J Biol Chem, Vol. 274, Issue 14, 9296-9304, April 2, 1999

Regulation of I-Branched Poly-N-Acetyllactosamine Synthesis
CONCERTED ACTIONS BY i-EXTENSION ENZYME, I-BRANCHING ENZYME, AND 1,4-GALACTOSYLTRANSFERASE I^*

Minoru Ujita, Joseph McAuliffe, Misa Suzuki, Ole Hindsgaul, Henrik Clausen^§, Michiko N. Fukuda, and Minoru Fukuda^¶

From the Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the ^§ School of Dentistry, University of Copenhagen, DK-2200 Copenhagen, Denmark

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

I-branched poly-N-acetyllactosamine is a unique carbohydrate composed of N-acetyllactosamine branches attached to linear poly-N-acetyllactosamine, which is synthesized by I-branching 1,6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewis^x, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching 1,6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers 1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various 1,4-galactosyltransferases (4Gal-Ts), 4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a 1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of 4Gal-TI and i-extension 1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Gal14GlcNAc13 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that 4Gal-TI adds a galactose poorly to 1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while 1,3-N-acetylglucosaminyltransferase and 4Gal-TI efficiently add N-acetyllactosamine to linear poly-N-acetyllactosamines. Together, these results strongly suggest that galactosylation of I-branch is a rate-limiting step in I-branched poly-N-acetyllactosamine synthesis, allowing poly-N-acetyllactosamine extension mostly along the linear poly-N-acetyllactosamine side chain. These findings are entirely consistent with previous findings that poly-N-acetyllactosamines in human erythrocytes, PA-1 embryonic carcinoma cells, and rabbit erythrocytes contain multiple, short I-branches.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Poly-N-acetyllactosamines are unique glycans having N-acetyllactosamine repeats, (Gal14GlcNAc13)_n (1). Poly-N-acetyllactosamines are attached to N-glycans (2-5), O-glycans (6, 7), and glycolipids (8-10) and can be digested by endo--galactosidase (11).

Poly-N-acetyllactosamines are often modified to express differentiation antigens and functional oligosaccharides. Among those oligosaccharides, sialyl Lewis^x (Le^x)¹ and its sulfated forms are ligands for E-, P-, and L-selectin (12-16). During inflammation, E- and P-selectin expressed on activated endothelial cells bind to sialyl Le^x oligosaccharides present on granulocytes and such initial binding leads to the extravasation of granulocytes. L-selectin on lymphocytes, on the other hand, recognizes sulfated sialyl Le^x expressed in high endothelial venules of blood vessels (15, 16). This L-selectin/counterreceptor interaction allows lymphocytes to migrate into lymphoid system, allowing lymphocytes to circulate fully in the body.

In human granulocytes, monocytes, and certain T lymphocytes, poly-N-acetyllactosamines contain Le^x, Gal14(Fuc13)GlcNAcR, and sialyl Le^x, NeuNAc23Gal14(Fuc13)GlcNAcR (17, 18). In contrast, poly-N-acetyllactosamines in human erythrocytes contain ABO blood group antigens, synthesized from the precursor structure, Fuc12Gal14GlcNAcR (5, 19, 20). In addition, poly-N-acetyllactosamines can contain I-branches, Gal14GlcNAc13(Gal14GlcNAc16)GalR. During development of human erythrocytes, the linear i antigen represented by Gal14GlcNAc13Gal14GlcNAcR is converted to those containing I-branches (21). In early mouse embryonic development, embryos express I antigen, which is gradually replaced with i antigen during development (2, 22).

The acquisition of I-branches is important, since two of the N-acetyllactosamine side chains can have functional terminal structures. It has been demonstrated that multivalent sialyl Le^x poly-N-acetyllactosamines inhibit L-selectin-mediated binding and the rejection of organ transplants with much better efficacy than monovalent sialyl Le^x poly-N-acetyllactosamines (23, 24). Similarly, blood group H antigens present at both termini in branched poly-N-acetyllactosamines were shown to have much better avidity to anti-ABO antibodies than linear poly-N-acetyllactosamines containing single antigenic structures (25). It was suggested that expression of i antigen in fetal erythrocytes minimizes a detrimental immune response when mother and fetus have incompatible blood group antigens (25).

These results, as a whole, indicate the significance of understanding how linear and I-branched poly-N-acetyllactosamines are synthesized. To this end, we have cloned cDNAs encoding 1,3-N-acetylglucosaminyltransferase (iGnT) that forms linear poly-N-acetyllactosamines (26) and 1,6-N-acetylglucosaminyltransferase (IGnT) that forms I-branches (27). The IGnT cloned was found to add 1,6-N-acetylglucosamine at the central galactose (underlined) of Gal14GlcNAc13Gal14GlcNAcR, thus termed as centrally acting IGnT (cIGnT) (28, 29). In addition, another IGnT, distally acting IGnT (dIGnT), was found to add 1,6-N-acetylglucosamine to peridistal galactose (underlined) of GlcNAc13Gal14GlcNAcR (29-33). No studies have been reported, however, to determine how I-branched poly-N-acetyllactosamine is synthesized by iGnT, IGnT, and 4Gal-T.

In the present study, we first describe how the IGnT cloned from PA-1 cells (27) adds I-branches to linear poly-N-acetyllactosamines containing multiple acceptor sites. We then demonstrate that 4Gal-TI is responsible for galactosylation in the synthesis of both linear and branched poly-N-acetyllactosamines. Finally, we reconstituted the synthesis of I-branched poly-N-acetyllactosamine, the structure of which resembles that present in human erythrocytes (5), PA-1 human embryonic carcinoma cells (34), and rabbit erythrocytes (10). The results demonstrate an intricate interaction between acceptor substrates and these glycosyltransferases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of cDNA Encoding iGnT and IGnT-- cDNA encoding iGnT was cloned into pcDNA3.1, resulting in pcDNA3.1-iGnT, as described previously (26). pcDNAI-A, harboring cDNA encoding a signal sequence and an IgG binding domain of Staphylococcus aureus protein A, was constructed as described before (35). The catalytic domain of iGnT was cloned into this vector, resulting in pcDNAI-A·iGnT.

cDNA encoding IGnT was cloned from a cDNA library constructed from human PA-1 embryonic carcinoma cells, resulting in pcDNAI-IGnT, as described previously (27). A catalytic domain of IGnT was prepared by polymerase chain reaction using pcDNAI-IGnT as a template. 5'- and 3'-primers for this polymerase chain reaction were 5'-GCGGATCCAAGCTTCCAAAGGCTA-3' and 5'-GGCTCGAGCTCAAAAATACCAGCTGGGT-3' (BamHI and XhoI sites are underlined). The polymerase chain reaction product encoding amino acid residues 30-400 of the IGnT was digested with BamHI and XhoI and then cloned into the same sites of pcDNAI-A, resulting in pcDNAI-A·IGnT.

Expression of the Protein A-iGnT and Protein A-IGnT Fusion Protein-- pcDNAI-A, pcDNAI-A·iGnT, and pcDNAI-A·IGnT were separately transfected with Lipofectamine Plus (Life Technologies) into COS-1 cells as described previously (36). The chimeric enzyme released into serum-free Opti-MEM was used after adsorbing the protein A chimeric enzymes to IgG-Sepharose 6FF (Amersham Pharmacia Biotech) as described previously (37). Alternatively, the culture medium was concentrated 100-fold by a Centricon 10 concentrator (Amicon) and directly used as an enzyme source. In most of the studies, the concentrated culture medium was used for iGnT, since IgG-Sepharose-bound enzymes had a low activity as seen for other glycosyltransferases (38, 39). Typically, the activities of iGnT and IGnT in the incubation mixture were 38.0 nmol/h/ml using 0.5 mM Gal14Glcp-nitrophenol (Toronto Research Chemicals) and 40.0 nmol/h/ml using 0.5 mM Gal14GlcNAc13Gal14GlcNAc16Man16Manoctyl (see below) as acceptors, respectively.

The medium from mock-transfected COS-1 cells contained less than of iGnT activity as described (26) or less than of IGnT activity compared with that derived from pcDNAI-A·iGnT- or pcDNAI-A·IGnT-transfected COS-1 cells.

Expression of cDNAs Encoding 4Gal-TII, -TIII, -TIV, and -TV-- Isolation of cDNAs encoding 4Gal-TII, -TIII, and -TIV was described previously (40, 41). 4Gal-TII, -TIII, and -TIV were expressed in insect cells, and the supernatants from these transfected insect cells were used as an enzyme source as described previously (36, 41). Human milk 4Gal-T preparation (Sigma) was directly used as 4Gal-TI (36).

4Gal-TV (42) was cloned and expressed in COS-1 cells as described previously (36). The supernatant from the transfected COS-1 cells was concentrated 100-fold as described above and used as an enzyme source. For comparing the enzymatic activities of different 4Gal-T samples, the final concentration of 4Gal-TI, -TII, -TIII, -TIV, and -TV was adjusted to 38.0 nmol/h/ml as measured using 0.5 mM GlcNAcp-nitrophenol (Sigma) as an acceptor.

Oligosaccharides-- (Gal14GlcNAc13)_nGal14GlcNAc16Man16ManO(CH₂)₇CH₃(octyl), where n = 0, 1, and 2, were synthesized, starting from the derivatives of Gal14GlcNAc and Man16Manoctyl, as described previously (36). GlcNAc13Gal14GlcNAc16Man16Manoctyl and GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man16Manoctyl were prepared by Escherichia coli -galactosidase treatment of Gal14GlcNAc13Gal14GlcNAc16Man16Manoctyl and Gal14GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man16Man octyl, respectively, as described previously (36).

Lacto-N-neo-tetraose (Gal14GlcNAc13Gal14Glc) and lacto-N-neo-hexaose (Gal14GlcNAc13(Gal14GlcNAc16)Gal14Glc) were pyridylaminated, as described previously (31, 43). Purification of the product was achieved by HPLC on a TSK gel ODS-80TS column (4.6 × 250 mm; TOSOH) equipped with a Gilson 306 HPLC apparatus. The column was equilibrated with 20 mM ammonium acetate buffer, pH 4.0, and eluted with the same buffer at a flow rate of 1.0 ml/min. Fluorescence was detected with a fluorescent spectrophotometer (Shimadzu, model RF-535) with excitation and emission wavelengths of 320 and 400 nm, respectively. The concentration of PA-oligosaccharides was estimated by comparing fluorescent intensity of synthesized PA-oligosaccharides and standard PA-glucose purchased from Takara Shuzo (PanVera, Madison, WI). Lacto-N-neo-tetraose, lacto-N-neo-hexaose, and lacto-N-hexaose (Gal13GlcNAc13(Gal14GlcNAc16)Gal14Glc) were purchased from Oxford GlycoSystems.

Addition of N-Acetylglucosamine by iGnT and IGnT-- To assay the transfer of N-acetylglucosamine residues by the iGnT, the reaction mixture was exactly the same as described previously (36). As acceptors, lacto-N-neo-tetraose, lacto-N-neo-hexaose, lacto-N-hexaose, pyridylaminated lacto-N-neo-hexaose (PA-lacto-N-neo-hexaose), and lacto-N-neo-tetraose (PA-lacto-N-neo-tetraose) were used. The assay products derived from the above oligosaccharides were purified by QAE-Sephadex A-25 gel and subjected to Bio-Gel P-4 gel filtration as described previously (36). The radioactivity of the aliquots was determined by a scintillation counter. The products derived from the PA-oligosaccharides were filtered through Ultrafree-MC (10-kDa cut; Millipore Corp.) and applied to the same ODS-80TS column and eluted as described above. Since PA-oligosaccharides can be detected by fluorescence, nonradioactive UDP-GlcNAc was used as a donor for the experiments using PA-oligosaccharides.

To assay the transfer of N-acetylglucosamine residues by IGnT, the reaction mixture contained 5 mM UDP-[³H]GlcNAc (2 × 10⁴ cpm/nmol; NEN Life Science Products), 10 mM EDTA, 20 µl of IGnT preparation as described above, 10 mM N-acetylglucosamino-1,5-lactone (Toronto Research Chemicals), 10 mM galactono-1,5-lactone, and various concentrations of an acceptor in 50 µl (final volume) of 100 mM cacodylate buffer, pH 7.0, modified from the previously described protocol (31). As acceptors, GlcNAc13Gal14GlcNAc16Man16Manoctyl, Gal14GlcNAc13Gal14GlcNAc16Man16Manoctyl, GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man16Manoctyl, and (Gal14GlcNAc13)₂Gal14GlcNAc16Man16Manoctyl were used. The incubation mixture was applied to a C18 reverse phase Sep-Pak column (Waters), and the product was eluted as described previously (36). The product was then analyzed by HPLC using NH₂-bonded silica column (Varian Micropak AX-5) as described previously (36). The radioactivity of aliquots was determined. In all of the above reactions, the reaction mixture was incubated for 10 h to analyze the products or for 1 h to obtain kinetic parameters.

Substrate Specificity of 4Gal-TI, -TII, -TIII, -TIV, and -TV-- Assays of 4Gal-Ts were performed exactly as described previously (36). As acceptors, GlcNAc13Gal14GlcPA and Gal14GlcNAc13(GlcNAc16)Gal14GlcPA were used. Gal14GlcNAc13(GlcNAc16)Gal14GlcPA was synthesized by incubating 5 mM Gal14GlcNAc13Gal14GlcPA with the recombinant IGnT and 5 mM UDP-GlcNAc for 10 h in 100 µl of the reaction mixture as described above. The product was purified by HPLC using the same ODS column as described above and used as an acceptor.

Poly-N-Acetyllactosamine Formation in I-branched Oligosaccharide-- To assay poly-N-acetyllactosamine formation, 0.5 mM Gal14GlcNAc13(GlcNAc16)Gal14GlcPA was incubated with 4Gal-TI (152 nmol/h/ml), iGnT (38 nmol/h/ml), 0.5 mM UDP-GlcNAc, and 0.5 mM UDP-Gal in 50 µl of 100 mM cacodylate buffer, pH 7.0, containing 20 mM MnCl₂ and 10 mM each of GlcNAc-1,5-lactone and Gal-1,5-lactone. After incubation at 37 °C for 4 h, the reaction mixture was filtered and subjected to HPLC as described above. In these experiments, the incubation condition was first determined using Gal14GlcNAc13Gal14Glc, where only one N-acetyllactosamine unit can be added. In addition, 4Gal-TI was 4-fold in excess over iGnT, the same ratio as present in HL-60 cells (44).

Analysis of Products by Endo--Galactosidase Digestion-- Products were digested with Escherichia freundii endo--galactosidase for 18 h at 37 °C (11). The digestion condition used allowed the cleavage of galactose linkage, where no -1,6-linked N-acetylglucosamine is attached (11, 45). The digests were subjected to HPLC using AX-5 column or TSK gel ODS-80TS.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Addition of I-branch to Various Poly-N-Acetyllactosaminyl Side Chains-- Recently, it was reported that the IGnT cloned from PA-1 cells exclusively adds 1,6-linked N-acetylglucosamine to a galactose residue in a central position as seen in Gal14GlcNAc13Gal14Glc(NAc)R (the underlined galactose is the accepting galactose (28)). To determine if the IGnT can add N-acetylglucosamine residues far from nonreducing terminals, acceptors with various numbers of N-acetyllactosamine repeats were used. As shown in Fig. 1, B and D, the oligosaccharide containing two potential acceptor sites, (Gal14GlcNAc13)₂Gal14GlcNAc16ManR, incorporated slightly more N-acetylglucosamine than that containing only one acceptor site. When the products obtained from (Gal14GlcNAc13)₂Gal14GlcNAc16ManR were analyzed by endo--galactosidase digestion, the majority of singly branched products contained 1,6-linked GlcNAc close to the nonreducing terminal (Fig. 1F and Fig. 2, compound e). These results indicate that the IGnT displays a preference for a galactose residue separated by one N-acetyllactosamine unit from the nonreducing terminal (Fig. 2, compounds b, e, and g). The results shown in Fig. 1, C and D, also indicate that the addition of two I-branches to neighboring galactose residues (compounds d and g in Fig. 2) occurs less efficiently than the addition of one branch.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Analysis of the products after incubation of poly-N-acetyllactosamine acceptors with IGnT. A, B, C, and D, HPLC analysis of the IGnT products derived from GlcNAc13Gal14GlcNAc16Man1R (A), Gal14GlcNAc13Gal14GlcNAc16Man1R (B), GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man1R (C), and Gal14GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man1R (D). The first and second peaks in C and D are the oligosaccharides containing one and two 1,6-linked GlcNAc residues, respectively. E and F, HPLC analysis of endo--galactosidase digestion of singly branched products from GlcNAc13Gal14GlcNAc13Gal14GlcNAc16Man1R shown in C (E) and (Gal14GlcNAc13)2Gal14GlcNAc16Man1R shown in D (F). The compounds eluted at fractions 28 and 45 in E and F are GlcNAc13([3H]GlcNAc1 6)Gal14GlcNAc16Man16Manoctyl and Gal14GlcNAc13([3H]GlcNAc16)Gal14GlcNAc13Gal, respectively. The numbers indicate the relative molar ratio of the IGnT products (A-D) or endo--galactosidase-digested products (E, F). In this HPLC, amino-bonded AX-5 column was used. The structures of the IGnT products are shown in Fig. 2.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Schematic representation of IGnT oligosaccharide products. Gal (), GlcNAc (), Man (), and 1,6-linked GlcNAc in I-branch () are denoted. The numbers indicate the relative molar ratio of the products. The vertical lines indicate the positions where endo--galactosidase cleaves.

Fig. 1, A and C, illustrate that oligosaccharides containing N-acetylglucosamine at nonreducing terminals can also serve as acceptors. In particular, the results shown in Fig. 1A demonstrate that the IGnT can add 1,6-linked GlcNAc to a peridistal galactose (Fig. 2, compound a), thus containing dIGnT activity. This finding is consistent with the results reported in our recent study (29). When two galactoses are available in the oligosaccharide containing nonreducing terminal N-acetylglucosamine, all of the singly branched product(s) remained radioactive after endo--galactosidase digestion (Fig. 1E), indicating its structure as GlcNAc13Gal14GlcNAc13(GlcNAc16)Gal14GlcNAc16ManR (Fig. 2, compound c). Peridistal galactose was utilized only when the central galactose was utilized (Fig. 2, compound d). These results indicate that a peridistal galactose is the least favored by the cIGnT cloned from PA-1 cells. When the preferred galactose is missed by the IGnT, the enzyme can still add an I-branch to that galactose after one N-acetylglucosamine (Fig. 2, compounds c and d) or N-acetyllactosamine is added (Fig. 2, compounds f and g).

The above results were obtained using the recombinant IGnT bound to IgG-Sepharose, the activity of which was 3.6 nmol/h/ml. Almost identical results were obtained when a concentrated culture medium from IGnT-transfected cells, of which activity was 40.0 nmol/h/ml, was used.

4Gal-TI Is Responsible for Galactosylation of Linear and Branched Poly-N-acetyllactosamine Synthesis-- In our previous study, we demonstrated that 4Gal-TI is involved in N-acetyllactosamine formation in N-glycans, while 4Gal-TIV forms N-acetyllactosamine in core 2 branched O-glycans (36). To determine which 4Gal-T is involved in galactosylation of I-branched oligosaccharides, Gal14GlcNAc13(GlcNAc16)Gal14GlcPA was enzymatically synthesized using the recombinant IGnT and Gal14GlcNAc13Gal14GlcPA and used as an acceptor. As shown in Fig. 3A, 4Gal-TI transfers a galactose most efficiently to this acceptor, whereas 4Gal-TII, -TIII, and -TV exhibit substrate inhibition at higher concentrations of the acceptor substrate, probably because -galactose in the acceptor competes with UDP-Gal (Fig. 3A). Although 4Gal-TIV does not exhibit substrate inhibition, it is less efficient than 4Gal-TI (Table I).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Dependence of iGnT and 4Gal-T activity on the concentration of linear and branched poly-N-acetyllactosamine acceptors. A and B, Gal14GlcNAc13(GlcNAc16)Gal14GlcPA (A) and GlcNAc13Gal14GlcPA (B) of various concentrations were incubated with 4Gal-TI (), -TII (), -TIII (), -TIV (), and -TV (). C and D, Gal14GlcNAc13(GlcNAc16)Gal14GlcPA (C) and Gal14GlcNAc13Gal14GlcPA (D) of various concentrations were incubated with the iGnT. The same amount of the enzyme, 38.0 nmol/h/ml, determined using 0.5 mM GlcNAcp-nitrophenol (A, B) or 0.5 mM Gal14Glcp-nitrophenol (C, D), was present in these experiments.

View this table: [in this window] [in a new window]   Table I Kinetic properties of 4Gal-Ts Arrows indicate where galactose is added.

It is also evident that 4Gal-TI, among these 4Gal-Ts, acts most efficiently on a linear poly-N-acetyllactosamine acceptor, GlcNAc13Gal14GlcPA (Fig. 3B, Table I). In both experiments, similar results were obtained when the concentration of these enzymes was increased 5-fold or decreased 5-fold. These results indicate that 4Gal-TI is mostly responsible for galactosylation of both branched and linear poly-N-acetyllactosamines.

Addition of N-Acetylglucosamine to I-branched Acceptors-- To determine how iGnT adds N-acetylglucosamine residues to I-branched oligosaccharides, the incorporation of N-acetylglucosamine was compared between different acceptors including linear and branched oligosaccharides. The results shown in Fig. 4 demonstrate that lacto-N-neo-hexaose did not incorporate twice the amount of N-acetylglucosamine compared with lacto-N-neo-tetraose, despite the fact that the former contains two acceptor sites (A and B). Moreover, lacto-N-hexaose containing only one acceptor galactose in the I-branch incorporated N-acetylglucosamine as much as lacto-N-neo-hexaose (Fig. 4C). The results strongly suggest that the addition of N-acetylglucosamine to one side chain precludes the addition of another GlcNAc to the other side chain in branched structures.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   iGnT activity on linear and branched N-acetyllactosaminyl oligosaccharides. Gal14GlcNAc13Gal14Glc (A), Gal14GlcNAc13(Gal14GlcNAc16)Gal14Glc (B), and Gal13GlcNAc13(Gal14GlcNAc16)Gal14Glc (C) were incubated with the iGnT and UDP-[3H]GlcNAc for 10 h. The products were analyzed by Bio-Gel P-4 gel filtration, and numbers shown in the upper right indicate the relative amount of incorporated radioactivity. A peak eluted at fraction 51 was due to a contaminant derived from UDP-[3H]GlcNAc.

To determine the structures of products, the enzymatic reaction products derived from PA-lacto-N-neo-hexaose were subjected to HPLC using an ODS column. Two products, peak A and B, were then separately digested with endo--galactosidase followed by exo--N-acetylglucosaminidase treatment (Fig. 5). The results indicate that peak A, the major product (75% of the total), contains N-acetylglucosamine in the I-branch, while peak B contains N-acetylglucosamine at the linear side chain (Fig. 6). These results indicate that iGnT prefers the Gal14GlcNAc16 branch over Gal14GlcNAc13, which was originally part of linear poly-N-acetyllactosamine. More importantly, no product containing two N-acetylglucosamines at both terminals was formed, supporting the above conclusion that N-acetylglucosamine can be added to only one of the terminals. This result indicates that a branched acceptor probably becomes a competitive inhibitor for iGnT as soon as one N-acetylglucosamine is added.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   HPLC analysis of the products after incubation of PA-lacto-N-neo-hexaose with iGnT. The products obtained after incubation of Gal14GlcNAc13(Gal14GlcNAc16)Gal14GlcPA with the iGnT and UDP-GlcNAc were applied to HPLC (A, E). Peak A (B-D) and peak B (F-H) were purified (B and F) and then sequentially digested with endo--galactosidase (C and G) and exo--N-acetylglucosaminidase (D and H) and analyzed by the same HPLC. Peak S corresponds to the starting material. ODS-80TS column was used in this HPLC.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Structures of the iGnT products from PA-lacto-N-neo-hexaose. The products after incubation of PA-lacto-N-neo-hexaose with the iGnT are shown. The products, obtained after sequential treatment of endo--galactosidase and -N-acetylglucosaminidase, are also shown. Peaks A and B correspond to A and B in Fig. 5, respectively.

Elongation of N-Acetyllactosamine Units on Branched Poly-N-acetyllactosamine-- To determine how N-acetyllactosamine elongation takes place after the IGnT adds a 1,6-linked N-acetylglucosamine, Gal14GlcNAc13(GlcNAc16)Gal14GlcPA was incubated with a mixture of 4Gal-TI and the iGnT, and the products were separated by reverse phase ODS column as shown in Fig. 7.

View larger version (10K): [in this window] [in a new window]   Fig. 7.   HPLC analysis of the products derived from Gal14GlcNAc13(GlcNAc16)Gal14GlcPA after incubation with iGnT and 4Gal-TI. Gal14GlcNAc13(GlcNAc16)Gal14GlcPA was incubated with the iGnT, 4Gal-TI, UDP-GlcNAc, and UDP-Gal (A). Peak E was purified (B) and then sequentially digested with endo--galactosidase (C) and exo--N-acetylglucosaminidase (D). Peak D was eluted at the same position as Gal14GlcNAc16Gal14GlcPA shown in Fig. 5H. Peaks S and F correspond to the starting material and PA-lacto-N-neo-hexaose, respectively.

The minor peak of the products, peak F, eluted shortly after the starting material and corresponds to Gal14GlcNAc13(Gal14GlcNAc16)Gal14GlcPA, which represents the addition of one galactose to the I-branch in the acceptor substrate. This structure was further confirmed by endo--galactosidase digestion and exoglycosidase digestion (data not shown). The major peak (84% of the total products), peak E, was digested by endo--galactosidase, followed by -N-acetylglucosaminidase (Fig. 7, C and D). This digested material was eluted at the position corresponding to Gal14GlcNAc16Gal14GlcPA (Fig. 7D), which was also obtained in the above experiment (Fig. 5H). These results indicate that the products are either those containing one N-acetyllactosamine extension at Gal14GlcNAc13Gal14Glc side chain (compound E in Fig. 8) or lacto-N-neo-hexaose (compound F in Fig. 8).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Proposed biosynthetic steps of I-branched poly-N-acetyllactosamine. 1,6-Linked N-acetylglucosamine is first added to a central galactose by cIGnT (B). This is followed by the addition of 1,3-linked N-acetylglucosamine by iGnT (C) and 1,4-linked galactose by 4Gal-TI (D), adding N-acetyllactosamine to the linear poly-N-acetyllactosamine side chain. This is followed by galactosylation of 1,6-linked N-acetylglucosamine, forming I-branch (E). As a minor biosynthetic pathway, galactosylation of I-branch may take place as soon as 1,6-linked N-acetylglucosamine is added (F). If compound F is formed, 1,3-linked N-acetylglucosamine is preferentially added to I-branch by iGnT, potentially leading to more complex poly-N-acetyllactosamines. E and F correspond to peaks E and F in Fig. 7, respectively. This biosynthetic pathway is based on the results obtained in the present study.

Since the majority of the product was compound E, the extension of N-acetyllactosamine units along the linear poly-N-acetyllactosamine side is favored over galactosylation of the I-branch forming compound F. These results are entirely consistent with the previous findings that I-branches are usually composed of only one N-acetyllactosamine unit in erythrocytes (5) and human PA-1 embryonic carcinoma cells (34) from which the cIGnT was cloned.

Galactosylation of 1,6-GlcNAc Branch Is a Rate-limiting Step-- The above results demonstrate that I-branch formed by cIGnT is not extended further and that N-acetyllactosamine extension takes place preferentially at linear poly-N-acetyllactosamine side chain. To understand how this is achieved, K_m and V_max values of 4Gal-TI and iGnT were obtained for linear and branched oligosaccharide acceptors. As shown in Table I, 4Gal-TI exhibits much lower affinity toward the branched acceptor Gal14GlcNAc13(GlcNAc16)Gal14Glc PA (K_m = 2.49 mM) than its linear counterpart GlcNAc13Gal14GlcPA (K_m = 0.31 mM). In contrast, iGnT exhibits higher affinity toward the branched acceptor Gal14GlcNAc13(GlcNAc16)Gal14GlcPA (K_m = 0.52 mM) than the linear acceptor Gal14GlcNAc13Gal14GlcPA (K_m = 1.09 mM) (Fig. 3, C and D, Table II).

View this table: [in this window] [in a new window]   Table II Kinetic properties of iGnT Arrows indicate where 1,3-linked N-acetylglucosamine is added.

These results indicate that 1,3-linked GlcNAc is added to the Gal14GlcNAc13Gal side chain before galactosylation of 1,6-linked GlcNAc branch (Fig. 8C). This reaction is, most likely, immediately followed by galactosylation of the GlcNAc13Gal14GlcNAc13Gal moiety (Fig. 8D), considering that the additions of GlcNAc and Gal to Gal14GlcNAc13Gal are favored by these two enzymes (Tables I and II). As the last step, galactosylation of I-branch takes place (Fig. 8E). Only as a minor biosynthetic pathway, galactosylation of I-branch precedes other reactions forming compound F in Fig. 8, which may lead to more complex poly-N-acetyllactosamines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study demonstrated that I-branch formation takes place more efficiently at sites closer to nonreducing termini than at internal sites, suggesting that I-branch is formed preferentially at the end of elongating poly-N-acetyllactosamine (Figs. 1 and 2). If this potential site is missed, the IGnT still utilizes the site with a lower efficiency after N-acetylglucosamine (Fig. 2, c and d) or N-acetyllactosamine is added (Fig. 2, f and g). It is noteworthy that the addition of I-branches is not facilitated by having two potential acceptor sites. Rather, the total amount of N-acetylglucosamine transferred is close to that in the compound containing one acceptor site (Fig. 2, b and e-g). The addition of one branch appears to inhibit the addition of another branch on the same side chain. These results strongly suggest that the second branch is usually formed when two acceptor sites are separated by more than one N-acetyllactosamine unit.

These results are consistent with the structures of branched poly-N-acetyllactosamines determined on human adult band 3 and PA-1 cells (5, 34). First, the majority of nonreducing termini contain I-branches. Second, two I-branches are mostly located more than one N-acetyllactosaminyl unit apart. Similar results were obtained on the ceramide pentadecasaccharide from rabbit erythrocytes (10). This spacing may be necessary for other modifications, since I-branch and A-blood group terminal structure are separated by two N-acetyllactosamine units in A^d glycolipid from human erythrocytes (8). The IGnT was cloned from a cDNA library of human PA-1 embryonic carcinoma cells (27). These results suggest that human and rabbit erythroid precursor cells contain IGnT that is the same as or similar to the IGnT cloned from PA-1 cells. Hog small intestine may have similar poly-N-acetyllactosamine structures as seen in erythrocytes and PA-1 cells, since a cIGnT was purified from this tissue (46).

The present study also demonstrated that intrinsic properties of 4Gal-TI and iGnT are critical in forming short I-branches. The addition of a galactose residue to 1,6-linked N-acetylglucosamine branch is a much slower process than the addition of N-acetylglucosamine or galactose to an elongating Gal14GlcNAc13 side chain, as demonstrated by kinetic data (Table I and II). The addition of 1,3-linked N-acetylglucosamine to the Gal14GlcNAc13 side chain is even more efficient when this acceptor has a 1,6-linked GlcNAc branch (Table II). Combined together, these results indicate that galactosylation of a GlcNAc16 branch takes place as a rate-limiting step in the synthesis of branched poly-N-acetyllactosamines. This is most likely the reason why no elongation of I-branch was observed in the present study when the acceptor containing 1,6-linked GlcNAc was incubated with a mixture of iGnT and 4Gal-TI (Figs. 7 and 8).

These results are entirely consistent with the structures determined on poly-N-acetyllactosamines from human erythrocyte band 3 (5), human PA-1 embryonic carcinoma cells (34), and rabbit erythrocytes (10). Only one N-acetyllactosamine unit is present in each I-branch of these glycans.

The results obtained in the present study predict that it takes longer for additional modifications of formed I-branch than the extension of Gal14GlcNAc13 side chain. If this is the case, I-branches in internal positions contain fewer modifications than I-branches at nonreducing termini. Similarly, additional modification at elongating linear poly-N-acetyllactosamines should take place faster than at I-branches even at nonreducing termini. In fact, more 1,2-fucosylated I-branch was found in those branches at nonreducing termini than those in internal positions in human band 3 (5). Moreover, Fuc12Gal14GlcNAc13(Gal14GlcNAc16)Gal1R ^<