Not core 2 ß1,6-N-acetylglucosaminyltransferase-2 or -3 but -1 regulates sialyl-Lewis x expression in human precursor B cells

Jiro Kikuchi2, Hirotaka Shinohara2,3, Chizu Nonomura2,4, Hidenobu Ando2, Shizuka Takaku2, Hisao Nojiri3 and Mitsuru Nakamura1,2,4

2 Cell Regulation Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-4, 1-1-1 Higashi, Tsukuba, 305-8562, Japan; 3 Department of Biochemical Oncology, School of Pharmaceutical Sciences, Teikyo University, Sagamiko 199-0195, Japan; and 4 Core Research for Evolution Science and Technology (CREST) of Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan


1 To whom correspondence should be addressed; e-mail: owlm.nakamura{at}aist.go.jp

Received on September 3, 2004; revised on October 7, 2004; accepted on October 8, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sialyl-Lewis x (sLeX), one of the major selectin ligands, is expressed on T and B cells in a differentiation or activation stage-specific manner. We have demonstrated before that sLeX expression and core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT) were simultaneously regulated during precursor B (pre-B) cell differentiation. Three C2GnT family genes, designated C2GnT-1, -2, and -3, were previously identified, but their roles have not been fully examined. In this study, we have investigated the roles of C2GnTs in the regulation of sLeX expression level during pre-B cell differentiation comparing with {alpha}1,3fucosyltransferase-VII (FucT-VII) and {alpha}2,3sialyltransferase-IV (ST3Gal-IV). Overexpression of not FucT-VII and ST3Gal-IV but C2GnT-1 blocked the down-regulation of sLeX expression by differentiation induction. Neither C2GnT-2 nor -3 but C2GnT-1 transcript was mainly expressed in B lineage cell lines and bone marrow–derived B lineage cells. Significant down-regulation of C2GnT-1 of the three C2GnTs was observed in KM3 cells during differentiation. The expression of C2GnT-1 correlated well to sLeX expression and differentiation stage. Furthermore, introduction of short interfering RNA against C2GnT-1 markedly reduced C2GnT-1 expression and resulted in down-regulation of sLeX expression. These results suggest that not the other glycosyltransferases but C2GnT-1 regulates sLeX expression level during differentiation of pre-B cells, providing the cells with substrate of sLeX structure biosynthesis.

Key words: siayl-Lewis x / core 2 GlcNAc transferase / precursor-B cell / differentiation / short intefering RNA


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Leukocyte rolling, mediated by selectins and their ligands, is a well-established initial step of cell adhesion to endothelial cells for lymphocyte homing and neutrophil recruitment in inflammation (McEver and Cummings, 1997Go). Sialyl-Lewis x (sLeX) structures, one of the physiologically relevant selectin ligands, are expressed on T and B lymphocytes in a differentiation stage-specific or an activation state-specific manner (Ohmori et al., 1993aGo) and synthesized through the essential action of {alpha}1,3fucosyltransferase VII ({alpha}1,3FucT; FucT-VII) (Maly et al., 1996Go; Sasaki et al., 1994Go). Although the terminal tetrasaccharide structure of sLeX is synthesized on N-acetyllactosamine unit repeats by sequential glycosyltransferase actions, it has been long believed that the expression of sLeX is controlled by up- or down-regulation of FucT-VII during lymphocyte differentiation or activation (Macher et al., 1991Go).

On the contrary, we have reported that there was no correlation between FucT-VII and sLeX expression level during differentiation of precursor B (pre-B) cells and activation of B cells (Nakamura et al., 1998Go, 1999aGo,bGo). We have also demonstrated that significant down-regulation of sLeX antigen expression during pre-B cell differentiation is accompanied by simultaneous decrease of core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT) expression level (Nakamura et al., 1998Go, 1999aGo). Particularly, the antigen is mainly located on O-linked sugar chains of a glycoprotein with molecular size of 150 kDa (gp150). However, it is not completely understood how far FucT-VII and {alpha}2,3sialyltransferase-IV (ST3Gal-IV) are involved in the regulation of sLeX expression level during pre-B cell differentiation. For {alpha}1,3FucTs, complementary enzymatic roles of FucT-IV and FucT-VII on sialylated or nonsialylated polylactosamines were revealed (Niemelä et al., 1998Go). Although FucT-IV was not detected at all, FucT-VII was definitely expressed in pre-B cell lines (Nakamura et al., 1998Go). For {alpha}2,3sialyltransferase, ST3Gal-IV (Kitagawa and Paulson, 1993Go; Sasaki et al., 1993Go) is responsible for the synthesis of {alpha}1,3FucT substrate and ST3Gal-IV expression was noticed in pre-B cell lines (Nakamura et al., 1998Go, 1999aGo). So it is of interest whether FucT-VII and ST3Gal-IV can regulate the sLeX expression level as well as C2GnT in our pre-B cell differentiation system.

C2GnT knockout mice were developed and characterized (Ellies et al., 1998Go). In their report, only partial deficiency of selectin ligands was recognized, and lymphocyte homing was not affected by the diminished presence of L-selectin ligands. Moreover, the other C2GnTs were found, and it was reported that the core 2 branch can be synthesized by at least three enzymes, C2GnT-1, -2, and -3 (Schwientek et al., 1999Go, 2000Go). In our B cell differentiation system, it was not clear yet which isoform plays a significant role in generating scaffolds for sLeX biosynthesis.

On the other hand, previous studies have been reported that pre-B leukemia often relapses in the central nervous system and other organs (Copelan and McGuire, 1995Go; Pui, 1995Go). This is in part attributable to the ability of pre-B leukemia cells to infiltrate into the liver, spleen, lymph nodes, and central nervous system. In the process of the infiltration, interaction between selectins and sLeX is thought to be the initial and essential step (Takada et al., 1993Go). sLeX expression levels on leukemia cells significantly correlated with their extravascular infiltration (Furukawa et al., 1994Go). As suppression of sLeX expression may inhibit the leukemia cell infiltration, it is of important to investigate the regulatory glycosyltransferase gene for sLeX biosynthesis in pre-B cells for the effective treatment of leukemia patients.

In this study, we have investigated the roles of C2GnT-1 by gain-of-function and loss-of-function analyses in pre-B cells, comparing with the other glycosyltransferases. In the gain-of-function analysis, sLeX expression was examined in human pre-B KM3 cell sublines transfected with FucT-VII, ST3Gal-IV, and C2GnT-1. For the loss-of-function analysis, we used lentiviral short interfering RNA (siRNA) for C2GnT-1 in KM3 cells. Remodeling of sLeX synthetic machinery revealed that C2GnT is a key regulator of the sLeX expression level during human B cell differentiation, and this regulatory role of C2GnT is solely played by C2GnT-1 and cannot be replaced by FucT-VII and ST3Gal-IV.


    Results
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Establishment of KM3 sublines transfected with FucT-VII, ST3Gal-IV, and C2GnT-1
We transfected plasmids containing the respective full-length cDNA driven by cytomegalovirus promoter into human pre-B leukemia cell line KM3. After selection by neomycin resistance, we established KM3 sublines transfected with FucT-VII, ST3Gal-IV, and C2GnT-1 and designated them KM3/f4b1, KM3/s4-4b3, and KM3/cl-6d1, respectively. First of all, very minor expression of endogenous C2GnT-1 was observed (arrows with asterisk), whereas FucT-VII and ST3Gal-IV expression was not recognized in the parental (Figure 1, lanes 1, 4, and 7) and mock-transfected KM3/m1e3 cells (Figure 1, lanes 2, 5, and 8). However, exogenous glycosyltransferase gene expression was confirmed in KM3/f4b1, KM3/s4-4b3, and KM3/cl-6d1 cells (arrows without asterisk in Figure 1, lanes 3, 6, and 9). The transfected KM3 sublines exhibited high glycosyltransferase enzymatic activities, respectively, compared with the mock transfectant (data not shown).



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Fig. 1. Expression of exogenous glycosyltransferase genes in KM3 cell sublines. Gene expression of FucT-VII (lanes 1–3), ST3Gal-IV (lanes 4–6), and C2GnT-1 (lanes 7–9) was examined in the parental KM3 (lanes 1, 4, and 7), mock-transfected KM3m1e3 (lanes 2, 5, and 8), and respective transfectants (lanes 3, 6, and 9) by northern blot analyses. Capillary transferred membranes were hybridized with [32P]-labeled probes for FucT-VII, ST3Gal-IV, and C2GnT-1. Control GAPDH gene expression was detected using the same membranes. Arrows indicate the positions of expressed glycosyltransferases and GAPDH. Arrows with asterisk (*) represent the positions of endogenous C2GnT-1 messages.

 
sLeX expression in KM3 cell sublines during differentiation induction
Subsequently, flow cytometry analysis was conducted for the reactivity against anti-sLeX monoclonal antibody (mAb), CSLEX1 and KM93, using the transfected sublines differentiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Representative results using CSLEX1 mAb were shown in Figure 2. Although the reactivity fluctuated from experiment to experiment, fluorescence intensity of FucT-VII-transfected KM3/f4b1 and ST3Gal-IV-transfected KM3/s4-4b3 cells was stronger than that of the mock and C2GnT-1-transfected cells before TPA treatment (top row). After differentiation induction, sLeX expression level was down-regulated to 1/8–1/12 of that at day 0 in the mock-transfected cells (left column; calculated by the mean fluorescence intensity). For KM3/f4b1 and KM3/s4-4b3 cells, the down-regulation of sLeX expression was blocked to a certain extent (middle two columns) compared with the mock transfectant. However, the block was far from sufficient compared with that in C2GnT-1-transfected KM3/cl-6d1 cells (right column). Namely, in FucT-VII and ST3Gal-IV-transfected cells, extent of the decreased CSLEX1-reactivity at day 3 was about 1/5 to 1/7 of that at day 0 (middle two columns), whereas the downward shift after treatment was only less than 20% in C2GnT-1-transfected cells (right column). In addition to KM3/cl-6d1, five independent C2GnT-1-transfectants out of six tested sublines possessed the same characteristics. The results using KM93 mAb were the same as CSLEX1 mAb (data not shown).



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Fig. 2. sLeX expression in KM3 cell sublines during differentiation induction. sLeX expression was detected on the transfected KM3 cell sublines during differentiation. Reactivity against anti-sLeX antibody was analyzed by flow cytometer using the mock-transfected KM3 cells and sublines transfected with FucT-VII, ST3Gal-IV, and C2GnT-1. The cells were differentiated by 8 nM TPA for 0–3 days (from top to bottom) and subjected to mAb (CSLEX1) staining followed by flow cytometry analysis. Solid and dotted lines represent the histograms of positive cells and control cells, respectively. Ordinate and abscissa represent cell numbers and relative fluorescence intensity, respectively.

 
Expression of anti-sLeX antibody-reactive glycoprotein in KM3 cell sublines
The expression of sLeX-bearing glycoproteins was examined by western blot analysis using CSLEX1 mAb. As shown in Figure 3, a major glycoprotein with molecular size of ~150 kDa, designated gp150, was recognized in nontreated cells transfected with FucT-VII, ST3Gal-IV, and vector alone (Figure 3, lanes 1, 3, 5, and 7). However, intensity of gp150 became much weaker after differentiation than that before TPA treatment (Figure 3, lanes 2, 4, and 6). On the other hand in C2GnT-1-transfected KM3/cl-6d1 cells, the intensity of gp150 did not decrease after differentiation induction (Figure 3, lane 8). These data clearly show that overexpression of not FucT-VII and ST3Gal-IV but C2GnT-1 blocks the down-regulation of sLeX expression level during pre-B KM3 cell differentiation.



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Fig. 3. Western blot analyses in KM3 sublines during differentiation induction. Western blot analyses were performed to detect anti-sLeX antibody-reactive glycoproteins in KM3 cell sublines. Forty micrograms of protein was prepared from the mock-transfected KM3 cells (lanes 1 and 2) and sublines, transfected with FucT-VII (lanes 3 and 4), ST3Gal-IV (lanes 5 and 6), and C2GnT-1 (lanes 7 and 8) before (–) and after (+; day 3) TPA treatment, and subjected to 5% polyacrylamide gel electrophoresis followed by transfer to Immobilon-P membrane and by staining with CSLEX1 mAb. Signal was detected by chemiluminescence method. An arrow indicates the position of gp150.

 
Expression of exogenous glycosyltransferase genes in KM3 cell sublines
To exclude the possible down-regulation of exogenous gene expression along with differentiation, northern blot analysis for FucT-VII, ST3Gal-IV, and C2GnT-1 was conducted using the transfected sublines before and after TPA treatment. Exogenous gene expression after differentiation was maintained as that before TPA treatment in the respective KM3 sublines as shown in Figure 4A. We examined the expression of C2GnT-1 by semi-quantitative reverse transcribed-polymerase chain reaction (PCR). Endogenous C2GnT-1 expression in FucT-VII-transfected KM3/f4b1 cells was suppressed to about 1/10-fold (Figure 4B). The down-regulation of endogenous C2GnT-1 was also confirmed in ST3Gal-IV-transfected KM3/s4-4b3 cells and mock-transfected cells (data not shown). Thus inability of complete block against sLeX suppression in these sublines was not caused by any simultaneous down-regulation of exogenous FucT-VII and ST3Gal-IV but by decrease of endogenous C2GnT-1 expression during differentiation.



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Fig. 4. Expression of exogenous glycosyltransferase genes in KM3 cell sublines. (A) Exogenous gene expression in the sublines, transfected with FucT-VII (left), ST3Gal-IV (center), and C2GnT-1 (right) before (–) and after (+; day 3) TPA treatement, was detected by northern blot analysis with respective probes. GAPDH expression analysis was conducted by rehybridization. (B) Endogenous C2GnT-1 gene expression was semi-quantitatively determined by reverse transcribed-PCR in FucT-VII-transfected KM3/f4b1 subline before (–) and after (+; day 3) TPA treatment.

 
Expression levels of C2GnT-1, -2, and -3 in KM3 cells during differentiation induction
Subsequently, expression of cell surface sLeX antigen and C2GnT transcript during pre-B cell differentiation was analyzed using KM3 cells. KM3 cells expressed significant sLeX structure before TPA treatment, and the expression was down-regulated along with differentiation (Figures 2 and 3, left panels). In addition, C2GnT-1 was strongly exhibited before differentiation, and the expression was markedly down-regulated during differentiation, as shown in Figure 5A. The nontreated cells expressed C2GnT-2 transcript as well as C2GnT-1, although C2GnT-2 expression level was significantly lower than that of C2GnT-1 (Figure 5A, day 0). Although expression of not only C2GnT-1 but also C2GnT-2 was down-regulated on days 1 and 2 after TPA treatment, C2GnT-2 level on day 3 returned to that on day 0. On the other hand, expression level of C2GnT-3 was extremely low before and after TPA treatment (Figure 5A). Furthermore, we conducted western blot analysis using specific antibody for C2GnT-1. C2GnT-1 protein was clearly detected, but the expression was significantly down-regulated during differentiation in KM3 cells (Figure 5B). However, C2GnT-2 and -3 were not detected (data not shown).



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Fig. 5. Expression of C2GnT family genes in KM3 cells during differentiation induction. (A) The amount of C2GnT transcripts was determined by real time-PCR analysis in KM3 cells during differentiation induction. The amount of GAPDH transcript was also detected to normalize the amount of RNA in each sample. The value of C2GnTs/GAPDH was exhibited. Solid bar indicates C2GnT-1; gray bar, C2GnT-2; and open bar, C2GnT-3. SEs of the mean of three independent experiments are shown. (B) C2GnT-1 protein was detected by western blot analysis using specific antibody in KM3 cells during differentiation induction. ß-actin protein was also detected to normalize the amount of protein in each sample. For peptide blocking (bl), antibodies were preincubated with antigen peptide and then used for staining of the sample on day 0.

 
Expression levels of C2GnT-1, -2, and -3 in B-lineage cell lines and bone marrow–derived pre-B cell
The transcript expression of C2GnT-1, -2, and -3 was examined by real time PCR analysis in human B lineage cell lines and primary bone marrow-derived B lineage cells. As shown in Figure 6A, the C2GnT-1 expression was the highest of the three C2GnT family genes in all B lineage cells including primary B lineage cells except for SSK-LCL cells. We have also detected the expression of sLeX, CD10, and CD21 in these cells by flow cytometry analysis (Figure 6B). Cell surface sLeX expression was higher in CD21low pre-B cell lines and bone marrow–derived pre-B cells than that of CD21high mature B cell lines and bone marrow–derived mature B cells. These results show that there is a strong correlation between sLeX expression level and C2GnT-1 transcript level in bone marrow-derived pre-B and mature B cells. This correlation suggests that loss of cell surface sLeX during pre-B cell differentiation is due to the change of C2GnT-1 level in bone marrow.



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Fig. 6. Expression of C2GnT family genes and cell surface sLeX in B lineage cell lines and bone marrow-derived B lineage cells. Human B cell lines were cultured as described in Materials and methods. Human primary B cells were prepared from bone marrow–derived mononuclear cells. Bone marrow-derived B cells (BM B cells) were fractioned into CD19+CD10+CD21-/pre-B and CD19+CD10-CD21+/mature-B cells by FACSaria. (A) The amount of C2GnT transcripts was determined in the various B lineage cell lines, bone marrow–derived pre-B, and bone marrow–derived mature-B cells. The amount of GAPDH transcript was detected by real time-PCR to normalize the amount of RNA in each sample. The value of C2GnTs/GAPDH was exhibited. Solid bar indicates C2GnT-1; gray bar, C2GnT-2; and open bar, C2GnT-3. SEs of the mean of three independent experiments are shown. (B) The expression levels of cell surface sLeX, CD10, and CD21 in B lineage cell lines and BM B cells are summarized. sLeX, CD10, and CD21 expression was determined by flow cytometry analysis. The expression was shown in a semi-quantitative manner; ++++, the positive cells were more than 80%; +++, 40–80% positive; ++, 20–40% positive; +, 5–20% positive; +/–, 1–5% positive; –, the positive cells were less than 1%.

 
Gene silencing analysis of C2GnT-1 in KM3 cells
We performed gene silencing analysis using lentiviral siRNA transduction system in KM3 cells (Rubinson et al., 2003Go). KM3 cells were suspended in 5 ml RPMI1640 medium supplemented with 10% fetal calf serum in the presence of 8 µg/ml polybrene. Lentiviral particles (multiplicity of infection of 5) including C2GnT-1-siRNA (Lenti-siC2GnT-1-pLL3.7) or mock (Lenti-pLL3.7) were added to the medium. About 70% of the cells were positive for green fluorescent protein (GFP) (data not shown). We collected 1–5 x 105 of GFP positive and alive cells for over 95% purity by FACS (data not shown), depleting dead cells by 1 µg/ml of propidium iodide staining. Then the expression of C2GnT-1 and ß-actin were detected by real time-PCR analysis. As shown in Figure 7A, Lenti-siC2GnT-1-pLL3.7 transduction caused marked reduction of C2GnT-1 expression levels to 40–30% relative to the Lenti-pLL3.7 transduced cells. Flow cytometry analysis revealed that sLeX expression was significantly down-regulated in siRNA introduced KM3 cells (Figure 7B). The mean fluorescent intensity (MFI) of the Lenti-siC2GnT-1-pLL3.7 transduced cells was 506, whereas that of the Lenti-pLL3.7 transduced was 3539.



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Fig. 7. Effect of gene silencing of C2GnT-1 by short interfering RNA on sLeX expression in KM3 cells. Lentivirus vector carrying siRNA for C2GnT-1 (Lenti-siC2GnT-1-pLL3.7) or mock (Lenti-pLL3.7) were infected to KM3 cells. After 4, 8, and 14 days culture, the GFP-positive cells were collected by flow cytometry. (A) C2GnT-1 expression was determined by real time-PCR analysis. The amount of ß-actin was also detected to normalize the amount of RNA in each samples. Relative C2GnT-1 expression was presented as fold increase compared with the C2GnT-1/ß-actin values for the Lenti-pLL3.7, which was set as x1. Data were obtained from triplicate experiments and error bars indicate the SEM. (B) sLeX expression was detected by flow cytometer using anti-sLeX antibody, CSLEX1, on day 8. The histogram plot analysis was exhibited in the GFP-positive fraction.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We have investigated roles of C2GnT-1 in the present study, comparing them with the other glycosyltransferases. The gain-of-function and loss-of-function analyses demonstrated that not the other glycosyltransferases but C2GnT-1 plays an important role in regulating sLeX expression level during differentiation of pre-B cell.

We used in this study CSLEX1 and KM93 as anti-sLeX antibodies. Although the specificity of KM93 has not been clarified yet, CSLEX1-reactive epitope is well established. We have already examined and revealed that pre-B cells and cell lines are positive for 2H5, FH6, and SNH3 as well as CSLEX1 (Nonomura et al., unpublished data). These suggest that pre-B cells express the conventional sLeX structures (Ohmori et al., 1993bGo). Moreover, we have already exhibited that pre-B cells and cell lines are reactive with E- and P-selectin/Ig chimeras (Nonomura et al., unpublished data). Although physiological roles of sLeX structures on pre-B cells are not elucidated yet, the sLeX structures may act as ligands of E- and P-selectins expressed on sinusoidal endothelial cells in bone marrow (Katayama et al., 2003Go; Xia et al., 2004Go). This is partly because pre-B cells are developed in bone marrow and differentiate therein into more mature B cells, which do not express sLeX antigen (LeBien, 2000Go). However, further elucidation should be required.

It has been long believed that sLeX expression level is controlled by FucT-VII expression during lymphocyte differentiation or activation (Hiraiwa et al., 1997Go, 2003Go; Smithson et al., 2001Go; Wagers et al., 1996Go). However, we have demonstrated that functional sugar structure synthesis is not regulated through terminal and intermediate glycosyltransferases during differentiation and transformation of human and murine myelogenous leukemia HL-60, K562, and NFS60 cells (Nakamura et al., 1991Go, 1992Go, 1996Go, 1998Go; Nojiri et al., 1988Go; Tsunoda et al., 1995Go). Instead, branching glycosyltransferases critically determine terminal carbohydrate structure expression by modulating total flow of glycoconjugate biosynthesis (Dennis et al., 1987Go; Nakamura et al., 1991Go, 1992Go, 1996Go, 1998Go; Nojiri et al., 1988Go; Tsuboi and Fukuda, 1997Go, Tsunoda et al., 1995Go).

The reactivity of anti-sLeX antibodies became high in the sublines transfected with FucT-VII and ST3Gal-IV before and after differentiation induction compared with the mock transfectants (Figure 2). Although the core 2 branches act as scaffolds of sLeX structure biosynthesis, it is suggested by the present study that the termini of core 2 branches are not completely sialylated and fucosylated in pre-B cells (middle two columns). The remaining C2GnT-1 activities may provide the other glycosyltransferases with minimal scaffolds of sLeX biosynthesis. Alternatively, the other core structure may be present in pre-B cells as well as high endothelial venule cells in which sialyl-6-sulfo-LeX is expressed not only on core 2 but also on extended core 1 structure (Yeh et al., 2001Go). However, these theories require further investigations.

Among C2GnT family, it has been reported that C2GnT-1 is widely expressed in tissues, including mucin-producing cells and lymphocytes. C2GnT-2 is expressed in colon and has broader substrate specificity than the other members; that is, C2GnT-2 has activity to synthesize core 4 and I-branching structures as well as core 2 (Yeh et al., 1999Go). By contrast, C2GnT-3 has been mentioned as thymus associated one and has the same type of substrate specificity as C2GnT-1 (Schwientek et al., 2000Go). In the present study, we showed that C2GnT-2 and C2GnT-3 were not significantly detected in B cell lines and primary B lineage cells. Mainly C2GnT-1 was expressed in pre-B cells. Remarkable down-regulation of C2GnT-1 but not C2GnT-2 and -3 was observed during differentiation induction of KM3 cells. C2GnT-1 expression correlated well with sLeX expression and differentiation stage.

In addition, we performed the loss-of-function analysis using lentiviral siRNA transduction system (Rubinson et al., 2003Go). RNA interference has emerged as rapid and efficient means to manipulate gene function in mammalian cells (Miyagishi and Taira, 2002Go; Ui-Tei et al., 2004Go). Glycosyltransferase genes have many isoforms and are regulated in cells or tissues specifically at the transcriptional level. For example, a large family of homologous UDP-GalNAc: polypeptide {alpha}GalNAc-transferases has been identified and the isoforms display distinct enzymatic properties and are differentially expressed (Cheng et al., 2004Go). In the large family of glycosyltransferases, gain-of-function analyses would be insufficient to determine the isoform playing the significant roles in cells or tissues. On the contrary, loss-of-function analyses using siRNA should be a useful and powerful method to determine the functional isoform. We have demonstrated that introduction of siRNA for C2GnT-1 significantly reduced the expression level of sLeX in pre-B KM3 cells and determined that C2GnT-1 is the functional glycosyltransferase among the C2GnT isoforms in pre-B cells.

We have demonstrated that not C2GnT-2 or -3 but -1 was a major regulator of sLeX biosynthesis in pre-B leukemia cells. As described in the Introduction, suppression of sLeX expression may inhibit leukemia cell infiltration. Therefore, siRNA targeting the C2GnT-1 may be useful for the prevention of extravascular infiltration of pre-B leukemia cells in vivo, and C2GnT-1 may be a candidate of effective therapeutic targets in the treatment of pre-B leukemia patients.


    Materials and methods
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 Materials and methods
 References
 
Cells, cell lines, and cultures
Human pre-B leukemia cell line KM3, NALL1, Nalm6, and Nalm1 cells; human Burkitt's lymphoma cell line Daudi; human lymphoblastoid cell line SSK-LCL; and human myeloma cell line U266 were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Differentiation of KM3 cells was induced by incubating the cells with 8 nM TPA at an initial density of 2–5 x 105 cells/ml (Nakamura et al., 1998Go).

Flow cytometry analysis and cell sorting
Flow cytometry analysis was carried out by FACScan (Becton Dickinson, San Jose, CA) as previously described (Nakamura et al., 1999aGo). The level of sLeX expression was detected by the reactivity of CSLEX1 (ATCC HB 8580) and KM93 (Seikagaku, Tokyo, Japan) antibodies. Biotin-conjugated anti-mouse IgM and allophycocyanin (APC)-Cy7-conjugated streptavidine (Beckman Coulter, Fullerton, CA) were used for the second and third antibodies, respectively. Cell sorting was carried out by FACSaria (Becton Dickinson). Human bone marrow–derived mononuclear cells were triple-stained with fluorescein isothiocyanate–conjugated anti-CD10, phycoerythrin-conjugated anti-CD21, and APC-conjugated anti-CD19 antibodies (Becton Dickinson). After staining, CD19 + CD10 + CD21-/pre-B cell fraction was sorted.

Preparation of glycosyltransferase cDNAs
Full-length human FucT-VII cDNA was prepared by PCR cloning. The cDNA was amplified by 32 cycles of PCR in 60 mM Tris–HCl buffer (pH 9.5), 15 mM (NH4)2SO4, 250 µM dATP, 250 µM dGTP, 250 µM dTTP, 250 µM dCTP, and 2.0 mM MgCl2. Used primers were as follows: forward, 5'-act-gat-cct-ggg-aga-ctg-tgg-atg-3' and reverse, 5'-agc-gga-tct-cag-gcc-tga-aac-caa-3'. The amplified FucT-VII cDNA was directly subcloned to pCR3 mammalian expression vector, and the sense-oriented clone was chosen and designated pCR3-hFucT-VII. The mouse full-length ST3Gal-IV cDNA (kindly provided from Dr. Takashi Kudo and Dr. Hisashi Narimatsu) (Kudo and Narimatsu, 1995Go) was excised by HindIII and NotI digestion and inserted into pCR3 vector to yield plasmid pCR3-mST3Gal-IV. Full-length human C2GnT-1 cDNA was prepared and inserted into pCR3 vector to yield plasmid pCR3-C2GnT-1 as described previously (Nakamura et al., 1998Go).

Northern blot analysis
We performed northern blot analysis to detect exogenous glycosyltransferase gene expression as described (Nakamura et al., 1998Go). Hybridizing probes were excised by endonuclease digestion using EcoRI, AflII, and HindIII from pCR3-hFucT-VII, pCR3-mST3Gal-IV, and pCR3-hC2GnT-1 plasmids, respectively. Glycelaldehyde-phosphate dehydrogenase (GAPDH) cDNA probe was obtained from Oncogene Science, and neoR cDNA was prepared by the reverse transcribed PCR method followed by subcloning to pCRII vector and excision by EcoRI. Used primer was the same as the previous report (Nakamura et al., 1999aGo).

Transfection of glycosyltransferase cDNAs in KM3 cells
The plasmids containing full-length glycosyltransferase cDNA were transfected into KM3 cells by electroporation method as described (Furukawa et al., 1999Go). Over 10 monoclonal transfectants for each cDNA were selected and established by limiting dilution in the presence of G418. Among them, a clone for FucT-VII, ST3Gal-IV, and C2GnT-1 expressing the highest level of exogenous transcript was chosen and designated as KM3/f4b1, KM3/s4-4b3, and KM3/cl-6d1, respectively. The transfected cell line with pCR3 vector alone was KM3m1e3 as reported (Nakamura et al., 1998Go).

Real time-PCR analysis
The amount of C2GnT-1, -2, and -3 in the human B lineage cells or cell lines was determined by real time-PCR analysis. The standard curves for the C2GnT-1, -2, -3, GAPDH, and ß-actin cDNAs were generated by serial dilution. Primer sets and probes for C2GnT-1, -2, -3, and GAPDH were as follows: forward primer for C2GnT-1, 5'-gaa gag ttg cct gtt cct gtc c-3'; reverse primer for C2GnT-1, 5'-gat atg ctg ctt ctt ttt tcc tgg t-3'; probe for C2GnT-1, 5'-FAM-cat tac tgc ctc ttc ctt ctc ctt ccc tac aat t-TAMRA-3'; forward primer for C2GnT-2, 5'-gac gtt gct gcg aag g-3'; reverse primer for C2GnT-2, 5'-cca agt gtc tga cac tta ca-3'; probe for C2GnT-2, 5'-FAM-tct ccg ttt taa gga ttc atc aaa agc ctg aat-TAMRA-3'; forward primer for C2GnT-3, 5'-gac gtt gct gcg aag g-3'; reverse primer for C2GnT-3, 5'-cca agt gtc tga cac tta ca-3'; probe for C2GnT-3, 5'-FAM-tct ccg ttt taa gga ttc atc aaa agc ctg aat-TAMRA-3'; forward primer for GAPDH, 5'-gaa ggt gaa ggt cgg agt c-3'; reverse primer for GAPDH, 5'-gaa gat ggt gat ggg att tc-3'; and probe for GAPDH, 5'-FAM-caa gct tcc cgt tct cag cc-TAMRA-3'. Primers and probe for ß-actin were purchased from Applied Biosystems (Foster City, CA). Primers, probes, and cDNAs were added to the TaqMan Universal PCR Master Mix (Applied Biosystems) that contained all reagents for PCR. The PCR conditions included 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 50 cycles at 95°C for 15 s and 60°C for 1 minute. PCR products were continuously measured with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The relative amount of C2GnT-1, -2, and -3 transcript was normalized to the amount of GAPDH or ß-actin transcript in the same cDNA. When using the endogenous control, absolute transcript expression values lower than 10 amol were thought to be under the detectable level, and the data were eliminated before the normalization.

Western blotting
Whole cell lysates (40 µg each) were separated on 5% or 10% polyacrylamide gels in the presence of sodium dodecyl sulfate and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated with the following antibodies: CSLEX1, anti-ß-actin mAb (Ab-1; Santa Cruz, Santa Cruz, CA), and specific antibodies for C2GnT-1, -2, or -3. Antibodies for C2GnT-1, -2, or -3 were purified from rabbit serum by affinity chromatography. For immunization of rabbit, synthetic peptides (Sawady Technology, Tokyo, Japan) were used and the sequences were as follows: C2GnT-1, MLPPLETPLFSC; C2GnT-2, ARWMPGSVPNHPKC; and C2GnT-3, RVPYEYVKLPIRC. The membranes were developed with the enhanced chemiluminescence system (Amersham, Uppsala, Sweden) after incubating with horseradish peroxidase–conjugated secondary antibody (Nakamura et al., 1999aGo). For peptide neutralization, all antibodies except for CSLEX1 antibody were incubated with a 10-fold weight excess of following blocking peptides: Ab-1P (Santa Cruz) for anti-ß-actin antibody and synthetic peptides used in the immunization for C2GnT-1, -2, and -3.

RNA interference lentivirus system
We used siRNA expression vector, pLL3.7 plasmid (kindly provided by Dr. Luk Van Parijs; Rubinson et al., 2003Go). This vector contains a cytomegalovirus promoter driving expression of enhanced GFP and the mouse U6 promoter with downstream restriction sites (HpaI and XhoI) to allow the efficient introduction of oligonucleotides encoding siRNAs. The siRNA sequences for C2GnT-1 were 5'-gaaaggtggaagaagcggt-3' for sense, 5'-accgcttcttccacctttc-3' for antisense. Computer analysis using the software developed by Ambion (Austin, TX) confirmed this sequence to be a good target. Oligonucleotides including siRNA and hairpin structure were chemically synthesized (GeneWorld-Exigen, Tokyo, Japan), and the sequences were as follows: forward for 5'-tgaaaggtggaagaagcggtttcaagagaaccgcttcttccacctttcttttttc-3', reverse for 5'-tcgagaaaaaagaaaggtggaagaagcggttctcttgaaaccgcttcttccacctttca-3'. The oligonucleotides were double-stranded and phosphorylated and inserted into pLL3.7 vector using HpaI and XhoI sites. We prepared lentivirus with packaging vectors into 293FT cells (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction (Dull et al., 1998Go). After 48 h, the resulting culture supernatant was recovered and ultracentrifuged for 1.5 h at 28,000 rpm in a S100AT5 rotor (Hitachi-Koki, Tokyo, Japan). Titers were determined by infecting 293FT cells with serial dilutions of concentrated lentivirus. For a typical preparation, the titer of lentivirus was ~5–10 x 108 infectious units (IFU) per ml (Kikuchi et al., 2004Go).


    Acknowledgements
 
We are indebted to Dr. Luk Van Parijs (MIT) for the generous gift of siRNA Lentivirus system and Dr. Takashi Kudo (University of Tsukuba, Japan) and Dr. Hisashi Narimatsu (Research Center for Glycoscience, National Institute of Advanced and Industrial Science and Technology, Japan) for the generous gift of full-length ST3Gal-IV cDNA. We thank Dr. Takashi Angata and Dr. Hidenori Ozaki for valuable comments on this study. We also thank Taeko Inageta, Yumi Nakamichi, and Kazunori Nakamura for their technical supports and Tomomi Yaguchi for her secretarial assistance. This work was supported in part by CREST, a Grant-in-Aid for Scientific Research on Priority Areas No. 12033215 and a Grant for General Scientific Research No. 11670147 from MEXT Japan (to M.N.) and by the Japan Leukemia Research Fund (to M.N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    Abbreviations
 
APC, allophycocyanin; GAPDH, Glycelaldehyde-phosphate dehydrogenase; GFP, green fluorescent protein; mAb, monoclonal antibody; PCR, polymerase chain reaction; siRNA, short interfering RNA; sLeX, sialylated Lewis antigen; TPA, 12-O-tetradecanoylphorbol-13-acetate


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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