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

Minoru UjitaDagger , 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 beta 1,6-N-acetylglucosaminyltransferase. I-branched poly-N-acetyllactosamine can carry bivalent functional oligosaccharides such as sialyl Lewisx, which provide much better carbohydrate ligands than monovalent functional oligosaccharides. In the present study, we first demonstrate that I-branching beta 1,6-N-acetylglucosaminyltransferase cloned from human PA-1 embryonic carcinoma cells transfers beta 1,6-linked GlcNAc preferentially to galactosyl residues of N-acetyllactosamine close to nonreducing terminals. We then demonstrate that among various beta 1,4-galactosyltransferases (beta 4Gal-Ts), beta 4Gal-TI is most efficient in adding a galactose to linear and branched poly-N-acetyllactosamines. When a beta 1,6-GlcNAc branched poly-N-acetyllactosamine was incubated with a mixture of beta 4Gal-TI and i-extension beta 1,3-N-acetylglucosaminyltransferase, the major product was the oligosaccharide with one N-acetyllactosamine extension on the linear Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3 side chain. Only a minor product contained galactosylated I-branch without N-acetyllactosamine extension. This finding was explained by the fact that beta 4Gal-TI adds a galactose poorly to beta 1,6-GlcNAc attached to linear poly-N-acetyllactosamines, while beta 1,3-N-acetylglucosaminyltransferase and beta 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, (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)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-beta -galactosidase (11).

Poly-N-acetyllactosamines are often modified to express differentiation antigens and functional oligosaccharides. Among those oligosaccharides, sialyl Lewisx (Lex)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 Lex 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 Lex 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 Lex, Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAcright-arrowR, and sialyl Lex, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAcright-arrowR (17, 18). In contrast, poly-N-acetyllactosamines in human erythrocytes contain ABO blood group antigens, synthesized from the precursor structure, Fucalpha 1right-arrow2Galbeta 1right-arrow4GlcNAcright-arrowR (5, 19, 20). In addition, poly-N-acetyllactosamines can contain I-branches, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galright-arrowR. During development of human erythrocytes, the linear i antigen represented by Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcright-arrowR 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 Lex poly-N-acetyllactosamines inhibit L-selectin-mediated binding and the rejection of organ transplants with much better efficacy than monovalent sialyl Lex 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 beta 1,3-N-acetylglucosaminyltransferase (iGnT) that forms linear poly-N-acetyllactosamines (26) and beta 1,6-N-acetylglucosaminyltransferase (IGnT) that forms I-branches (27). The IGnT cloned was found to add beta 1,6-N-acetylglucosamine at the central galactose (underlined) of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcright-arrowR, thus termed as centrally acting IGnT (cIGnT) (28, 29). In addition, another IGnT, distally acting IGnT (dIGnT), was found to add beta 1,6-N-acetylglucosamine to peridistal galactose (underlined) of GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcright-arrowR (29-33). No studies have been reported, however, to determine how I-branched poly-N-acetyllactosamine is synthesized by iGnT, IGnT, and beta 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 beta 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 Galbeta 1right-arrow4Glcbeta right-arrowp-nitrophenol (Toronto Research Chemicals) and 40.0 nmol/h/ml using 0.5 mM Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl (see below) as acceptors, respectively.

The medium from mock-transfected COS-1 cells contained less than <FR><NU>1</NU><DE>5</DE></FR> of iGnT activity as described (26) or less than <FR><NU>1</NU><DE>24</DE></FR> of IGnT activity compared with that derived from pcDNAI-A·iGnT- or pcDNAI-A·IGnT-transfected COS-1 cells.

Expression of cDNAs Encoding beta 4Gal-TII, -TIII, -TIV, and -TV-- Isolation of cDNAs encoding beta 4Gal-TII, -TIII, and -TIV was described previously (40, 41). beta 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 beta 4Gal-T preparation (Sigma) was directly used as beta 4Gal-TI (36).

beta 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 beta 4Gal-T samples, the final concentration of beta 4Gal-TI, -TII, -TIII, -TIV, and -TV was adjusted to 38.0 nmol/h/ml as measured using 0.5 mM GlcNAcbeta right-arrowp-nitrophenol (Sigma) as an acceptor.

Oligosaccharides-- (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)nGalbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowO(CH2)7CH3(octyl), where n = 0, 1, and 2, were synthesized, starting from the derivatives of Galbeta 1right-arrow4GlcNAc and Manalpha 1right-arrow6Manbeta right-arrowoctyl, as described previously (36). GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl and GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl were prepared by Escherichia coli beta -galactosidase treatment of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrow octyl, respectively, as described previously (36).

Lacto-N-neo-tetraose (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc) and lacto-N-neo-hexaose (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glc) 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 (Galbeta 1right-arrow3GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glc) 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, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl, and (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl 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 beta 4Gal-TI, -TII, -TIII, -TIV, and -TV-- Assays of beta 4Gal-Ts were performed exactly as described previously (36). As acceptors, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA were used. Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA was synthesized by incubating 5 mM Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA was incubated with beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc, where only one N-acetyllactosamine unit can be added. In addition, beta 4Gal-TI was 4-fold in excess over iGnT, the same ratio as present in HL-60 cells (44).

Analysis of Products by Endo-beta -Galactosidase Digestion-- Products were digested with Escherichia freundii endo-beta -galactosidase for 18 h at 37 °C (11). The digestion condition used allowed the cleavage of galactose linkage, where no beta -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 beta 1,6-linked N-acetylglucosamine to a galactose residue in a central position as seen in Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc(NAc)right-arrowR (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, (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manright-arrowR, incorporated slightly more N-acetylglucosamine than that containing only one acceptor site. When the products obtained from (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manright-arrowR were analyzed by endo-beta -galactosidase digestion, the majority of singly branched products contained beta 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.


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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 GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR (A), Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR (B), GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR (C), and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR (D). The first and second peaks in C and D are the oligosaccharides containing one and two beta 1,6-linked GlcNAc residues, respectively. E and F, HPLC analysis of endo-beta -galactosidase digestion of singly branched products from GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR shown in C (E) and (Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3)2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrowR shown in D (F). The compounds eluted at fractions 28 and 45 in E and F are GlcNAcbeta 1right-arrow3([3H]GlcNAcbeta 1right-arrow 6)Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manalpha 1right-arrow6Manbeta right-arrowoctyl and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3([3H]GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Gal, respectively. The numbers indicate the relative molar ratio of the IGnT products (A-D) or endo-beta -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.


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Fig. 2.   Schematic representation of IGnT oligosaccharide products. Gal (open circle ), GlcNAc (), Man (), and beta 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-beta -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 beta 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-beta -galactosidase digestion (Fig. 1E), indicating its structure as GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Manright-arrowR (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.

beta 4Gal-TI Is Responsible for Galactosylation of Linear and Branched Poly-N-acetyllactosamine Synthesis-- In our previous study, we demonstrated that beta 4Gal-TI is involved in N-acetyllactosamine formation in N-glycans, while beta 4Gal-TIV forms N-acetyllactosamine in core 2 branched O-glycans (36). To determine which beta 4Gal-T is involved in galactosylation of I-branched oligosaccharides, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA was enzymatically synthesized using the recombinant IGnT and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA and used as an acceptor. As shown in Fig. 3A, beta 4Gal-TI transfers a galactose most efficiently to this acceptor, whereas beta 4Gal-TII, -TIII, and -TV exhibit substrate inhibition at higher concentrations of the acceptor substrate, probably because beta -galactose in the acceptor competes with UDP-Gal (Fig. 3A). Although beta 4Gal-TIV does not exhibit substrate inhibition, it is less efficient than beta 4Gal-TI (Table I).


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Fig. 3.   Dependence of iGnT and beta 4Gal-T activity on the concentration of linear and branched poly-N-acetyllactosamine acceptors. A and B, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA (A) and GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA (B) of various concentrations were incubated with beta 4Gal-TI (), -TII (Delta ), -TIII (), -TIV (open circle ), and -TV (black-square). C and D, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA (C) and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA (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 GlcNAcbeta right-arrowp-nitrophenol (A, B) or 0.5 mM Galbeta 1right-arrow4Glcbeta right-arrowp-nitrophenol (C, D), was present in these experiments.

                              
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Table I
Kinetic properties of beta 4Gal-Ts
Arrows indicate where galactose is added.

It is also evident that beta 4Gal-TI, among these beta 4Gal-Ts, acts most efficiently on a linear poly-N-acetyllactosamine acceptor, GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA (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 beta 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.


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Fig. 4.   iGnT activity on linear and branched N-acetyllactosaminyl oligosaccharides. Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc (A), Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glc (B), and Galbeta 1right-arrow3GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glc (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-beta -galactosidase followed by exo-beta -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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 branch over Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3, 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.


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Fig. 5.   HPLC analysis of the products after incubation of PA-lacto-N-neo-hexaose with iGnT. The products obtained after incubation of Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA 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-beta -galactosidase (C and G) and exo-beta -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.


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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-beta -galactosidase and beta -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 beta 1,6-linked N-acetylglucosamine, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA was incubated with a mixture of beta 4Gal-TI and the iGnT, and the products were separated by reverse phase ODS column as shown in Fig. 7.


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Fig. 7.   HPLC analysis of the products derived from Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA after incubation with iGnT and beta 4Gal-TI. Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA was incubated with the iGnT, beta 4Gal-TI, UDP-GlcNAc, and UDP-Gal (A). Peak E was purified (B) and then sequentially digested with endo-beta -galactosidase (C) and exo-beta -N-acetylglucosaminidase (D). Peak D was eluted at the same position as Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Galbeta 1right-arrow4Glcright-arrowPA 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA, which represents the addition of one galactose to the I-branch in the acceptor substrate. This structure was further confirmed by endo-beta -galactosidase digestion and exoglycosidase digestion (data not shown). The major peak (84% of the total products), peak E, was digested by endo-beta -galactosidase, followed by beta -N-acetylglucosaminidase (Fig. 7, C and D). This digested material was eluted at the position corresponding to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6Galbeta 1right-arrow4Glcright-arrowPA (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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glc side chain (compound E in Fig. 8) or lacto-N-neo-hexaose (compound F in Fig. 8).


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Fig. 8.   Proposed biosynthetic steps of I-branched poly-N-acetyllactosamine. beta 1,6-Linked N-acetylglucosamine is first added to a central galactose by cIGnT (B). This is followed by the addition of beta 1,3-linked N-acetylglucosamine by iGnT (C) and beta 1,4-linked galactose by beta 4Gal-TI (D), adding N-acetyllactosamine to the linear poly-N-acetyllactosamine side chain. This is followed by galactosylation of beta 1,6-linked N-acetylglucosamine, forming I-branch (E). As a minor biosynthetic pathway, galactosylation of I-branch may take place as soon as beta 1,6-linked N-acetylglucosamine is added (F). If compound F is formed, beta 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 beta 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, Km and Vmax values of beta 4Gal-TI and iGnT were obtained for linear and branched oligosaccharide acceptors. As shown in Table I, beta 4Gal-TI exhibits much lower affinity toward the branched acceptor Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrow PA (Km = 2.49 mM) than its linear counterpart GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA (Km = 0.31 mM). In contrast, iGnT exhibits higher affinity toward the branched acceptor Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA (Km = 0.52 mM) than the linear acceptor Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4Glcright-arrowPA (Km = 1.09 mM) (Fig. 3, C and D, Table II).

                              
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Table II
Kinetic properties of iGnT
Arrows indicate where beta 1,3-linked N-acetylglucosamine is added.

These results indicate that beta 1,3-linked GlcNAc is added to the Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Gal side chain before galactosylation of beta 1,6-linked GlcNAc branch (Fig. 8C). This reaction is, most likely, immediately followed by galactosylation of the GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Gal moiety (Fig. 8D), considering that the additions of GlcNAc and Gal to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Gal 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 Ad 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 beta 4Gal-TI and iGnT are critical in forming short I-branches. The addition of a galactose residue to beta 1,6-linked N-acetylglucosamine branch is a much slower process than the addition of N-acetylglucosamine or galactose to an elongating Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3 side chain, as demonstrated by kinetic data (Table I and II). The addition of beta 1,3-linked N-acetylglucosamine to the Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3 side chain is even more efficient when this acceptor has a beta 1,6-linked GlcNAc branch (Table II). Combined together, these results indicate that galactosylation of a GlcNAcbeta 1right-arrow6 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 beta 1,6-linked GlcNAc was incubated with a mixture of iGnT and beta 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 Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3 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 alpha 1,2-fucosylated I-branch was found in those branches at nonreducing termini than those in internal positions in human band 3 (5). Moreover, Fucalpha 1right-arrow2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrowR but not Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(Fucalpha 1right-arrow2Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrowR was found in monofucosylated termini of human band 3 (5). Thus, all of these structural data are consistent with the results predicted from the present study, demonstrating that galactosylation of I-branch is a rate-limiting step.

It is noteworthy that the iGnT can act very efficiently on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA (Fig. 3C and Table II) but not on Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6)Galbeta 1right-arrow4Glcright-arrowPA (Fig. 5, peak A). This result suggests that short GlcNAcbeta 1right-arrow6 branch may not be recognized by the iGnT, while the terminal GlcNAc residue in the extended GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6 branch may be recognized by the iGnT, preventing the addition of GlcNAc to the other side chain due to a substrate inhibition. It is thus tempting to speculate that the short GlcNAcbeta 1right-arrow6 branch attached to linear poly-N-acetyllactosamines may be difficult for beta 4Gal-TI to recognize because of its conformation. In fact, NMR studies on the pentadecaglycolipid indicate that the anomeric proton of beta 1,6-GlcNAc linked to the internal galactose is not detected, suggesting that it is conformationally inaccessible (10). These results, combined together, indicate that intricate interaction between these glycosyltransferases and I-branched acceptors play a critical role in the synthesis of I-branched poly-N-acetyllactosamines.

Previously, it was determined that core 2 branch GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow3)GalNAcalpha right-arrowR is galactosylated most efficiently by beta 4Gal-TIV (36). Other galactosyltransferases such as beta 4Gal-TI, -TII, -TIII, and -TV exhibit a substrate inhibition toward the core 2 acceptor, probably because of competition between beta -galactose in the acceptor and the donor substrate, UDP-Gal. Similarly, beta 4Gal-TII, -TIII, and -TV exhibited a substrate inhibition toward the I-branched acceptor (Fig. 3A). beta 4Gal-TI and beta 4Gal-TIV did not show a substrate inhibition toward the I-branched acceptor. However, the kinetic efficiency (Vmax/Km) for beta 4Gal-TIV is less than a half of that for beta 4Gal-TI (Table I), indicating that beta 4Gal-TI is probably dominant in I-branch formation. beta 4Gal-TI is most efficient in galactosylation of a linear poly-N-acetyllactosamine as well (Fig. 3B and Table I). Moreover, beta 4Gal-TI is most efficient in the synthesis of N-glycan poly-N-acetyllactosamine as shown in the previous study (36). Overall, these results indicate that beta 4Gal-TI plays a major role in poly-N-acetyllactosamine extension and I-branch formation in N-glycans. It is noteworthy that beta 4Gal-TI knock-out mice survive during development (47), and those mutant mice express polysialic acid (35) and the HNK-1 carbohydrate epitope (48) in brain glycoproteins (49). These results suggest that a beta 4Gal-T other than beta 4Gal-TI partly compensates for the loss of beta 4Gal-TI in the knock-out mice and is possibly involved in N-acetyllactosamine synthesis under normal conditions as well.

The experiments carried out in the present study were designed to mimic cellular biosynthetic pathways. The biosynthetic oligosaccharide products are also a result of the balance between the amount of glycosyltransferases present and the movement of glycoproteins in the Golgi apparatus during biosynthesis (50). For large scale synthesis of oligosaccharides in vitro, however, enzymatic synthesis can be achieved despite the fact that such a reaction is unlikely in vivo. For example, Renkonen et al. (51) synthesized highly branched poly-N-acetyllactosaminyl oligosaccharides containing four sialyl Lex termini using the cIGnT. In this oligosaccharide, every possible acceptor site was occupied by I-branch and all of the I-branches contained sialyl Lex. It was also reported that galactosylation of core 2 branch GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow3)GalNAc could be achieved using an excess amount of beta 4Gal-TI (36, 52), although beta 4Gal-TI is unlikely to be involved in its galactosylation in vivo. These results strongly suggest that the results obtained by in vitro studies need to be evaluated regarding how these findings reflect the biosynthesis taking place in cells.

The present study reveals the biosynthetic pathway involving the cIGnT that adds beta 1,6-linked GlcNAc to central galactose residues. It has been demonstrated that there is an additional IGnT, dIGnT, which adds beta 1,6-linked GlcNAc at peridistal galactose residues, forming GlcNAcbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)Galright-arrowR at nonreducing termini (30-33). In this situation, galactosylation at I-branch may not be a rate-limiting step, since no substrate inhibition takes place. Recently, we have cloned a novel beta 1,6-N-acetylglucosaminyltransferase that has more dIGnT activity than cIGnT activity (29). Future studies will be of significance to determine the structures of I-branched poly-N-acetyllactosamines synthesized by this newly cloned enzyme.

    ACKNOWLEDGEMENTS

We thank Drs. Kiyohiko Angata for pcDNAI-A, Jiunn-Chern Yeh for useful discussion, Edgar Ong for critical reading of the manuscript, and we thank Susan Greaney and Sanae Ujita for organizing the manuscript.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health, Grants RO1 CA48737 and PO1 CA71932.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.

Dagger Recipient of a Toyobo Biotechnology Fellowship.

To whom correspondence should be addressed: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3144; Fax: 619-646-3193; E-mail: minoru{at}burnham-inst.org.

    ABBREVIATIONS

The abbreviations used are: Lex, Lewisx; iGnT, i-extension beta 1,3-N-acetylglucosaminyltransferase; IGnT, I-branching beta 1,6-N-acetylglucosaminyltransferase; cIGnT and dIGnT, centrally and distally acting IGnT, respectively; HPLC, high performance liquid chromatography; PA, pyridylamino; beta 4Gal-T, beta 1,4-galactosyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Fukuda, M. (1994) in Molecular Glycobiology (Fukuda, M., and Hindsgaul, O., eds), pp. 1-52, Oxford University Press, Oxford
  2. Muramatsu, T., Gachelin, G., Damonneville, M., Delarbre, C., and Jacob, F. (1979) Cell 18, 183-191[Medline] [Order article via Infotrieve]
  3. Yoshima, H., Takasaki, S., and Kobata, A. (1980) J. Biol. Chem. 255, 10793-10804[Abstract/Free Full Text]
  4. Cummings, R. D., and Kornfeld, S. (1984) J. Biol. Chem. 259, 6253-6260[Abstract/Free Full Text]
  5. Fukuda, M., Dell, A., Oates, J. E., and Fukuda, M. N. (1984) J. Biol. Chem. 259, 8260-8273[Abstract/Free Full Text]
  6. Fukuda, M., Carlsson, S. R., Klock, J. C., and Dell, A. (1986) J. Biol. Chem. 261, 12796-12806[Abstract/Free Full Text]
  7. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J. Biol. Chem. 271, 18732-18742[Abstract/Free Full Text]
  8. Fukuda, M. N., and Hakomori, S. (1982) J. Biol. Chem. 257, 446-455[Abstract/Free Full Text]
  9. Zdebska, E., Krauze, R., and Koscielak, J. (1983) Carbohydr. Res. 120, 113-130[CrossRef][Medline] [Order article via Infotrieve]
  10. Dabrowski, U., Hanfland, P., Egge, H., Kuhn, S., and Dabrowski, J. (1984) J. Biol. Chem. 259, 7648-7651[Abstract/Free Full Text]
  11. Fukuda, M. N. (1981) J. Biol. Chem. 256, 3900-3905[Abstract/Free Full Text]
  12. Lowe, B. J. (1994) in Molecular Glycobiology (Fukuda, M., and Hindsgaul, O., eds), pp. 163-205, Oxford University Press, Oxford
  13. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270, 11025-11028[Abstract/Free Full Text]
  14. Rosen, S. D., and Bertozzi, C. R. (1996) Curr. Biol. 6, 261-264[Medline] [Order article via Infotrieve]
  15. Imai, Y., Lasky, L. A., and Rosen, S. D. (1993) Nature 361, 555-557[CrossRef][Medline] [Order article via Infotrieve]
  16. Tsuboi, S., Isogai, Y., Hada, N., King, J. K., Hindsgaul, O., and Fukuda, M. (1996) J. Biol. Chem. 271, 27213-27216[Abstract/Free Full Text]
  17. Fukuda, M., Spooncer, E., Oates, J. E., Dell, A., and Klock, J. C. (1984) J. Biol. Chem. 259, 10925-10935[Abstract/Free Full Text]
  18. Mizoguchi, A., Takasaki, S., Maeda, S., and Kobata, A. (1984) J. Biol. Chem. 259, 11949-11957[Abstract/Free Full Text]
  19. Krusius, T., Finne, J., and Rauvala, H. (1978) Eur. J. Biochem. 92, 289-300[Abstract]
  20. Järnefelt, J., Rush, J., Li, Y. T., and Laine, R. A. (1978) J. Biol. Chem. 253, 8006-8009[Abstract]
  21. Fukuda, M., Fukuda, M. N., and Hakomori, S. (1979) J. Biol. Chem. 254, 3700-3703[Abstract]
  22. Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D., and Evans, M. J. (1981) Nature 292, 156-158[Medline] [Order article via Infotrieve]
  23. Turunen, J. P., Majuri, M.-L., Seppo, A., Tiisala, S., Paavonen, T., Miyasaka, M., Lemström, K., Penttilä, L., Renkonen, O., and Renkonen, R. (1995) J. Exp. Med. 182, 1133-1142[Abstract]
  24. Toppila, S., Lauronen, J., Mattila, P., Turunen, J. P., Penttila, L., Paavonen, T., Renkonen, O., and Renkonen, R. (1997) Eur. J. Immunol. 27, 1360-1365[Medline] [Order article via Infotrieve]
  25. Romans, D. G., Tilley, C. A., and Dorrington, K. J. (1980) J. Immunol. 124, 2807-2811[Abstract/Free Full Text]
  26. Sasaki, K., Kurata-Miura, K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T., and Fukuda, M. (1997) Proc. Nati. Acad. Sci. U. S. A. 94, 14294-14299[Abstract/Free Full Text]
  27. Bierhuizen, M. F. A., Mattei, M.-G., and Fukuda, M. (1993) Genes Dev. 7, 468-478[Abstract]
  28. Mattila, P., Salminen, H., Hirvas, L., Niittymäki, J., Salo, H., Niemelä, R., Fukuda, M., Renkonen, O., and Renkonen, R. (1998) J. Biol. Chem. 273, 27633-27639[Abstract/Free Full Text]
  29. Yeh, J.-C., Ong, E., and Fukuda, M. (1999) J. Biol. Chem. 274, 3215-3221[Abstract/Free Full Text]
  30. Piller, F., Cartron, J. P., Maranduba, A., Veyrieres, A., Leroy, Y., and Fournet, B. (1984) J. Biol. Chem. 259, 13385-13390[Abstract/Free Full Text]
  31. Gu, J., Nishikawa, A., Fujii, S., Gasa, S., and Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999[Abstract/Free Full Text]
  32. Ropp, P. A., Little, M. R., and Cheng, P. W. (1991) J. Biol. Chem. 266, 23863-23871[Abstract/Free Full Text]
  33. Schachter, H., and Brockhausen, I. (1992) in Glycoconjugates: Composition, Structure, and Function (Allen, H. J., and Kisailus, E. C., eds), 1st Ed., pp. 263-332, Marcel Dekker, Inc., New York
  34. Fukuda, M. N., Dell, A., Oates, J. E., and Fukuda, M. (1985) J. Biol. Chem. 260, 6623-6631[Abstract/Free Full Text]
  35. Angata, K., Suzuki, M., and Fukuda, M. (1998) J. Biol. Chem. 273, 28524-28532[Abstract/Free Full Text]
  36. Ujita, M., McAuliffe, J., Schwientek, T., Almeida, R., Hindsgaul, O., Clausen, H., and Fukuda, M. (1998) J. Biol. Chem. 273, 34843-34849[Abstract/Free Full Text]
  37. Nakayama, J., and Fukuda, M. (1996) J. Biol. Chem. 271, 1829-1832[Abstract/Free Full Text]
  38. Bakker, H., Van Tetering, A., Agterberg, M., Smit, A. B., Van den Eijnden, D. H., and Van Die, I. (1997) J. Biol. Chem. 272, 18580-18585[Abstract/Free Full Text]
  39. Cho, S. K., and Cummings, R. D. (1997) J. Biol. Chem. 272, 13622-13628[Abstract/Free Full Text]
  40. Almeida, R., Amado, M., David, L., Levery, S. B., Holmes, E. H., Merkx, G., van Kessel, A. G., Rygaard, E., Hassan, H., Bennett, E., and Clausen, H. (1997) J. Biol. Chem. 272, 31979-31991[Abstract/Free Full Text]
  41. Schwientek, T., Almeida, R., Levery, S., Holmes, E., Bennett, E., and Clausen, H. (1998) J. Biol. Chem. 273, 29331-29340[Abstract/Free Full Text]
  42. Sato, T., Furukawa, K., Bakker, H., Van den Eijnden, D. H., and Van Die, I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 472-477[Abstract/Free Full Text]
  43. Hase, S., Ibuki, T., and Ikenaka, T. (1984) J. Biochem. (Tokyo) 95, 197-203[Abstract]
  44. Lee, N., Wang, W.-C., and Fukuda, M. (1990) J. Biol. Chem. 265, 20476-20487[Abstract/Free Full Text]
  45. Fukuda, M. N. (1992) in CRC Handbook of Endoglycosidases and Glycoamidases (Takahashi, N., and Muramatsu, T., eds), pp. 55-103, CRC Press, Inc., Boca Raton, FL
  46. Sakamoto, Y., Taguchi, T., Tano, Y., Ogawa, T., Leppanen, A., Kinnunen, M., Aitio, O., Parmanne, P., Renkonen, O., and Taniguchi, N. (1998) J. Biol. Chem. 273, 27625-27632[Abstract/Free Full Text]
  47. Asano, M., Furukawa, K., Kido, M., Matsumoto, S., Umesaki, Y., Kochibe, N., and Iwakura, Y. (1997) EMBO J. 16, 1850-1857[Abstract/Free Full Text]
  48. Ong, E., Yeh, J. C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 273, 5190-5195[Abstract/Free Full Text]
  49. Kido, M., Asano, M., Iwakura, Y., Ichinose, M., Miki, K., and Furukawa, K. (1998) Biochem. Biophys. Res. Commun. 245, 860-864[CrossRef][Medline] [Order article via Infotrieve]
  50. Wang, W. C., Lee, N., Aoki, D., Fukuda, M. N., and Fukuda, M. (1991) J. Biol. Chem. 266, 23185-23190[Abstract/Free Full Text]
  51. Renkonen, O., Toppila, S., Penttila, L., Salminen, H., Helin, J., Maaheimo, H., Costello, C. E., Turunen, J. P., and Renkonen, R. (1997) Glycobiology 7, 453-461[Abstract]
  52. Srivastava, G., and Hindsgaul, O. (1992) Carbohydr. Res. 224, 83-93[CrossRef][Medline] [Order article via Infotrieve]


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