Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity*

Junya MitomaDagger , Bronislawa Petryniak§, Nobuyoshi HiraokaDagger , Jiunn-Chern YehDagger ||, John B. Lowe§**, and Minoru FukudaDagger DaggerDagger

From the Dagger  Glycobiology Program, Cancer Research Center, the Burnham Institute, La Jolla, California 92037 and the § Howard Hughes Medical Institute, Department of Pathology, and the Life Sciences Institute, University of Michigan Medical School, Ann Arbor, Michigan 48104

Received for publication, December 16, 2002, and in revised form, January 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core O-glycans, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3(sulforight-arrow6))GlcNAcbeta 1right-arrow 3Galbeta 1right-arrow3GalNAcalpha 1right-arrowSer/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta 1,3-N-acetylglucosaminyltransferase-3 (beta 3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta 3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta 3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta 1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta 3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mucin-type O-glycans are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine residues in a polypeptide (1). These attached O-glycans can be classified into several different groups according to the core structure (2). In many cells, a structure called core 1, Galbeta 1right-arrow3GalNAc, is the major constituent of O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides, Galbeta 1right-arrow3(GlcNAcbeta 1right-arrow6)GalNAc, when core 2 beta 1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT)1 is present (3, 4). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lewis x in mucin-type glycoproteins of blood cells can be found in core 2 branched oligosaccharides such as NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAcbeta 1right-arrow6(NeuNAcalpha 2right-arrow 3Galbeta 1right-arrow3)GalNAcalpha 1right-arrowSer/Thr (see Fig. 1) (5, 6).

When leukocytes are recruited to inflammatory sites, E- and P-selectins expressed in activated endothelial cells recognize alpha 2right-arrow3-sialylated, alpha 1right-arrow3-fucosylated O-glycans, allowing leukocytes to eventually extravasate (7-9). It has also been demonstrated that L-selectin in neutrophils mediates neutrophil rolling on neutrophils adherent to activated endothelial cells (10, 11). PSGL-1 (P-selectin glycoprotein ligand-1) contributes to this process, although the nature of the glycan moieties that decorate this and other potential L-selectin ligands in neutrophils has not been well characterized. On the other hand, L-selectin in lymphocytes recognizes 6-sulfosialyl Lewis x in core 2 branched O-glycans, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3(sulforight-arrow6))GlcNAcbeta 1right-arrow 6(Galbeta 1right-arrow3)GalNAcalpha 1right-arrowSer/Thr, which are expressed in high endothelial venules (HEV) in lymph nodes (12-14). Such recognition leads to lymphocyte adhesion to HEV, allowing lymphocyte movement from the vascular to the lymphatic system. The formation of 6-sulfosialyl Lewis x depends on 6-sulfotransferase, designated L-selectin ligand sulfotransferase (LSST) or high endothelial cell N-acetylglucosamine 6-O-sulfotransferase (12, 15, 16).

The roles of sialyl Lewis x in core 2 branched O-glycans have been demonstrated by analyzing mutant mice with deficient Core2GlcNAcT-I, obtained through homologous recombination (17). Leukocytes derived from null mutant mice display significantly reduced adhesion to L-, P-, and E-selectins, demonstrating that ligands for these selectins are mainly carried by core 2 branched O-glycans. By contrast, in these mice, lymphocyte adhesion to HEV in lymph nodes is only marginally impaired, and MECA-79 antibody staining that decorates HEV is not reduced (17). Recent studies reveal that L-selectin ligand activity remaining after abrogation of Core2GlcNAcT-I is due to the activity of 6-sulfosialyl Lewis x in extended core O-glycans, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4[Fucalpha 1right-arrow3(sulforight-arrow6)]GlcNAcbeta 1right-arrow 3Galbeta 1right-arrow3GalNAcalpha 1right-arrowSer/Thr (18). Moreover, a minimum MECA-79 epitope was found to be a 6-sulfo structure in the extended core 1 O-glycan, and MECA-79 antibody binds efficiently to 6-sulfosialyl Lewis x-containing extended core 1 O-glycans (18). These findings are consistent with previous findings that MECA-79 antibody inhibits lymphocyte adhesion to HEV without removal of sialic acid (19) or fucose and that MECA-79 staining remains after expression of fucose is abrogated by inactivation of fucosyltransferase VII (FucT-VII) (20).

Extended core 1 structure is synthesized by core 1 beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAcT), which adds beta 1,3-linked N-acetylglucosamine to Galbeta 1right-arrow3GalNAcalpha 1right-arrowR (Fig. 1). A cDNA encoding beta 3GlcNAcT was first cloned by expression cloning, and the encoded protein was designated both i-antigen forming beta 1,3-N-acetylglucosaminyltransferase (21) and beta 3GlcNAcT-1 (22, 23). Expression of beta 3GlcNAcT-1 does not, however, result in the synthesis of extended core 1 structure (18). By screening expressed sequence tag data bases using the cDNA encoding the now designated beta 1,3-galactosyltransferase-6 as a probe, cDNA encoding core 1-beta 3GlcNAcT was identified (18). This cloning was possible because it was reported that beta 1,3-galactosyltransferase and beta 3GlcNAcT are highly homologous proteins (24). In parallel, at least five additional beta 3GlcNAcTs have been molecularly identified based on similarity to the previously cloned beta 1,3-galactosyltransferase or to beta 3GlcNAcT-2 (22, 25-28). These results indicate that core 1-beta 3GlcNAcT belongs to the beta 3GlcNAcT gene family, and it is thus designated beta 3GlcNAcT-3.


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Fig. 1.   Biosynthetic schemes of core 1 and core 2 O-glycans. Core 2 branch can be added to core 1 by Core2GlcNAcT-I, and this branch is galactosylated by beta 1,4-galactosyltransferase IV (beta 4GalT-IV) (54). This is followed by sialylation and fucosylation by alpha 2,3-sialyltransferase and FucT-VII, forming sialyl Lewis x in core 2 branch (left). Core 1 is also modified by core 1 beta 3GlcNAcT, which is also known as beta 3GlcNAcT-3, forming extended core 1. Extended core 1 is then galactosylated (most likely by beta 1,4-galactosyltransferase I), sialylated, and fucosylated, forming sialyl Lewis x in extended core 1 structure (middle). Core 1 can be sialylated by beta -galactoside alpha 2,3-sialyltransferase I (ST3Gal I) (50) and then by N-acetylgalactosamine alpha 2,6-sialyltransferase (ST6GalNAc), forming disialosyl core 1 O-glycan (right). This biosynthetic pathway precludes either core 1 extension or core 2 branching.

beta 3GlcNAcT-3 transcripts are highly expressed in the small intestine, colon, and placenta and are moderately expressed in various tissues, including the liver, kidney, pancreas, and prostate (18). It is not known whether blood cells express beta 3GlcNAcT-3.

In this study, we address the function and expression pattern of beta 3GlcNAcT-3. First, we show that beta 3GlcNAcT-3 was the only enzyme that significantly formed extended core 1 among highly related beta 3GlcNAcTs. We then show that beta 3GlcNAcT-3 transcripts were present in both human neutrophils and lymphocytes, but that these cells lacked LSST. Transfection studies using FucT-VII and beta 3GlcNAcT-3 or Core2GlcNAcT-I showed that extended core 1 could be synthesized in Chinese hamster ovary (CHO) cells and that extended core 1 structure was fucosylated by FucT-VII more efficiently than core 2 branches. Finally, we show that sialyl Lewis x synthesized in extended core 1 served as an L-selectin ligand, although it is apparently less potent than sialyl Lewis x in core 2 branches.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of cDNA Encoding beta 3GlcNAcTs-- cDNA cloning of beta 3GlcNAcT-3 was described previously (18). cDNAs encoding human beta 3GlcNAcT-1 (21), beta 3GlcNAcT-2 (22), beta 3GlcNAcT-4 (22), beta 3GlcNAcT-5 (25, 26), beta 3GlcNAcT-6 (27), and beta 3GlcNAcT-7 (28) were cloned by reverse transcription (RT)-PCR using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) (29). RT-PCR primers were as follows: beta 3GlcNAcT-1, 5'-CGAGAGCCATGCAGATGTCCTAC-3' (5'-primer) and 5'-AAGGGCTCAGCAGCGTCGGGGAG-3' (3'-primer); beta 3GlcNAcT-2, 5'-GACAAGATATGAGAAATGAGTGTTGG-3' (5'-primer) and 5'-TTTTAGCATTTTAAATGAGCACTCTGC-3' (3'-primer); beta 3GlcNAcT-4, 5'-AGCACGGAGACAGTCTCCAGCTG-3' (5'-primer) and 5'-AGGCATCAATTTCGCATCACGATAG-3' (3'-primer); beta 3GlcNAcT-5, 5'-AGACTTGAGTGGATATGAGAATGTTG-3' (5'-primer) and 5'-AAGTACTATTAGATAAACGCAGCCCT-3' (3'-primer); beta 3GlcNAcT-6, 5'-ACGCTCAAGCACCTGCACTTGCT-3' (5'-primer) and 5'-ACTGGCCTCAGGAGACCCGGTG-3' (3'-primer); and beta 3GlcNAcT-7, 5'-GCCGCCATGTCGCTGTGGAAGA-3' (5'-primer) and 5'GGGTCAGAGCACCTGGAGCTTG-3' (3'-primer). As templates, we used a human fetal brain cDNA library in pcDNA1 (30) for beta 3GlcNAcT-1, human esophagus cDNA in a human digestive system multiple tissue cDNA panel (BD Biosciences) for beta 3GlcNAcT-2, an SK-N-MC cell cDNA library (see below) for beta 3GlcNAcT-4, human pituitary gland Marathon-Ready cDNA (BD Biosciences) for beta 3GlcNAc-5, human stomach cDNA in a human digestive system multiple tissue cDNA panel (BD Biosciences) for beta 3GlcNAcT-6, and human colon Marathon-Ready cDNA (BD Biosciences) for beta 3GlcNAcT-7. A cDNA library of SK-N-MC neuroblastoma cells was prepared by isolation of total RNA with TRIzol (Invitrogen), followed by first-strand cDNA synthesis using SuperScript II RNase H- reverse transcriptase (Invitrogen).

PCR products were first inserted into pCR2.1-TOPO (Invitrogen). In the case of beta 3GlcNAcT-1, -2, -4, -6, and -7, cDNAs in pCR2.1-TOPO were digested with EcoRI and inserted into the dephosphorylated EcoRI site of pcDNA3.1(N). pcDNA3.1(N) is a vector created by the digestion of pcDNA3.1/Zeo with SphI and BspLU11I (Roche Molecular Biochemicals), followed by filling in and self-ligation to remove the Zeocin resistance gene and f1 origin. For beta 3GlcNAcT-5, cDNA in pCR2.1-TOPO was digested with EcoRI and ScaI and subcloned into the EcoRI-EcoRV sites of pcDNA3.1(N). Similarly, beta 3GlcNAcT-3 was cloned into the HindIII-XhoI sites of pcDNA1.1, resulting in pcDNA1.1-beta 3GlcNAcT-3. cDNA encoding LSST was cloned as described previously (12, 15).

Expression of i Antigen in HeLa Cells by Different beta 3GlcNAcTs-- To determine whether all of beta 3GlcNAcTs direct the synthesis of poly-N-acetyllactosamine synthesis, HeLa cells were transiently transfected with one of the pcDNA3.1(N)-beta 3GlcNAcTs or pcDNA1.1-beta 3GlcNAcT-3. Thirty-six hours after transfection, cells were dissociated into monodispersed cells using an enzyme-free cell dissociation solution (Hanks' balanced saline solution-based) purchased from Cell and Molecular Technologies. Monodispersed cells were incubated with human anti-i serum (Dench) (31), followed by affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgM antibodies (Pierce). The stained cells were subjected to FACS analysis using a FACScan (BD Biosciences) as described previously (32).

HeLa cells were chosen as recipient cells for transfection because the molecular mass of lysosome-associated membrane protein-1, a major carrier of poly-N-acetyllactosamines (33), is the smallest among Namalwa, HL-60, CHO, HepG2, and HeLa cells. The results indicated that HeLa cells express minimum amounts of beta 3GlcNAcTs because poly-N-acetyllactosaminylated lysosome-associated membrane protein-1 displays a higher molecular mass than lysosome-associated membrane protein-1 containing minimum amounts of poly-N-acetyllactosamine (33).

Expression of MECA-79 in Lec2 Cells by LSST and beta 3GlcNAcT-- To determine which beta 3GlcNAcT directs expression of MECA-79 antigen, Lec2 cells were transiently transfected with pcDNA1-LSST and one of the pcDNA3.1(N)-beta 3GlcNAcTs or pcDNA1.1-beta 3GlcNAcT-3. Thirty-six hours after transfection, cells were dissociated into monodispersed cells using the cell dissociation solution as described above. Monodispersed cells were incubated with MECA-79 antibody (BD Biosciences) (19), followed by affinity-purified FITC-conjugated goat anti-rat IgM antibody (ICN Biochemicals). The stained cells were subjected to FACS analysis as described above. CHO mutant Lec2 cells lack a functional Golgi CMP-sialic acid transporter; and therefore, sialylation is absent in Lec2 cells (34). The absence of sialylation facilitates core 1 extension because core 1 extension and sialylation compete with each other for the same acceptor, Galbeta 1right-arrow3GalNAcalpha 1right-arrowR.

Core 1 Extension in PSGL-1 by beta 3GlcNAcTs-- To determine which beta 3GlcNAcT can add beta 1,3-N-acetylglucosamine to core 1, Galbeta 1right-arrow3GalNAcalpha 1right-arrowR, Lec2 cells were transiently transfected with pZeoSV-PSGL-1 (kindly provided by Dr. Richard Cummings) and vectors encoding beta 3GlcNAcT using LipofectAMINE as described previously (12). The ratio of pZeoSV-PSGL-1 and beta 3GlcNAcT cDNA was 1:5 (w/w) to achieve efficient modification of PSGL-1 by beta 3GlcNAcT. Forty-eight hours after transfection, cells were harvested in phosphate-buffered saline with a cell scraper. The cells were subjected to three cycles of freezing and thawing to disrupt the plasma membrane. The membrane fraction was collected by centrifugation at 12,000 × g for 10 min. The resultant membrane fraction was first resuspended in 10 mM Tris-HCl and 1 mM EDTA (pH 8.0), and then 10% Triton X-100 was added to a final concentration of 1%. After gentle rocking at 4 °C for 15 min, the Triton X-100-soluble membrane fraction, containing PSGL-1, was obtained by centrifugation at 12,000 × g for 10 min.

The membrane fraction was then lysed in sample buffer and subjected to SDS-PAGE. After blotting onto a polyvinylidene difluoride membrane filter, the blot was reacted with anti-PSGL-1 antibody (KPL-1, BD Biosciences), followed by secondary antibody; and immunoreactive PSGL-1 was visualized using enhanced Luminol reagent (PerkinElmer Life Sciences).

RT-PCR of beta 3GlcNAcT-3, Core2GlcNAcT-I, LSST, FucT-VII, and PSGL-1 on RNA Isolated from Neutrophils and Lymphocytes-- Human neutrophils and lymphocytes were isolated from the peripheral blood of a volunteer as described previously (35). Briefly, blood was drawn in a syringe containing heparin, and erythrocytes were sedimented by dextran/saline solution, obtaining leukocyte-rich plasma in the upper layer. Ficoll-Paque Plus solution (Amersham Biosciences) was added beneath this layer and centrifuged. Mononuclear cells enriched with lymphocytes were isolated from the saline/Ficoll interface. The pellet from the above centrifugation was enriched with neutrophils, which were isolated after hypotonic lysis of erythrocytes. Total RNA was isolated from neutrophils and lymphocytes using TRIzol. RT-PCR of beta 3GlcNAcT-3 (18), Core2GlcNAcT-I (4), LSST (12, 15), FucT-VII (20), and PSGL-1 (36, 37) was carried out as follows. Using isolated RNA from respective cells as a template, first-strand cDNA was synthesized using RNase H- reverse transcriptase. PCR was carried out using AmpliTaq DNA polymerase (Applied Biosystems) and the following PCR primers: beta 3GlcNAcT-3, 5'-TTCTTCAACCTCACGCTCAAGCAG-3' (5'-primer) and 5'-AGCATCTCATAAGGTAGGAAGCGG-3' (3'-primer); Core2GlcNAcT-I, 5'-TGAAATGCTTGACAGGCTGCTGAG-3' (5'-primer) and 5'-GGTGTTTCGAGTGGAGGAAGCATT-3' (3'-primer); LSST, 5'-GGGAGATCTCATGATTGACAGTCG-3' (5'-primer) and 5'-TAGTGGATTTGCTCAGGGACAGTC-3' (3'-primer); FucT-VII, 5'-GTCAGCAACTTCCAGGAGCGGCA-3' (5'-primer) and 5'-TCAAGGTCCTCATAGACTTGGCTG-3' (3'-primer); PSGL-1, 5'-CCCTGTCCACAGAACCCAGTGC-3' (5'-primer) and 5'-GAAGCTGTGCAGGGTGAGGTCAT-3' (3'-primer); and glyceraldehyde-3-phosphate dehydrogenase, 5'-CCTGGCCAAGGTCATCCATGACA-3' (5'-primer) and 5'-ATGAGGTCCACCACCCTGTTGCT-3' (3'-primer) or 5'-GACCCCTTCATTGACCTCAACTACA-3' (5'-primer) and 5'-ACATGGCCTCCAAGGAGTAAGA-3' (3'-primer). The last primer pair was included in the human digestive system multiple tissue cDNA panel. PCR products were separated by electrophoresis on 1% agarose gels. To confirm that the PCR products were derived from respective transcripts, RT-PCR products were digested with restriction enzymes and subjected to electrophoresis on 2% agarose.

Stable Expression of beta 3GlcNAcT-3 and Core2GlcNAcT-I in CHO Cells-- CHO cells were first transfected with pZeoSV-PSGL-1 and selected in the presence of 100 µg/ml Zeocin (Invitrogen). CHO colonies stably expressing PSGL-1 were selected after staining with anti-PSGL-1 antibody KPL-1, establishing CHO-PSGL-1 cells. CHO-PSGL-1 cells were stably cotransfected with pcDNA1.1-beta 3GlcNAcT-3 (core 1 beta 3GlcNAcT) and pSV-hygromycin and selected in 100 µg/ml Zeocin and 400 µg/ml hygromycin B (Calbiochem). Expression of beta 3GlcNAcT-3 was shown by the expression of larger forms of PSGL-1 that contains extended core 1 O-glycans (see also "Results"). The resultant CHO-PSGL-1/C1 cells were then cotransfected with pCDM8-FucT-VII and pcDNA3 and cultured in the presence of 400 µg/ml Geneticin (Invitrogen). CHO-PSGL-1/C1 cells stably expressing FucT-VII were selected after staining with anti-sialyl Lewis x antibody (CSELX-1), resulting in CHO-PSGL-1/F7/C1 cells.

In parallel, CHO-PSGL-1 cells were transfected with pcDNA1.1-Core2GlcNAcT-I together with pcDNA3 and cultured in the presence of 100 µg/ml Zeocin and 400 µg/ml Geneticin. Cells expressing Core2GlcNAcT-I were chosen for expressing larger forms of PSGL-1 that contains core 2 branched O-glycans. Expression of core 2 O-glycans was confirmed after transient transfection of CD43 (also called leukosialin) and staining with T305 antibody, resulting in CHO-PSGL-1/C2 cells. As reported previously, T305 reacts with core 2 branched O-glycans attached to CD43 (4, 38). CHO-PSGL-1/C2 cells were stably transfected with pCDM8-FucT-VII and pSV-hygromycin and cultured in the presence of Zeocin, hygromycin B, and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7/C2 cells.

As a control, CHO-PSGL-1 cells were stably transfected with pCDM8-FucT-VII and pcDNA3 and cultured in the presence of Zeocin and Geneticin. Cells expressing FucT-VII were selected after staining with CSELX-1 antibody, resulting in CHO-PSGL-1/F7 cells.

Isolation of PSGL-1/IgG Chimeric Protein from Transfected CHO Cells-- cDNA encoding PSGL-1/IgG chimeric protein was constructed using pZeoSV-PSGL-1 and pcDNA3.1-IgG essentially as described previously (12), resulting in pcDNA3.1-PSGL-1/IgG. CHO-PSGL-1/F7/C1, CHO-PSGL-1/F7/C2, and CHO-PSGL-1/F7 cells were transfected with pcDNA3.1-PSGL-1/IgG as described previously (12). Twenty-four hours after transfection, cells were cultured in glucose-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum, 100 µM sodium pyruvate, 2 mM glutamine, 25 mM HEPES, and 20 µCi/ml [3H]glucosamine (PerkinElmer Life Sciences). PSGL-1/IgG chimeric protein was isolated using ImmunoPure immobilized protein A (Pierce) from the medium obtained after 48 h of additional culture as described previously (12, 18).

Structural Analysis of Oligosaccharides Attached to PSGL-1/IgG-- PSGL-1/IgG isolated as described above was subjected to alkaline borohydride treatment to release O-glycans as described (39). Released O-glycans were recovered after Sephadex G-50 gel filtration (12, 18). The isolated O-glycans were then applied to a column (1.0 × 150 cm) of Bio-Gel P-4 (200-400 mesh) equilibrated with 0.1 M NH4HCO3. Those oligosaccharide fractions, separated by Bio-Gel P-4 gel filtration, were then desialylated by treatment in 2 M acetic acid at 80 °C for 4 h (40). After neutralization of the sample with ammonium hydroxide and Sephadex G-25 gel filtration in 7% 1-propanol to remove salt, the sample was again subjected to Bio-Gel P-4 gel filtration under the same conditions. Neutral oligosaccharides separated by Bio-Gel P-4 gel filtration were subjected to HPLC using an amino-bonded column (Asahipak NH2 P50-4E, 4.6 × 250 mm) that was equilibrated with solution A (75% acetonitrile, 20% H2O, and 5% 0.25 M KH2PO4/H2O) and that was attached to a Gilson 306 HPLC apparatus. The sample was eluted for 40 min with solution A and then eluted with a linear gradient from solution A to a 60:40 mixture of solution A and solution B (50% acetonitrile, 45% H2O, and 5% 0.25 M KH2PO4/H2O) over the next 50 min. The sample was finally eluted with 100% of solution B over the last 30 min.

Standard O-glycans were obtained from CD34/IgG glycans synthesized in the presence of beta 3GlcNAcT-3 and Core2GlcNAcT-I as described previously (12, 18). Oligosaccharides were digested with Streptomyces sp. alpha 1,3/4-fucosidase (Panvera/Takara, Madison, WI) and jack bean beta -galactosidase (Sigma) as described previously (39, 41, 42). The digest was desalted by Sephadex G-25 gel filtration in 7% 1-propanol before HPLC analysis.

Measurement of L-selectin-mediated Rolling in CHO Cells Expressing Sialyl Lewis x in Extended Core 1 or Core 2 Branched O-Glycans-- CHO cells stably expressing PSGL-1, FucT-VII, and beta 3GlcNAcT-3 or Core2GlcNAcT-I were established as described above. These cells maintained similar amounts of sialyl Lewis x and PSGL-1 as assessed by FACS analysis using anti-PSGL-1 antibody KPL-1 and anti-sialyl Lewis x antibody CSELX-1 (see also "Structural Analysis of PSGL-1 O-glycans Synthesized in the Presence of beta 3GlcNAcT-3 or Core2GlcNAcT-I"). These stably transfected cells seeded on dishes were used as the bottom plate of a parallel flow chamber as described previously (18).

Neutrophils or lymphocytes were initially introduced into the flow chamber at a wall sheer stress of 5 dynes/cm2 for 15 s, followed by the termination of flow to allow the cells to adhere under static conditions (17). Flow rate was then initiated at different shear forces. Image analysis was performed and analyzed as described previously (17).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta 3GlcNAcT-3 Acts on Core 1 O-Glycans, Galbeta 1right-arrow3GalNAcalpha 1right-arrowSer/Thr-- Previously, we showed that, of beta 3GlcNAcT-1, -2, -3, and -4, beta 3GlcNAcT-3 can act on Galbeta 1right-arrow3GalNAcalpha 1right-arrowR, forming GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcalpha 1right-arrowR (18). This extended core 1 structure can be converted to Galbeta 1right-arrow4(sulforight-arrow6)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcalpha 1right-arrowR in the presence of the 6-sulfotransferase LSST and beta 1,4-galactosyltransferase. Because Galbeta 1right-arrow4(sulforight-arrow6)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcalpha 1right-arrowR is a minimum epitope for MECA-79 antigen, the formation of extended core 1 can be detected by immunostaining with MECA-79 antibody when LSST is also expressed.

After the initial report on core 1 beta 3GlcNAcT (or beta 3GlcNAcT-3) (18), three additional beta 3GlcNAcTs highly related to beta 3GlcNAcT-3 were molecularly cloned: beta 3GlcNAcT-5 (25, 26), beta 3GlcNAcT-6 (27), and beta 3GlcNAcT-7 (28). We thus determined whether extended core 1 can be formed by these more recently identified members of the beta 3GlcNAcT gene family.

First, we tested whether all of the cloned beta 3GlcNAcTs are active in synthesizing i antigen, Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcright-arrowR (21, 31). The synthesis of i antigen is dependent on beta 3GlcNAcT, which adds beta 1,3-linked GlcNAc to N- acetyllactosamine. HeLa cells were thus transiently transfected with one of the beta 3GlcNAcTs in mammalian expression vectors, and the transfected cells were stained with anti-i antibody, followed by FITC-conjugated secondary antibody. Fig. 2 shows that the cells transfected with any of the beta 3GlcNAcTs tested displayed increased amounts of i antigen compared with mock-transfected cells. The results also indicate that the expression efficiency of different beta 3GlcNAcTs is essentially invariable because similar amounts of i antigen were detected in HeLa cells transfected with different beta 3GlcNAcTs.


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Fig. 2.   Expression of i antigen after transient transfection with one of the beta 3GlcNAcTs. HeLa cells, which express minimum amounts of i antigen, were transiently transfected with one of the beta 3GlcNAcTs. Transfected cells were dissociated, stained with anti-i antibody, which recognize poly-N-acetyllactosamine, followed by FITC-conjugated antibody, and subjected to FACS (thick solid lines). Control experiments were performed by transfection with pcDNA3.1(N) without a cDNA insert, followed by staining with (thin solid lines) or without (broken line in Vector panel) anti-i antibody.

As shown previously, CHO cells and the CHO mutant Lec2 cell line do not synthesize core 2 O-glycans (43) or extended core 1 oligosaccharides (18). Lec2 cells were used as a reporter cell line to test beta 3GlcNAcT-dependent reconstitution of MECA-79 antigen expression because Lec2 cells are deficient in core 1 extension and thus MECA-79 antigen expression and because the sialylation defect in Lec2 cells will not inhibit formation of MECA-79 antigen by sialylation of Galbeta 1right-arrow3GalNAcalpha 1right-arrowR. Lec2 cells were thus transiently transfected with LSST and one of the beta 3GlcNAcTs. As shown in Fig. 3, Lec2 cells transfected with beta 3GlcNAcT-3, also called core 1-beta 3GlcNAcT, expressed significant amounts of MECA-79 antigen, but none of the other enzymes formed MECA-79 antigen. Because all of the beta 3GlcNAcTs tested were expressed in similar amounts in HeLa cells, it is reasonable to assume that these beta 3GlcNAcTs were expressed in similar amounts in Lec2 cells as well (see also Fig. 4). These results indicate that only beta 3GlcNAcT-3 can form extended core 1 structure.


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Fig. 3.   Expression of MECA-79 antigen after transient transfection with LSST and one of the beta 3GlcNAcTs. Lec2 cells, which are deficient in sialylation, were transiently transfected with LSST and one of the beta 3GlcNAcTs. Transfected cells were dissociated, stained with MECA-79 antibody, followed by FITC-conjugated secondary antibody, and subjected to FACS (thick solid lines). Control experiments omitted MECA-79 antibody (broken lines). As a mock transfection, pcDNA3.1(N) without a cDNA insert was transfected (Vector panel).

Modification of PSGL-1 O-Glycans by beta 3GlcNAcTs-- To confirm the above conclusions, Lec2 cells were transiently cotransfected with vectors encoding PSGL-1 and one of the beta 3GlcNAcTs. The transfected Lec2 cells were then subjected to Western blot analysis using anti-PSGL-1 antibody KPL-1.

Fig. 4 illustrates that PSGL-1 in mock-transfected Lec2 cells migrated at ~125 kDa. By contrast, beta 3GlcNAcT-3 converted PSGL-1 into polydisperse higher molecular mass glycoforms of ~150-200 kDa. Apparently, beta 3GlcNAcT-2 can form similar products with very low efficacy, and beta 3GlcNAcT-6 forms small amounts of extended core 1, but its product is smaller than that formed by beta 3GlcNAcT-3 or beta 3GlcNAcT-2. These results indicate that beta 3GlcNAcT-3 is almost exclusively responsible for extending core 1. 


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Fig. 4.   Extension of core 1 O-glycans attached to PSGL-1 by different beta 3GlcNAcTs. Lec2 cells were transiently transfected with vectors encoding PSGL-1 and one of the beta 3GlcNAcTs. PSGL-1 in the membrane fraction was separated by SDS-PAGE, followed by Western blot analysis using anti-PSGL-1 monoclonal antibody KPL-1 and secondary antibody. Control experiments (-) were obtained by transfection with a cDNA insert-free vector. PSGL-1 migrated as a monomer and a dimer.

Core 1 Extension Enzyme (beta 3GlcNAcT-3) Is Also Expressed in Neutrophils and Lymphocytes-- Previously, we showed that beta 3GlcNAcT-3 is expressed in HEV in peripheral lymph nodes and forms L-selectin ligand critical for lymphocyte homing (18). To determine whether beta 3GlcNAcT-3 is also expressed in human neutrophils and lymphocytes, neutrophils and lymphocytes were isolated from the peripheral blood, and RT-PCR was used to assay for beta 3GlcNAcT-3 transcripts. The results shown in Fig. 5 demonstrate that the beta 3GlcNAcT-3 transcript was expressed in neutrophils and lymphocytes, although the amount of the transcript was much less than that of the Core2GlcNAcT-I transcript. In addition, the level of Core2GlcNAcT-I transcripts was less than that of PSGL-1. Notably, neutrophils, but not lymphocytes, contained a significant amount of FucT-VII transcripts. To confirm that these transcripts are derived from the proper corresponding GlcNAcT locus, the RT-PCR products were digested with restriction enzymes and analyzed by agarose gel electrophoresis. The results shown in Fig. 5B demonstrate that the transcripts from neutrophils and lymphocytes produced the same restriction digest products as those derived from plasmids encoding those proteins, supporting our conclusion that the transcripts derived from these cells represent beta 3GlcNAcT-3, Core2GlcNAcT-I, FucT-VII, and PSGL-1, respectively.


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Fig. 5.   Detection of beta 3GlcNAcT-3, Core2GlcNAcT-I, FucT-VII, LSST, and PSGL-1 transcripts in human neutrophils and lymphocytes. A, cDNAs were synthesized by reverse transcriptase using total RNA isolated from human neutrophils and lymphocytes. PCR was carried out using the cDNAs as templates and oligonucleotide primers specific to each transcript (+). Control reactions employed total RNA that was not reverse-transcribed (-). The products were separated by electrophoresis on 1% agarose gels. Transcripts encoding glyceraldehyde-3-phosphate dehydrogenase (G3PDH) served as a positive control. B, PCR products derived from neutrophils (N) and lymphocytes (L) were digested with the indicated restriction enzymes and separated by electrophoresis on 2% agarose gel. Positive controls employed PCR using plasmids (P) containing respective cDNAs as templates. The migration positions of molecular mass markers are shown on the left.

On the other hand, LSST transcripts were barely detected in neutrophils or lymphocytes, indicating that LSST is expressed, if at all, in very low quantities in these cells (Fig. 5). These results indicate that neutrophils express each of the enzymes necessary to form sialyl Lewis x in extended core 1 structure, whereas lymphocytes express trace amounts of the sialyl Lewis x moiety due to negligible expression of FucT-VII.

Structural Analysis of PSGL-1 O-Glycans Synthesized in the Presence of beta 3GlcNAcT-3 or Core2GlcNAcT-I-- Because PSGL-1 is expressed in neutrophils and lymphocytes as indicated, the above results suggest that beta 3GlcNAcT-3 can form extended core 1 structure in PSGL-1, a counter-receptor for L-, P-, and E-selectins in neutrophils. Such expression led to the formation of sialyl Lewis x structure in extended core 1 O-glycans when FucT-VII was also present. Fig. 6 illustrates that either beta 3GlcNAcT-3 (C1) or Core2GlcNAcT-I (C2) could convert PSGL-1/IgG chimeric protein into a polydisperse high molecular mass glycoform, which could be metabolically labeled with [3H]glucosamine (second and third lanes). By contrast, PSGL-1 chimeric protein migrated as sharper bands when isolated from control CHO cells expressing only FucT-VII (first lane).


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Fig. 6.   Analysis of PSGL-1/IgG chimeric proteins isolated from CHO cells expressing beta 3GlcNAcT-3 or Core2GlcNAcT-I. CHO cells expressing PSGL-1 and FucT-VII (PSGL-1·F7); PSGL-1, FucT-VII, and beta 3GlcNAcT-3 (PSGL-1·F7·C1); or PSGL-1, FucT-VII, and Core2GlcNAcT-I (PSGL-1·F7·C2) were transiently transfected with pcDNA3.1-PSGL-1/IgG in the presence of [3H]glucosamine. [3H]Glucosamine-labeled PSGL-1/IgG was isolated from the culture medium and subjected to SDS-PAGE, followed by fluorography. The migration positions of molecular mass markers are shown on the left. CHO-PSGL-1/F7 cells produced two discrete bands of PSGL-1/IgG, and a low molecular mass form may be produced due to proteolytic digestion of the high molecular mass form.

These samples were treated with alkaline borohydride to release O-glycans and subjected to Sephadex G-50 gel filtration. Released O-glycans (Fig. 7, A and E, horizontal bars) were subjected then to Bio-Gel P-4 gel filtration. As shown in Fig. 7 (B and F), PSGL-1 O-glycans derived from CHO cells expressing FucT-VII and beta 3GlcNAcT-3 or Core2GlcNAcT-I mainly produced two or three peaks (I, I', and II). After desialylation and Bio-Gel P-4 gel filtration, peak I produced peaks IA and IB (Fig. 7, C and G). As shown in Fig. 7 (C and G), peak C2-IA from Core2GlcNAcT-I-expressing CHO cells eluted slightly later than peak C1-IA from beta 3GlcNAcT-3-expressing CHO cells.


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Fig. 7.   Analysis of O-glycans isolated from PSGL-1/IgG chimeric proteins released from transfected CHO cells. PSGL-1/IgG isolated from CHO-PSGL-1/F7/C1 or CHO-PSGL-1/F7/C2 cells, shown in Fig. 6, was subjected to Sephadex G-50 gel filtration after alkaline degradation (A and E). Released O-glycans were then fractionated by Bio-Gel P-4 gel filtration (B and F). Peaks C1-I (C), C1-II (D), C2-I (G), C2-I' (H), and C2-II (not shown) were hydrolyzed with mild acid to remove sialic acid, and desialylated oligosaccharides were subjected to the same Bio-Gel P-4 filtration. Oligosaccharides subjected to further analysis are shown by horizontal bars.

Peaks C1-IA and C2-IA were then analyzed by HPLC (Fig. 8). Both peaks C1-IA and C2-IA produced two peaks. The first peaks C1-IA1 and C2-IA1 eluted corresponding to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcOH and Galbeta 1right-arrow 4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow3)GalNAcOH, respectively. After alpha 1,3-specific fucosidase digestion, the second peaks C1-IA2 and C2-IA2 were converted to peaks C1-IA1 and C2-IA1, respectively (Fig. 8, B and D), indicating that peaks C1-IA2 and C2-IA2 were derived from NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow 3GalNAcOH and NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAcbeta 1right-arrow6(NeuNAcalpha 2right-arrow3Galbeta 1right-arrow3)GalNAcOH, respectively.


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Fig. 8.   Separation of non-fucosylated and fucosylated O-glycans by HPLC. Peaks C1-IA (A) and C2-IA (C) were subjected to HPLC using an amino-bonded column (Asahipak NH2 P50-4E). The sample was eluted isocratically in the first 40 min and then eluted with a linear gradient by decreasing acetonitrile concentration. In B and D, the peaks that eluted at 64-67 ml in A and at 68-71 ml in C were separately digested with alpha 1,3-fucosidase and subjected to the same HPLC step. In a separate experiment, a mixture of peaks C1-IA and C2-IA was subjected to the same HPLC step, demonstrating that peaks from C1-IA and C2-IA can be separated under these elution conditions (data not shown). The main peaks in B and D correspond to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcOH and Galbeta 1right-arrow4GlcNAcbeta 1right-arrow 6(Galbeta 1right-arrow3)GalNAcOH, respectively.

After desialylation, peaks C1-I and C2-I (Fig. 7, B and F) also produced peaks C1-IB and C2-IB, which eluted at the same positions as Galbeta 1right-arrow3GalNAcOH and sialic acid monomer (Fig. 7, C and G). These results indicate that peaks C1-I and C2-I (Fig. 7, B and F) also contained NeuNAcalpha 2right-arrow6(NeuNAcalpha 2right-arrow3Galbeta 1right-arrow3)GalNAcOH, which was recovered in peaks C1-IB and C2-IB after desialylation. After desialylation, peak C1-II (monosialylated fraction in Fig. 7B) produced peak C1-IIB and a small amount of peak C1-IIA (Fig. 7D). Peak C1-IIA did not change its elution position after desialylation and corresponds to Galbeta 1right-arrow4(±Fucalpha 1right-arrow3)GlcNAcbeta 1right-arrow3Galbeta 1right-arrow3GalNAcOH. Peak C1-IIB was, on the other hand, found to be a mixture of Galbeta 1right-arrow3GalNAcOH and sialic acid. Similarly, peak C2-II (Fig. 7F) produced sialic acid and Galbeta 1right-arrow3GalNAcOH (data not shown). The amount of Galbeta 1right-arrow3GalNAcOH in peaks IB and IIB was determined after sialic acid was removed by QAE-Sephadex A-25 column chromatography.

On the other hand, peak C2-I' produced two peaks corresponding to Galbeta 1right-arrow4GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow3)GalNAcOH (peak C2-I'A) and GlcNAcbeta 1right-arrow6(Galbeta 1right-arrow3)GalNAcOH (peak C2-I'B), in addition to sialic acid (peak C2-I'C) (Fig. 7H and Table I). The latter oligosaccharide (peak C2-I'B) was also obtained previously in CHO cells transfected with Core2GlcNAcT-I (38).

                              
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Table I
Structures and relative molar ratios of O-linked oligosaccharides isolated from CHO cells expressing beta 3GlcNAcT-3 or Core2GlcNAcT-I

These combined results indicate that extended core 1 O-glycans can be fucosylated more efficiently than core 2 branched O-glycans (Fig. 8, compare A and C; and Table I). However, the conversion of core 1 structure to extended core 1 is less efficient than the conversion of core 1 structure to core 2 branched structure (Fig. 7, B versus F, compare peaks I and II; and Table I). These results as a whole indicate that sialyl Lewis x in core 2 branched O-glycans is expressed at levels equivalent to sialyl Lewis x in extended core 1 O-glycans (Table I).

Sialyl Lewis x in Extended Core 1 Functions as an L-selectin Ligand-- To determine the role of sialyl Lewis x in extended core 1 structure, tethering and rolling of neutrophils and lymphocytes were assayed in CHO cells expressing PSGL-1 and sialyl Lewis x in extended core 1 O-glycans or core 2 branched O-glycans. Because CHO cells lack endogenous beta 3GlcNAcT-3 (18) or Core2GlcNAcT (4, 39), CHO cells expressing only PSGL-1 and FucT-VII lack sialyl Lewis x in mucin-type O-glycans. As shown in Fig. 9A, neutrophil tethering and rolling were not detected in this CHO cell line lacking both beta 3GlcNAcT-3 and Core2GlcNAcT-I (open circles). By contrast, CHO cells expressing sialyl Lewis x in extended core 1 (closed circles) or core 2 (open triangles) oligosaccharides supported neutrophil tethering and rolling under shear. Essentially identical results were obtained when lymphocyte tethering and rolling were assayed in the same transfected CHO cells (Fig. 9B). These combined results indicate that non-sulfated sialyl Lewis x in extended core 1 or core 2 oligosaccharides functions as an L-selectin ligand, although sialyl Lewis x in core 2 branched O-glycans functions as a more efficient L-selectin ligand than does sialyl Lewis x in extended core 1 O-glycans (Fig. 9, compare open triangles and closed circles).


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Fig. 9.   L-selectin-mediated adhesion of neutrophils and lymphocytes in CHO cells expressing sialyl Lewis x in extended core 1 or core 2 O-glycans. CHO cells stably expressing PSGL-1, FucT-VII, and beta 3GlcNAcT-3 (closed circles); PSGL-1, FucT-VII, and Core2GlcNAcT-I (open triangles); or PSGL-1 and FucT-VII only (open circles) were grown on plastic dishes and used as the bottom plate of a parallel flow chamber. The number of tethering and rolling neutrophils (A) and lymphocytes (B) at different shear forces is shown (means ± S.D.). Data are from one experiment, representative of two nearly identical ones.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have demonstrated that beta 3GlcNAcT-3, formerly called core 1 beta 3GlcNAcT, is almost exclusively responsible for adding beta 1,3-linked GlcNAc to Galbeta 1right-arrow3GalNAcalpha 1right-arrowR, forming extended core 1 oligosaccharide. Among other beta 3GlcNAcTs, beta 3GlcNAcT-2 may add beta 1,3-linked GlcNAc to core 1 O-glycans with low efficiency, whereas beta 3GlcNAcT-6 apparently forms small amounts of extended core 1 structure. beta 3GlcNAcT-2 is the most efficient enzyme to add N-acetyllactosamine repeats (22),2 whereas beta 3GlcNAcT-6 acts on GalNAcalpha 1right-arrowSer/Thr (27). These results suggest that beta 3GlcNAcT-2 and beta 3GlcNAcT-6 may act as core 1 extension enzymes with very low efficiency under certain conditions. Indeed, the amino acid sequence of beta 3GlcNAcT-3 is highly related to that of beta 3GlcNAcT-6 (51.6 8% identify), which acts on GalNAcalpha 1right-arrowR.

This study unexpectedly demonstrated that extended core 1 is most likely present in neutrophils and lymphocytes, although the amount of extended core 1 structure is less than that of core 2 branched structure. Interestingly, LSST, which is required to form 6-sulfo-GlcNAc structure, is apparently not expressed in neutrophils and lymphocytes. This finding is consistent with the fact that neutrophils and lymphocytes are negative for MECA-79 antigen. beta 3GlcNAcT-3 belongs to the beta 3GlcNAcT gene family, which consists of at least eight different beta 3GlcNAcTs. The members of this gene family include beta 3GlcNAcTs encoded by fringe and brainiac (44-47). Fringe was identified as a protein that regulates Notch signaling by adding beta 1,3-linked GlcNAc to alpha -fucose attached to the extracellular domain of Notch (44, 45). By contrast, Brainiac apparently acts on glycolipids and thereby modulates Notch activity by another mechanism (46, 47). Although beta 3GlcNAcT-1 does not have discernible homology to the beta 3GlcNAcT gene family, recent studies show that one protein predicted by DNA sequence within the human or mouse Large locus has some homology to beta 3GlcNAcT-1 (48). Premature translation termination of this putative glycosyltransferase within the Large locus in mice results in myodystrophy (48), whereas the LARGE locus is deleted in human patients with meningioma, a tumor of the meninges of the central nervous system (49). These result suggest that beta 3GlcNAcT-1 may play an important role in development and cancer because beta 3GlcNAcT-1 is ubiquitously expressed (21).

This study demonstrated that, in transfected CHO cells, fucosylation of N-acetyllactosamine in extended core 1 structure takes place more efficiently than does fucosylation in core 2 branched O-glycans. This finding is consistent with the previous finding that a significant portion of extended core 1 structure is fucosylated in HEV (18). Our results show that extended core 1 structure is efficiently fucosylated once core 1 structure is formed. On the other hand, the amount of extended core 1 O-glycans is less than that of core 2 branched O-glycans (Table I). As an aggregate, extended core 1 and core 2 branched O-glycans contain similar amounts of sialyl Lewis x. Synthesis of both extended core 1 and core 2 branches competes with alpha 2,3-sialylation of core 1, which is catalyzed by beta -galactoside alpha 2,3-sialyltransferase I (50). It is thus possible that both the levels of expressed beta 3GlcNAcT-3 and its catalytic activity are not as great as those of expressed Core2GlcNAcT-I in transfected CHO cells.

It is also possible that localization of beta 3GlcNAcT-3, Core2GlcNAcT-I, and beta -galactoside alpha 2,3-sialyltransferase I in different Golgi compartments is a key factor in determining the amount of oligosaccharides synthesized by beta 3GlcNAcT-3 or Core2GlcNAcT-I (51, 52). Previously, we have shown that Core2GlcNAcT-I resides in the cis- to medial-Golgi, whereas the majority of N- and O-glycan sialyltransferases are thought to reside in the medial- to trans-Golgi (51). This difference allows Core2GlcNAcT-I to add core 2 branch before core 1 oligosaccharide is sialylated. If core 1 oligosaccharide is sialylated first, it becomes unavailable for Core2GlcNAcT-I action (see Fig. 1 in Ref. 2). It is tempting to speculate that beta 3GlcNAcT-3 resides in later compartments of the Golgi than does Core2GlcNAcT-I, thus directly competing with beta -galactoside alpha 2,3-sialyltransferase I for the same acceptor, Galbeta 1right-arrow 3GalNAcalpha right-arrowSer/Thr. Such direct competition, if it occurs, should lead to moderate synthesis of extended core 1 structure.

Previously, we showed that 6-sulfosialyl Lewis x in extended core 1 O-glycans serves as an L-selectin ligand as efficiently as 6-sulfosialyl Lewis x in core 2 branched O-glycans (18). In this study, we extended this finding by showing that sialyl Lewis x in extended core 1 O-glycans also functions as an L-selectin ligand, although it is not as efficient as sialyl Lewis x in core 2 branched O-glycans. Our results are also consistent with previous reports showing that sialyl Lewis x functions as an L-selectin ligand, although extended core 1 structure was not evaluated in that study (53). In our previous study, we found that the sialyl Lewis x structure is present in extended core 1 O-glycans of HEV-derived GlyCAM-1, which were converted to neutral oligosaccharides after desialylation (18). These results combined indicate that sialyl Lewis x in extended core 1 serves as an L-selectin ligand in HEV. L-selectin-mediated neutrophil rolling was shown to take place in adherent neutrophils bound to activated endothelial cells (10, 11). Very recently, we obtained mice heterozygous for beta 3GlcNAcT-3 deficiency and knock-in of green fluorescent protein under the control of the beta 3GlcNAcT-3 promoter. Analysis of these mice indicated that beta 3GlcNAcT-3 is expressed in neutrophils and T lymphocytes because neutrophils and T lymphocytes stained with markers CD11b (Mac-1) and CD3, respectively, were also positive for green fluorescent protein as determined by FACS analysis.2 These studies did not, however, inform at to how much sialyl Lewis x in extended core 1 structure is present in neutrophils. Further studies on the knockout mice are important to determine the degree to which sialyl Lewis x moieties in extended core 1 structure contribute to L-selectin-mediated adhesion in HEV and neutrophil-neutrophil interaction.

    ACKNOWLEDGEMENTS

We thank Dr. Richard Cummings for pZeoSV-PSGL-1, Dr. Shigeru Tsuboi and Misa Suzuki for helpful discussion, Dr. Elise Lammer for critical reading of the manuscript, and Tracy Keeton and Thu Gruenberg for organizing the manuscript.

    FOOTNOTES

* This work was supported by NCI Grant R01CA428737 and in part by NCI Grant P01CA71932 from the National Institutes of Health.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.

Present address: Pathology Div., National Cancer Center Research Inst., 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

|| Present address: La Jolla Bioengineering Inst., 505 Coast Blvd. South, La Jolla, CA 92037.

** Investigator of the Howard Hughes Medical Institute.

Dagger Dagger To whom correspondence should be addressed: The Burnham Inst., 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax: 858-646-3193; E-mail: minoru@burnham.org.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212756200

2 J. Mitoma and M. Fukuda, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Core2GlcNAcT-I, core 2 beta 1,6-N-acetylglucosaminyltransferase I; HEV, high endothelial venule(s); LSST, L-selectin ligand sulfotransferase; FucT-VII, fucosyltransferase VII; beta 3GlcNAcT, beta 1,3-N-acetylglucosaminyltransferase; CHO, Chinese hamster ovary; RT, reverse transcription; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting; HPLC, high performance liquid chromatography.

    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. Schachter, H., and Brockhausen, I. (1992) in Glycoconjugates: Composition, Structure, and Function (Allen, H. J. , and Kisailus, E. C., eds) , pp. 263-332, Marcel Decker, Inc., New York
3. Piller, F., Piller, V., Fox, R. I., and Fukuda, M. (1988) J. Biol. Chem. 263, 15146-15150[Abstract/Free Full Text]
4. Bierhuizen, M. F., and Fukuda, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9326-9330[Abstract]
5. Fukuda, M., Carlsson, S. R., Klock, J. C., and Dell, A. (1986) J. Biol. Chem. 261, 12796-12806[Abstract/Free Full Text]
6. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J. Biol. Chem. 271, 18732-18742[Abstract/Free Full Text]
7. Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484[Medline] [Order article via Infotrieve]
8. Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singhai, A. K., Hakomori, S. I., and Paulson, J. C. (1990) Science 250, 1130-1132[Medline] [Order article via Infotrieve]
9. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science 250, 1132-1135[Medline] [Order article via Infotrieve]
10. Bargatze, R. F., Kurk, S., Butcher, E. C., and Jutila, M. A. (1994) J. Exp. Med. 180, 1785-1792[Abstract]
11. Alon, R., Fuhlbrigge, R. C., Finger, E. B., and Springer, T. A. (1996) J. Cell Biol. 135, 849-865[Abstract]
12. Hiraoka, N., Petryniak, B., Nakayama, J., Tsuboi, S., Suzuki, M., Yeh, J.-C., Izawa, D., Tanaka, T., Miyasaka, M., Lowe, J. B., and Fukuda, M. (1999) Immunity 11, 79-89[Medline] [Order article via Infotrieve]
13. Tangemann, K., Bistrup, A., Hemmerich, S., and Rosen, S. D. (1999) J. Exp. Med. 190, 935-942[Abstract/Free Full Text]
14. Mitsuoka, C., Sawada-Kasugai, M., Ando-Furui, K., Izawa, M., Nakanishi, H., Nakamura, S., Ishida, H., Kiso, M., and Kannagi, R. (1998) J. Biol. Chem. 273, 11225-11233[Abstract/Free Full Text]
15. Bistrup, A., Bhakta, S., Lee, J. K., Belov, Y. Y., Gunn, M. D., Zuo, F. R., Huang, C. C., Kannagi, R., Rosen, S. D., and Hemmerich, S. (1999) J. Cell Biol. 145, 899-910[Abstract/Free Full Text]
16. Hemmerich, S., Bistrup, A., Singer, M., van Zante, A., Lee, J. K., Tsay, D., Peters, M., Carminati, J. L., Brennan, T. J., Carver-Moore, K., Leviten, M., Fuentes, M., Ruddle, N. H., and Rosen, S. D. (2001) Immunity 15, 237-247[Medline] [Order article via Infotrieve]
17. Ellies, L. G., Tsuboi, S., Petryniak, B., Lowe, J. B., Fukuda, M., and Marth, J. D. (1998) Immunity 9, 881-890[Medline] [Order article via Infotrieve]
18. Yeh, J.-C., Hiraoka, N., Petryniak, B., Nakayama, J., Ellies, L. G., Rabuka, D., Hindsgaul, O., Marth, J. D., Lowe, J. B., and Fukuda, M. (2001) Cell 105, 957-969[CrossRef][Medline] [Order article via Infotrieve]
19. Streeter, P. R., Rouse, B. T., and Butcher, E. C. (1988) J. Cell Biol. 107, 1853-1862[Abstract]
20. Maly, P., Thall, A., Petryniak, B., Rogers, C. E., Sith, P. L., Marks, R. M., Kelly, R. J., Gersten, K. M., Cheng, G., Saunders, T. L., Camper, S. A., Camphausen, R. T., Sullivan, F. X., Isogai, Y., Hindsgaul, O., von Andrian, U. H., and Lowe, J. B. (1996) Cell 86, 643-653[Medline] [Order article via Infotrieve]
21. Sasaki, K., Kurata-Miura, K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T., and Fukuda, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14294-14299[Abstract/Free Full Text]
22. Shiraishi, N., Natsume, A., Togayachi, A., Endo, T., Akashima, T., Yamada, Y., Imai, N., Nakagawa, S., Koizumi, S., Sekine, S., Narimatsu, H., and Sasaki, K. (2001) J. Biol. Chem. 276, 3498-3507[Abstract/Free Full Text]
23. Fukuda, M. (2002) in Handbook of Glycosyltransferases and Related Genes (Taniguchi, N. , Honke, K. , and Fukuda, M., eds) , pp. 114-124, Springer-Verlag, Berlin
24. Zhou, D., Dinter, A., Gutierrez Gallego, R., Kamerling, J. P., Vliegenthart, J. F., Berger, E. G., and Hennet, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 406-411[Abstract/Free Full Text]
25. Togayachi, A., Akashima, T., Ookubo, R., Kudo, T., Nishihara, S., Iwasaki, H., Natsume, A., Mio, H., Inokuchi, J., Irimura, T., Sasaki, K., and Narimatsu, H. (2001) J. Biol. Chem. 276, 22032-22040[Abstract/Free Full Text]
26. Henion, T. R., Zhou, D., Wolfer, D. P., Jungalwala, G. B., and Hennet, T. (2001) J. Biol. Chem. 276, 30261-30269[Abstract/Free Full Text]
27. Iwai, T., Inaba, N., Naundorf, A., Zhang, Y., Gotoh, M., Iwasaki, H., Kudo, T., Togayachi, A., Ishizuka, Y., Nakanishi, H., and Narimatsu, H. (2002) J. Biol. Chem. 277, 12802-12809[Abstract/Free Full Text]
28. Kataoka, K., and Huh, N. (2002) Biochem. Biophys. Res. Commun. 294, 843-848[CrossRef][Medline] [Order article via Infotrieve]
29. Barnes, W. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2216-2220[Abstract]
30. Nakayama, J., Fukuda, M. N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7031-7035[Abstract]
31. Feizi, T., Childs, R. A., Watanabe, K., and Hakomori, S. I. (1979) J. Exp. Med. 149, 975-980[Abstract]
32. Ohyama, C., Tsuboi, S., and Fukuda, M. (1999) EMBO J. 18, 1516-1525[Abstract/Free Full Text]
33. Fukuda, M. (1991) J. Biol. Chem. 266, 21327-21330[Free Full Text]
34. Deutscher, S. L., Nuwayhid, N., Stanley, P., Briles, E. I., and Hirschberg, C. B. (1984) Cell 39, 295-299[Medline] [Order article via Infotrieve]
35. Clark, R. A., and Nuaseef, W. M. (1994) in Current Protocols in Immunology (Coligan, J. E., ed) , pp. 7.23.1-7.23.17, John Wiley & Sons, Inc., New York
36. Sako, D., Chang, X. J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., and Furie, B. (1993) Cell 75, 1179-1186[Medline] [Order article via Infotrieve]
37. Moore, K. L., Patel, K., Dbruehl, R. E., Fugang, L., Johnson, D. A., Lichenstein, H. S., Cummings, R. D., Bainton, D. F., and McEver, R. P. (1995) J. Cell Biol. 128, 661-671[Abstract]
38. Piller, F., Le Deist, F., Weinberg, K., Parkman, R., and Fukuda, M. (1991) J. Exp. Med. 173, 1501-1510[Abstract]
39. Bierhuizen, M. F., Maemura, K., and Fukuda, M. (1994) J. Biol. Chem. 269, 4473-4479[Abstract/Free Full Text]
40. Wang, W. C., and Cummings, R. D. (1988) J. Biol. Chem. 263, 4576-4585[Abstract/Free Full Text]
41. Lee, N., Wang, W. C., and Fukuda, M. (1990) J. Biol. Chem. 265, 20476-20487[Abstract/Free Full Text]
42. Maemura, K., and Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386[Abstract/Free Full Text]
43. Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076[Abstract/Free Full Text]
44. Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J. H., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S., and Vogt, T. F. (2000) Nature 406, 369-375[CrossRef][Medline] [Order article via Infotrieve]
45. Bruckner, K., Perex, L., Clausen, H., and Cohen, S. (2000) Nature 406, 411-415[CrossRef][Medline] [Order article via Infotrieve]
46. Müller, R., Altmann, F., Zhou, D., and Hennet, T. (2002) J. Biol. Chem. 277, 32417-32420[Abstract/Free Full Text]
47. Schwientek, T., Keck, B., Levery, S. B., Jensen, M. A., Pedersen, J. W., Wandall, H. H., Stroud, M., Cohen, S. M., Amado, M., and Clausen, H. (2002) J. Biol. Chem. 277, 32421-32429[Abstract/Free Full Text]
48. Grewal, P. K., Holsfeind, P. J., Bittner, R. E., and Hewitt, J. E. (2001) Nat. Genet. 28, 151-154[CrossRef][Medline] [Order article via Infotrieve]
49. Peyrard, M., Seroussi, E., Sandberg-Nordqvist, A.-C., Xie, Y.-G., Han, F.-Y., Fransson, I., Collins, J., Dunham, I., Kost-Alimova, M., Imreh, S., and Dumanski, J. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 598-603[Abstract/Free Full Text]
50. Priatel, J. J., Chui, D., Hiraoka, N., Simmons, C. J. T., Richardson, K. B., Page, D. M., Fukuda, M., Varki, N. M., and Marth, J. D. (2000) Immunity 12, 273-283[Medline] [Order article via Infotrieve]
51. Skrincosky, D., Kain, R., El-Battari, A., Exner, M., Kerjaschki, D., and Fukuda, M. (1997) J. Biol. Chem. 272, 22695-22702[Abstract/Free Full Text]
52. Whitehouse, C., Burchell, J., Gschmeissner, S., Brockhausen, I., Loyd, K. O., and Taylor-Papadimitriou, J. (1997) J. Cell Biol. 137, 1229-1241[Abstract/Free Full Text]
53. Foxall, C., Watson, S. R., Dowbenko, D., Fennie, C., Lasky, L. A., Kiso, M., Hasegawa, A., Asa, D., and Brandley, B. K. (1992) J. Cell Biol. 177, 895-902
54. 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]


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