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
Received on September 3, 2004; revised on October 7, 2004; accepted on October 8, 2004
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Abstract |
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Key words: siayl-Lewis x / core 2 GlcNAc transferase / precursor-B cell / differentiation / short intefering RNA
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Introduction |
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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., 1998, 1999a
,b
). 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., 1998
, 1999a
). 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
2,3sialyltransferase-IV (ST3Gal-IV) are involved in the regulation of sLeX expression level during pre-B cell differentiation. For
1,3FucTs, complementary enzymatic roles of FucT-IV and FucT-VII on sialylated or nonsialylated polylactosamines were revealed (Niemelä et al., 1998
). Although FucT-IV was not detected at all, FucT-VII was definitely expressed in pre-B cell lines (Nakamura et al., 1998
). For
2,3sialyltransferase, ST3Gal-IV (Kitagawa and Paulson, 1993
; Sasaki et al., 1993
) is responsible for the synthesis of
1,3FucT substrate and ST3Gal-IV expression was noticed in pre-B cell lines (Nakamura et al., 1998
, 1999a
). 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., 1998). 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., 1999
, 2000
). 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, 1995; Pui, 1995
). 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., 1993
). sLeX expression levels on leukemia cells significantly correlated with their extravascular infiltration (Furukawa et al., 1994
). 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.
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Results |
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Discussion |
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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., 1993b). 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., 2003
; Xia et al., 2004
). 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, 2000
). 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., 1997, 2003
; Smithson et al., 2001
; Wagers et al., 1996
). 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., 1991
, 1992
, 1996
, 1998
; Nojiri et al., 1988
; Tsunoda et al., 1995
). Instead, branching glycosyltransferases critically determine terminal carbohydrate structure expression by modulating total flow of glycoconjugate biosynthesis (Dennis et al., 1987
; Nakamura et al., 1991
, 1992
, 1996
, 1998
; Nojiri et al., 1988
; Tsuboi and Fukuda, 1997
, Tsunoda et al., 1995
).
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., 2001). 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., 1999). 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., 2000
). 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., 2003). RNA interference has emerged as rapid and efficient means to manipulate gene function in mammalian cells (Miyagishi and Taira, 2002
; Ui-Tei et al., 2004
). 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
GalNAc-transferases has been identified and the isoforms display distinct enzymatic properties and are differentially expressed (Cheng et al., 2004
). 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.
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Materials and methods |
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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., 1999a). 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 marrowderived mononuclear cells were triple-stained with fluorescein isothiocyanateconjugated 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 TrisHCl 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, 1995) 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., 1998
).
Northern blot analysis
We performed northern blot analysis to detect exogenous glycosyltransferase gene expression as described (Nakamura et al., 1998). 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., 1999a
).
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., 1999). 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., 1998
).
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 peroxidaseconjugated secondary antibody (Nakamura et al., 1999a). 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., 2003). 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., 1998
). 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
510 x 108 infectious units (IFU) per ml (Kikuchi et al., 2004
).
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Acknowledgements |
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Abbreviations |
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References |
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