Significant decrease in a 1 , 3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing

Lymphocyte homing is mediated by binding of L-selectin on lymphocytes with L-selectin ligands present on highendothelial venules (HEV) of peripheral and mesenteric lymph nodes. L-selectin ligands are specific O-linked carbohydrates, 6-sulfo sialyl Lewis X, composed of sialylated, fucosylated, and sulfated glycans. Abrogation of fucosyltransferase-VII (FucT-VII) results in almost complete loss of lymphocyte homing, but structural analysis of carbohydrates has not been carried out on FucT-VII null mice. To determine whether functional losses seen in FucT-VII null mice are caused by structural changes in carbohydrates, we elucidated the carbohydrate structure of GlyCAM-1, a major L-selectin counter-receptor. Our results show that most a1,3-fucosylated structures in 6-sulfo sialyl Lewis X are absent and 6-sulfo N-acetyllactosamine is increased in the mutant mice. Surprisingly, the amount of 60-sulfated galactose (Gal) that bound to Sumbucus nigra agglutinin column was also increased. We found that structures of those oligosaccharides containing 60-sulfated Gal are almost identical to those synthesized by keratan sulfate sulfotransferase (KSST). We then showed that overexpression of KSST suppresses the expression of sialyl Lewis X on Chinese hamster ovary (CHO) cells engineered to express sialyl Lewis X. Moreover, KSST expression in those cells suppressed lymphocyte rolling compared with mock-transfected CHO cells expressing 6-sulfo sialyl Lewis X. 60-Sulfo sialyl Lewis X can neither be found in GlyCAM-1 from CHO cells expressing both KSST and FucT-VII nor be found in GlyCAM-1 from HEV of mice. These results combined together suggest that KSST competes with FucT-VII for the same acceptor substrate and downregulates the synthesis of L-selectin ligand by inhibiting a1,3-fucosylation.


Introduction
Lymphocyte recirculation through lymph nodes and Peyer's patches is required for the immune system to detect antigens and activate processes that neutralize them (Marchesi and Gowans 1964).Lymphocyte recirculation critically depends on interaction between the leukocyte adhesion molecule, L-selectin, and its counter-receptors, peripheral lymph node addressins, which are restricted to specialized postcapillary venules, high-endothelial venules (HEV) in secondary lymphoid organs (Arbones et al. 1994).The counter-receptors on the luminal surface of HEV capture circulating lymphocytes via L-selectin-dependent adhesive interactions that lead, in turn, to lymphocyte tethering and rolling, chemokine-dependent activation, integrin-mediated firm attachment, and lymphocyte transmigration (Springer et al. 1994;Butcher and Picker 1996;von Andrian and Mempel 2003).HEV-like microvasculature is also induced on endothelium in association with insulitis seen in the nonobese diabetic mouse and the rejection of heart transplants in humans (Hanninen et al. 1993;Faveeuw et al. 1994;Toppila et al. 1999).Similarly, HEV-like structures are observed in inflammatory bowel diseases, rheumatoid arthritis, lymphocytic thyroiditis, the hyperplastic thymus characteristic of the AKR mouse, and Helicobacter pylori-infected stomach (van Dinther-Janssen et al. 1990;Michie et al. 1993Michie et al. , 1995;;Salmi et al. 1994;Kobayashi et al. 2004).It has been suggested that recruitment of lymphocytes by induced L-selectin ligand may contribute to the pathogenesis of these chronic inflammatory diseases characterized by induced HEV-like microvasculature.
Mice with targeted deletion of the fucosyltransferase (FucT)-VII exhibit approximately an 80% reduction in lymphocyte homing to peripheral lymph nodes.FucT-VII knockout mice also exhibit significant reduction in T-cell trafficking to inflamed cutaneous sites in vivo, indicative of a significant reduction in the cutaneous immune response (Maly et al. 1996).When fucosyltransferase (FucT)-IV is also inactivated, almost all L-selectin ligands are abrogated in the double knockouts as assessed by lymphocyte homing assays (Homeister et al. 2001).In contrast to GlcNAc6ST-1 and GlcNAc6ST-2 double knockouts, no structural studies of Lselectin ligands on HEV have been reported for FucT-VII or FucT-VII/IV knockout mice.Thus, changes in carbohydrate structures of L-selectin ligands have not been correlated with loss of FucT-VII function in vivo.
In the present study, we first analyzed O-linked oligosaccharides attached to GlyCAM-1 derived from wild-type and FucT-VII knockout mice.We found more than 80% of reduction in a1,3-linked fucose (Fuc) and a significant increase in O-glycans containing the 6-sulfo galactose group (6 0 -sulfo galactose) in FucT-VII-deficient mice.Since 6 0 -sulfo galactose can be synthesized by keratan sulfate 6 0 -sulfo transferase (KSST; Fukuta et al. 1997;Torii et al. 2000), we overexpressed KSST in Chinese hamster ovary (CHO) cells expressing 6-sulfo sialyl Lewis X.We found that the expression of 6 0 -sulfo galactose reduced the amount of sialyl Lewis X and 6-sulfo sialyl Lewis X, L-selectin ligand.These results indicate that KSST inhibits the synthesis of 6-sulfo sialyl Lewis X by competing with FucT-VII, and an increased in the amount of 6 0 -sulfo galactose, potentially synthesized by KSST, in FucT-VII-deficient mice does not lead to an enhanced L-selectin ligand activity.

Sulfated oligosaccharide increase in FucT-VII-deficient mutant mice
It has been previously shown that an inactivation of FucT-VII by homologous recombination results in more than 80% decrease in lymphocyte homing.Although L-selectin immunoglobulin M (IgM) chimeric protein scarcely bound to HEV of these mutant mice (Maly et al. 1996), the MECA-79 antigen was expressed as strongly as in wild-type HEV, since Fuc is not required for MECA-79 recognition (Yeh et al. 2001).To determine how changes in the structure of L-selectin ligand oligosaccharides lead to such functional consequences, we undertook structural analysis of oligosaccharides attached to the GlyCAM-1, a major glycoprotein carrying L-selectin ligands.Lymph nodes were cultured in the presence of [ 3 H]-galactose and GlyCAM-1 was isolated from the culture medium.Upon SDS-gel electrophoresis, GlyCAM-1 from FucT-VII-deficient mice migrated slightly faster than GlyCAM-1 from wild-type mice (Figure 1A), suggesting that GlyCAM-1 from the mutant mice contains slightly less amount of carbohydrates.
O-Glycans were released from GlyCAM-1 by mild alkaline treatment and recovered by Sephadex G-50 gel filtration.The majority of O-glycans are sialylated and were separated by QAE-Sephadex column chromatography before and after removal of sialic acid by mild acid hydrolysis (Figure 1B).All sialic acids were also removed by a2,3-specific sialidase and after desialylation, O-glycans from wild-type GlyCAM-1 are primarily composed of mono-sulfated or nonsulfated oligosaccharides.On the contrary, a significant portion of O-glycans from GlyCAM-1 of FucT-VII-deficient mice were mono-sulfated, di-sulfated, and multi-sulfated oligosaccharides, indicating that the loss of fucosylation by FucT-VII led to an increase in the sulfation (Figure 1B).
Since our structural studies showed only a small amount of 6 0 -sulfated galactosyl oligosaccharide in smaller oligosaccharides, we subjected oligosaccharides containing five to seven monosaccharides (hexasaccharide fraction) to SNA column chromatography.Figure 4 (top) illustrates that oligosaccharides from FucT-VII-deficient mice contained much more SNA-binding oligosaccharides (65.1% of the total 3 H-galactose) than does wild-type mice (22.4% of the total 3 H-galactose).
These results combined together indicate that core 2 O-glycans containing 6-sulfo sialyl Lewis X are highly enriched in wild-type GlyCAM-1 (Figure 3).On the contrary, Fuc-free O-glycans such as peak f in Figure 2A constitute the majority of O-glycans in GlyCAM-1 from FucT-VII null mice.Interestingly, O-glycans containing 6 0 -sulfo galactose increased substantially upon inactivation of FucT-VII and was accompanied by the absence of 6-sulfo sialyl Lewis X.One of those O-glycans was recovered ( peak h).

Expression of KSST reduces the synthesis of sialyl Lewis X
The above results show that oligosaccharides containing 6 0 -sulfo galactose are increased in FucT-VII null mice.Those increased oligosaccharides are almost identical to oligosaccharides synthesized in the presence of KSST.Since this increase in 6 0 -sulfo galactose took place in the absence of FucT-VII, it suggests that FucT-VII and KSST may compete for the same acceptor molecules.
To test this hypothesis, we expressed FucT-VII, Core2GlcNAcT-I, or Core1-b3GlcNAcT in CHO cells stably expressing KSST.Since KSST and EGFP-F are expressed in a bi-cistronic vector, stable expression of KSST in CHO cells could be detected by cytoplasmic-and membrane-bound EGFP-F expression.Three cloned lines of CHO cells expressing KSST exhibited a similar level of EGFP-F expression, as assessed by fluorescent activated cell sorting (FACS) analysis (Figure 6, left panel).By contrast, control parental CHO cells showed no EGFP-F expression.KSST activity in CHO cells was measured using keratan sulfate as an acceptor (Torri et al. 2000) and 35 S-labeled keratan sulfate was recovered after Sephadex G-25 gel filtration (Figure 6, right panel).Three cloned lines, K1-A6, K1-B6, and K2-D5, exhibited almost identical KSST activity.Mammalian expression vectors harboring FucT-VII, Core2GlcNAcT-I, or Core1-b3GlcNAcT were transiently introduced into these cells.Compared with control cells, fewer CHO cells stably expressing KSST are strongly positive for sialyl Lewis X expression, as assessed by CSLEX-1 antibody staining (Figure 7A).Almost identical results were obtained when HECA-452 antibody was used.These results are summarized in Figure 7B and C.These observations establish that expression of KSST significantly reduces sialyl Lewis X expression.

Expression of KSST inhibits lymphocyte rolling
To determine whether overexpression of KSST inhibits lymphocyte rolling, CHO cells stably expressing CD34, Core2GlcNAcT-1, Core1-b3GlcNAcT, GlcNAc6ST-2, and FucT-VII were transiently transfected with KSST, and rolling over these cells was measured.In two independent experiments, lymphocyte rolling was significantly decreased in CHO cells expressing KSST compared with mock-transfected CHO parental cells.Similarly, CHO cells expressing CD34, GlcNAc6ST-2, FucT-VII, and Core2GlcNAcT-1 were transiently transfected with KSST.Lymphocyte rolling was also significantly decreased in these cells compared with mock-transfected CHO cells (Figure 8).As shown above, KSST expression results in a significant decrease in the amount of expressed sialyl Lewis X. Taken together, all of these results indicate that expression of KSST reduces the amount of sialyl Lewis X and 6-sulfo sialyl Lewis X, leading to decrease in lymphocyte rolling.

Discussion
FucT-VII plays a major role in the synthesis of sialyl Lewis X and 6-sulfo sialyl Lewis X. Genetic studies have demonstrated that abrogation of FucT-VII in mutant mice results in an 80% decrease in lymphocyte homing and an 80% decrease in lymphocyte counts in the peripheral lymph nodes (Maly et al. 1996).The present study showed that this functional loss is associated with loss of 6-sulfo sialyl Lewis X and an increase in sialyl 6-sulfo N-acetyllactosamine and 6 0 -sulfo galactose in GlyCAM-1 derived from HEV of FucT-VII mutant mice.These results suggest that addition of a 6 0 -sulfo group to galactose competes with a1,3-fucosylation of N-acetylglucosamine.
Although 6 0 -sulfo galactose is present in various O-glycans of GlyCAM-1, none of those branches terminated with 6 0 -sulfo galactose contain a1,3-linked Fuc at the penultimate N-acetylglucosamine.This situation contrasts with that of 6sulfo N-acetylglucosamine, which is mostly fucosylated through a1,3-linkage.In the present study, we also found that 6 0 -sulfo galactose is present in core 1 side-chains, and SO 3 !6Galb1 !3GalNAca !R was detected in various O-glycans.Similar to these findings, oligosaccharides formed by KSST do not contain a1,3-linked Fuc in the same branch and 6'-sulfo Lewis X (shown as X in Figure 5) is absent.On the other hand, 6'-sulfo galactose in the core 1 side, sulfo !6Galb1 !3GalNAc !R, is found in many O-glycans synthesized in CHO cells transfected with KSST and in GlyCAM-1 derived from wild-type and FucT-VII-deficient mice.This finding is consistent with the previous report that KSST prefers negatively charged acceptors to nonsulfated oligosaccharides (Torii et al. 2000).
These findings indicate that 6 0 -sulfated oligosaccharides found in FucT-VII mutant mice are almost identical to those synthesized by KSST.Indeed, the results shown here indicate that overexpression of KSST leads to a decrease in sialyl Lewis X in CHO cells expressing CD34, FucT-VII, and Core2GlcNAcT, as assessed by anti-sialyl Lewis X antibodies, either CSLEX-1 or HECA-452.These results are consistent with our conclusion that FucT-VII competes with KSST under physiological conditions and that overexpression of KSST reduces the FucT-VII product, while FucT-VII expression inhibits formation of 6 0 -sulfo galactose.This is consistent with previous report that KSST acts very efficiently on sialic acida2 !3Galb1 !4GlcNAc structures, but not on sialic acida2 !3Galb1 !4(Fuca1 !3)GlcNAc (Torii et al. 2000).On the other hand, FucT-VII acts efficiently on sialic acida2 !3Galb1 !4GlcNAc, but not on sialic acida2 !3(SO 3 !6)Galb1 !4GlcNAc (Britten et al. 1998;Neimela et al. 1998;Shinoda et al. 1998).It has been shown that KSST is expressed in HEV of peripheral lymph nodes (Kawashima et al. 2005).Considering that FucT-VII null mice contain an increased amount of 6 0 -sulfo galactose, these results are consistent with our conclusion that KSST playing a major role in synthesizing 6 0 -sulfo galactose in HEV.
In GlcNAc6ST-1 and GlcNAc6ST-2 double knockout mice, 6-sulfo sialyl Lewis X is almost absent.As a result, sialyl Lewis X is significantly increased, and the amount of (Kawashima et al. 2005).KSST and GlcNAc6ST do not compete for the same acceptor due to their substrate specificities.The increase in 6 0 -sulfo galactose in GlcNAc6ST-1 and GlcNAc6ST-2 doubly knockout mice is therefore likely due to an increase in the availability of the donor substrate, PAPS, and of acceptor substrates for KSST.
Sialic acida2 !3Galb1 !4(Fuca1 !3)GlcNAcb1 !6[sialic acida2 !3(6-sulfo) Galb1 !3]GalNAc was detected as a minor component in mono-sulfated oligosaccharides in FucT-VII knockout mice.It has been previously reported that expression of KSST in COS-1 leads to an increase in L-selectin ligands (Bistrup et al. 1999;Tangemann et al. 1999).This increase cannot be due to an increase in 6 0 -sulfo sialyl Lewis X, since KSST cannot form 6 0 -sulfo sialyl Lewis X.It is possible that the above oligosaccharide containing sialyl Lewis X on core 2 branch and 6 0 -sulfo galactose in the core 1 chain may function as an L-selectin ligand and that the amount of this oligosaccharide is what is increased in KSST-transfected cells.Further studies will be important in determining whether this is the case.
CHO cells stably expressing KSST Core2GlcNAcT-1 and FucT-VII were transiently transfected with pcDNAI-GlyCAM † IgG and labeled with [ 3 H]-galactose.Chimeric GlyCAM † IgG protein released into the medium was isolated by a protein A-Agarose column.O-glycans were released from GlyCAM † IgG and fractionated by Bio-Gel-4 gel filtration and HPLC as described subsequently.
Sulfated O-glycans were separated using an NH 2 -bonded HPLC column (Asahipack NH 2 P50E-4E, 4.6 Â 25 mm, Asahichemical Industry, Tokyo, Japan) with elution conditions modified from those previously reported (Hiraoka et al. 1999;Hiraoka et al. 2000;Yeh et al. 2001;Kawashima et al. 2005).Solvent A (64% acetonitrile, 36% H 2 O), solvent B (25 mM NaH 2 PO 4 in solvent A), and solvent C (50 mM NaH 2 PO 4 in solvent A) were used for HPLC as follows: (i) Mono-sulfated tetrasaccharide core O-glycans were eluted with a linear gradient from 0% to 30% solvent B in solvent A for 10 min, from 30% to 65% for 40 min, and from 65% to 100% for 5 min, followed by 100% solvent B for 5 min.(ii) Mono-sulfated hexasaccharide core O-glycans were eluted with a linear gradient from 0% to 40% solvent B in solvent A for 10 min, from 40% to 80% for 40 min, and from 80% to 100% for 5 min, followed by 100% solvent B for 5 min.(iii) Di-sulfated O-glycans were eluted with a linear gradient from 0% to 40% solvent C in solvent A for 10 min, from 40% to 80% for 40 min, and from 80% to 100% for 5 min, followed by 100% solvent C for 15 min.
Lectin column chromatography SNA from Elderberry (EY Lab, San Mateo, CA) was immobilized to make lectin-conjugated beads (5 mg/mL beads) using UltraLink Biosupport Medium (PIERCE).Oligosaccharides were applied to SNA-conjugated beads column (1 mL gel) equilibrated in 10 mM Tris -HCl ( pH 7.4), 150 mM NaCl, and 0.02% NaN 3 , and incubated for 10 min, then eluted with the same Tris buffer followed by the buffer containing 0.1 M lactose.All procedures were performed at 48C.

Antibody staining for flow cytometry
Transfected CHO cells were dissociated with enzyme-free dissociation solution (Ca 2þ -and Mg 2þ -free Specialty Media, Phillipsburg, NJ) and suspended in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (incubation solution).Cells were then incubated with an anti-sialyl Lewis X antibody, either CSLEX-1 or HECA-452 (Duijvestijn et al. 1988), in the incubation solution.After washing with the incubation solution, the cells were incubated with phycoerythrin-conjugated anti-mouse IgM antibody or phycoerythrinconjugated anti-rat IgM antibody (Pharmingen, San Diego, CA) dissolved in the incubation solution.After washing in the incubation solution, stained cells were assessed by flow cytometry using FACScan and analyzed with Cell Quest (BD Biosciences).

CHO cells stably expressing KSST
The cDNA fragment encoding enhanced green fluorescent protein (EGFP) plus a farnesylation signal (EGFP-F) was excised from pEGFP-F (Clontech, Mountain View, CA) using NcoI and SmaI and subcloned into the same sites of IRES1/pBluescriptII.A DNA fragment harboring both IRES and EGFP-F was digested by XbaI and XhoI and cloned into the same site of pcDNA3.1/Hyg.A DNA fragment harboring IRES and EGFP-F that had been subcloned into pcDNA3.1 was excised using XbaI and NheI, and cloned into the XbaI site of pcDNA3.1 -human KSST and pcDNA3.1 -mouse KSST as described previously (Hiraoka et al. 1999), resulting in pcDNA3.1 -human KSST -IRES -EGFP-F and pcDNA3.1 -mouse KSST -IRES -EGFP-F.
To obtain CHO cells stably expressing KSST, cells stably expressing CD34 were transfected with pcDNA3.1 -KSST -IRES -EGFP-F and selected in 200 mg/mL of hygromycin B. Several clones showed high EGFP expression as detected by flow cytometry.KSST expression in these cells was confirmed by assaying KSST activity.

Assay of KSST activity
Attached CHO cells stably expressing KSST were washed with PBS, scraped and homogenized in 10 mM Tris -HCl, pH 7.2, containing 0.5% Triton X-100, 0.25 M sucrose, a protease inhibitor mixture, and 1 mM aprotinin as described previously (Torri et al. 2000).The homogenate was mixed by rotation for 1 h and centrifuged at 10 000 g for 15 min.Supernatant derived from transfected and mock-transfected cells were used as the enzyme source.
KSST activity was assayed as described previously (Torri et al. 2000).Briefly, the reaction mixture (50 mL) contained 50 mM imidazol -HCl, pH 6.4, 10 mM CaCl 2 , 2 mM dithiothreitol, 50 mg of keratan sulfate, 2 mM [ 35 S]phospho adenosyl 5 0 -phospho sulfate (PAPS) (approximately 5 Â 10 5 cpm), and 25 mL of an enzyme solution.After incubation for 20 min at 378C, the reaction mixture was boiled for 2 min, and 0.1 volume of 4 M potassium acetate and 3 volumes of ethanol were added.Reaction products were precipitated by brief centrifugation and subjected to Sephadex G-25 gel filtration in 0.1 M (NH 4 ) 4 HCO 3 to separate high molecular weight products from the remaining [ 35 S] PAPS and its degradation products.
Measurement of L-selectin-mediated rolling on CHO cells expressing 6-sulfo sialyl Lewis X CHO cells stably expressing CD34, GlcNAc6ST-2, FucT-VII, Core2GlcNAcT-1, Core1-b3GlcNAcT (CHO-CD34/GlcNA c6ST-2/F7/C2/C1) were established.Establishment of CHO-CD34/GlcNAc6ST-2/F7/C2 cells was described previously (Yeh et al. 2001;Mitoma et al. 2003).Both lines were transiently transfected with pcDNAI-1 KSST and a rolling assay performed 72 h after transfection.The parental and transfected cell lines seeded on dishes were used as the bottom plate of a parallel wall flow chamber as described previously (Hiraoka et al. 1999;Yeh et al. 2001;Kawashima et al. 2005).Lymphocytes were initially introduced into the flow chamber at a wall sheer stress of 5 dyne/cm 2 for 15 s, followed by the termination of flow to allow the cells to adhere under static conditions (Kawashima et al. 2005).A flow rate was then initiated at different sheer forces.Image analysis was performed and analyzed as described previously (Maly et al. 1996).

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Fig. 1 .
Fig. 1. Isolation of O-linked oligosaccharides from GlyCAM-1.(A) Peripheral lymph nodes were metabolically labeled with [ 3 H]-galactose, and 3 H-galactose-labeled GlyCAM-1 was isolated by immunoadsorption.Isolated GlyCAM-1 was subjected to SDS-gel electrophoresis and fluorography.(B) O-glycans were released from GlyCAM-1 by alkaline borohydride and isolated by Sephadex G-50 gel filtration.O-glycans were then subjected to QAE-Sephadex column chromatography before and after desialylation.Mono-sulfated to tetra-sulfated O-glycans were eluted in a step-wise manner with an increasing concentration of NaCl.Arrows indicate points where NaCl concentration is increased.Detailed elution conditions are described in Materials and methods section.GlyCAM-1 from wild-type (WT) and FucT-VII-deficient (FucT-VII 2/2 ) mice were analyzed.O-glycans in FucT-VII-deficient mice

Fig. 2 .
Fig. 2. HPLC analysis of mono-sulfated tetrasaccharide fraction.The mono-sulfated fraction (Mono-S in Fig. 1B) from both wild-type (left and middle panels) and FucT-VII-deficient (right panel) mice was separated into tetrasaccharide and hexasaccharide (the major components) and even higher oligosaccharides by Bio-Gel P-4 gel filtration.(A) The tetrasaccharide fraction was digested with b-galactosidase and subjected to HPLC using the elution condition (i) described in Materials and methods section.Numbers indicated the elution position correspond to the elution positions for oligosaccharides shown in (C).Peak d was digested with a1,3/4 fucosidase (Fucosidase panel) followed by b-galactosidase (b-Galase) (middle low panel) and each product was analyzed by HPLC.Peak d2 was eluted at the same position of peak b.Peak a eluted at the same position of oligosaccharides containing SO 3 !6Galb1 !4GlcNAcb1 !3Galb1 !3GalNAcOH and GlcNAcb1 !6(SO 3 !6Galb1 !3)GalNAcOH.It was split into two peaks a1 and a2 after hexosaminidase A (Hexase A) digestion.Peak a2 was eluted at the same elution position of peak a. Peak 2 was eluted at the same positions of oligosaccharides containing GlcNAcb1 !6(SO 3 !6Galb1 !3)GalNAcOH and SO 3 !6GlcNAcb1 !3Galb1 !3GalNAcOH, and was split into two peaks e1 and e2 after hexaminidase B (Hexas B) digestion.Peak f was the same elution position as peak b containing SO 3 !6GlcNAcb1 !6(Galb1 !3)GalNAcOH.Peak g was eluted at the same positions of peak c and was split into two peaks g1 and g2 after a1 !3/a1 ! 4 fucosidase digestion.Peaks g1 and g2 were the same elution position of peaks c1 and c2, respectively.(B) The hexasaccharide fraction was digested with b-galactosidase and subjected to HPLC using the elution condition (ii) described in Materials and methods section.Numbers indicated the elution position correspond to the elution positions for oligosaccharides shown in (C) and (D).[(C) and (D)] The elution profiles of oligsaccharides from CHO cells expressing FucT-VII, GlcNAc6ST-2 and Core2GlcNAcT and Core1-b3GlcNAcT.In addition, the elution positions obtained by KSST transfection (Figure 5) are added.In determination of the radioactivity, 5-10% of the elate were subjected to scintillation counting.

Fig. 7 .
Fig. 7. Expression of sialyl Lewis X is suppressed by expression of KSST.[(A) and (B)] CHO cells stably expressing KSST (clone K2-D5 in Fig.6) (right panels) and control cells (CHO/CD34) (left panels) were transiently transfected with a combination of FucT-VII and Core2GlcNAcT, or Core1-b3GlcNAcT, or FucT-VII alone.Forty-eight hour after transfection, cells were stained with the anti-sialyl Lewis X antibody CSELX-1 and subjected to cytometric analysis.Compared to mock-transfected cells, the number of sialyl Lewis X-positive cells was significantly reduced and this is quantified in (B) and (C).Results obtained using another anti-sialyl Lewis X antibody, HECA-452 are shown.

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