed the formation of the branched pentasaccharide 2 from the linear tetrasaccharide 1 in the presence of exogenous UDP-GlcNAc (For the structures of the saccharides, see Table I). In addition, heptasaccharides 4 and 5 were formed from the linear hexasaccharide 3. Evidence was also provided supporting the notion that a second beta 1,6-GlcNAc branch was transferred during incubation of a mixture of the heptasaccharides 4 and 5 with PA1 cell lysates and UDP-GlcNAc. We call the activity responsible for these reactions as cIGnT6 to emphasize the site-specificity of the reaction in the central area of the acceptor and the formation of precursors of the blood group I antigen.

Because PA1 cells are known to express branched polylactosamine backbones (11, 27), it is reasonable to assume that the in vitro reactions described in the present experiments are similar to those responsible for the synthesis of the multiply branched polylactosamines in vivo.

Also lysates from murine embryonal carcinoma cells of line PC13 contained cIGnT6 activity. The PC13 cells are also known to express large amounts of branched polylactosamines (15). Hence, the cIGnT6 reactions observed in vitro in the present experiments are likely to occur also in vivo during the synthesis of PC13 glycans.

The action of dIGnT6, too, leads in vitro to the formation of branched polylactosamines (14), and this enzyme has been suggested to be responsible for the in vivo biosynthesis of branched polylactosamine backbones (5, 11, 12). However, in the present experiments we were unable to observe any dIGnT6 activity in lysates of PA1 cells or PC13 cells that would have converted the linear trisaccharide GlcNAcbeta 1-3Galbeta 1-4GlcNAc into the branched tetrasaccharide GlcNAcbeta 1-3(GlcNAcbeta 1-6)Galbeta 1-4GlcNAc. Taken together, our present data imply that the cIGnT6 activity rather than the dIGnT6 activity may be responsible for the in vivo synthesis of branched polylactosamine backbones in embryonal carcinoma cells.

The dominant role of cIGnT6 in the branch generation, combined with the data showing that the branches of glycans in PA1 cells are short along the entire backbone chain (11), suggests that the biosynthesis of branched polylactosamine backbones in PA1 cells occurs in rather distinct stages as shown in Scheme 2: First, alternating action of GnT3 and GalT4 elongates the linear backbone chains to their final size. Second, the linear backbones are branched by cIGnT6 at different sites along the chains. Third, the GlcNAc branches are finally galactosylated by GalT4. A process of this kind is likely to produce rather similar branches along the entire primary backbone chain. By contrast, participation of dIGnT6 in the branching process would generate branches in association with chain elongation, probably leading to more complex branches in the proximal than the distal parts of the mature backbones.


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Scheme 2.   Proposed biosynthetic pathway leading to branched polylactosaminoglycan backbones in PA1 cells. The number of branches will vary depending on the relative amounts of IGnT6, UDP-GlcNAc, and acceptor sites, but all internal galactoses of the primer chain are inherently able to react with the cIGnT6 activity. A similar pathway appears to exist in murine embryonal carcinoma cells.

The concept that linear polylactosamine chains are precursors of the branched backbones is not new. The relationship was proposed already in 1979 when the developmentally regulated expression of small i (linear chains) and big I (branched backbones) as blood group antigens in human and bovine red blood cells was described (28, 29). The present data merely provide the underlying mechanism of the interconversion in EC cells. Scheme 2 suggests also that cIGnT6 is localized in the Golgi compartment of PA1 cells in a more restricted manner than Gal T4 and more distally than GnT3.

The presence of the cIGnT6 activity in the murine EC cell lysates suggests that the polylactosamine backbones of these cells may also consist of primary linear chains that carry short branches. Such arrays may be important scaffolds for "presenting" the binding epitopes of cell adhesion saccharides in multivalent, high affinity modes. This notion is supported by the finding that functionally active sperm receptor saccharides are successfully assembled to ZP3 protein of murine zona pellucida in "appropriately" transfected murine embryonal carcinoma cells but not in a number of other cells similarly transfected (30); the failing cells probably did not express sufficient amounts of enzymes required for synthesis of branched polylactosamines. Recently, a polylactosamine backbone decorated by several sialyl Lewis X-bearing branches has actually proven to be a highly potent antagonist of lymphocyte L-selectin (31).

We note that the cDNA directing the expression of branched polylactosamines has already been isolated from the cDNA expression library from PA1 cells (32). This cDNA probably codes the cIGnT6 observed in the present experiments.

    FOOTNOTES

* This work was supported by Academy of Finland Grants 38042 and 40901 and Technology Development Center of Finland Grant TEKES 40057/97.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 should be addressed. Tel.: 358-9-708 59375; Fax: 358-9-708 59563; E-mail: ossi.renkonen{at}helsinki.fi.

1 The abbreviations used are: GnT3, beta 1,3-N-acetylglucosaminyltransferase (GlcNAc to Gal); GalT4, beta 1,4-galactosyltransferase (Gal to GlcNAc); cIGnT6, centrally acting beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to Gal); dIGnT6, distally acting beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to Gal); EC, embryonal carcinoma; Gal (or G), D-galactose; GlcNAc (or Gn), N-acetyl-D-glucosamine; Lac, lactose; LacNAc, N-acetyllactosamine (Galbeta 1-4GlcNAc); MALDI-TOF mass spectroscopy, matrix-assisted laser desorption-ionization mass spectrometry with time-of-flight detection; MH, maltoheptaose [Glcalpha 1-4(Glcalpha 1-4)5Glc]; MP, maltopentaose [Glcalpha 1-4(Glcalpha 1-4)3Glc]; MT, maltotriose (Glcalpha 1-4Glcalpha 1-4Glc); MTet, maltotetraose [Glcalpha 1-4(Glcalpha 1-4)2Glc].

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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