UDP-GlcNAc:Gal[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase holds a key role on the control of CD15s expression in human pre-B lymphoid cell lines

Mitsuru Nakamura1,10, Yusuke Furukawa1, Ryuhei Sasaki5, Jun-ichi Masuyama3, Jiro Kikuchi1,6, Satsuki Iwase1,7, Takashi Kudo4, Hisashi Narimatsu4, Shinji Asakura2, Shigeyoshi Fujiwara8 and Jin-ichi Inokuchi9

1Division of Hemopoiesis and 2Division of Hemostasis and Thrombosis Research, Institute of Hematology, and 3Department of Allergy and Collagen Disease, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan, 4Division of Cell Biology, Institute of Life Science, Soka University, 1-236 Tangi, Hachioji, Tokyo 192, Japan, 5The Attached Clinic, International University of Health and Welfare, Kitakanemaru, Otawara, Tochigi 324, Japan, 6Katsuta Research Laboratory, Hitachi Koki Co., Ltd., Ibaraki 312, Japan, 7Department of Internal Medicine, Jikei University School of Medicine, Tokyo 105, Japan, 8Department of Microbiology, Nihon University School of Medicine, Oyaguchikami-machi, Itabashi,Tokyo 173, Japan and 9Seikagaku Corporation, Tokyo Research Institute, 1253 Tateno 3-chome, Higashiyamato, Tokyo 207, Japan

Received on January 28, 1998; revised on June 17, 1998; accepted on June 19, 1998

Expression mechanism of CD15s (sialyl-Lex, sLex) antigen has been investigated using human B lymphoid cell lines. sLex structures were not expressed in mature B lymphoids but highly expressed in pre-B leukemia and pre-B lymphoma cell lines. The expression site was mainly on the O-linked oligosaccharide chains and E-selectin mediated-cell adhesion capability of sLex-positive cells were significantly suppressed by benzyl-[alpha]-GalNAc treatment. Subsequently, the bases of the sLex expression control mechanism were examined at the levels of enzymatic activities and transcripts of glycosyltransferases. (1) The activities of [alpha]1->3fucosyltransferase, [alpha]2->3sialyltransferase, [beta]1->4Gal-transferase, and elongation [beta]1->3GlcNAc-transferase, did not correlate with sLex expression levels. (2) The transcripts of Fuc-TVII were not parallel with sLex expression, and those of ST3Gal IV and [beta]1->4Gal-transferase were constitutively detected in all cell lines tested. (3) There was no detectable enzyme activity for core 3 and 4 backbone structure synthesis in human B cell lines. (4) By contrast, the enzyme activities and transcripts of UDP-GlcNAc:Gal[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase (Core2GnT) had significant correlation with the cell surface expression of sLex antigen. (5) Moreover, Western blot analysis revealed the presence of a major ~150 kDa glycoprotein that carries O-linked oligosaccharides recognized by anti-sLex monoclonal antibody in sLex-positive pre-B leukemia cell lines. This correlation of Core2GnT with CD15s expression suggests that Core2GnT is a regulator of the cell surface expression of sLex in human pre-B lymphoid cells.

Key words: B cell precursor leukemia/glycosyltransferases/O-glycan

Introduction

Majority of the leukocyte differentiation markers are glycoproteins and each protein has a unique primary sequence that may direct the function of the molecule. In a proportion of the antigens, however, the protein portion may serve as a carrier that presents cell surface carbohydrate antigens. In addition, some established differentiation antigens are expressed on the carbohydrate chains of glycosphingolipids. A recent account of such carbohydrate antigens includes CD15, CD15s, CDw17, CD57, CDw60, CDw65, CDw75, and CD76 (Schlossman et al., 1995). The carbohydrate antigens, like the protein antigens, may be expressed in a lineage specific manner or a differentiation stage specific manner.

CD15s antigen (sialyl-Lex; sLex)3 is a well-established ligand of cell adhesion molecules, CD62E, CD62P, and CD62L also known as E-, P-, and L-selectins, respectively (Lowe et al., 1990; Phillips et al., 1990; Walz et al., 1990). The antigen mediates the cell adhesion with E-selectin on the endothelial cells, and this adhesion is considered as the initial and essential step of cancer cell metastasis and leukemia cell infiltration (Takada et al., 1993). In human leukocytes, sLex is expressed on myeloids, lymphoids, and monocytes (Schlossman et al., 1995). The formation of sLex structures as well as the other terminal carbohydrate structures has been demonstrated to be accomplished by sequential actions of glycosyltransferases. Expression of sLex structures was proved to be through the action of [alpha]1->3fucosyltransferase VII (Fuc-TVII) (Knibbs et al., 1996). On the other hand, regulation mechanism of sLex expression during differentiation and transformation has not been fully elucidated. It is true that most of the previous studies have been concentrated on the terminal fucosylation or sialylation in their investigations. However, during differentiation and transformation of human and murine myelogenous leukemia cells, we have demonstrated that the most up-stream glycosyltransferases markedly influenced the expression levels of the terminal carbohydrate structures by modulating the total metabolic flow of the glycosphingolipid biosynthesis at the upstream branching step (Nojiri et al., 1988; Kitagawa et al., 1989; Nakamura et al., 1991, 1992, 1996; Tsunoda et al., 1995).

In the present study, we have tried to investigate the biosynthetic mechanism of CD15s antigen recognized by KM93 monoclonal antibody (mAb) using human B lymphoid cell lines. We present here our results that KM93-reactive sLex structures of B lymphoids are mainly expressed on O-linked oligosaccharide chains and the O-glycans are located on a ~150 kDa glycoprotein. In addition, it is suggested that the up-stream branching UDP-GlcNAc:Gal[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase (Core2GnT or C2GnT) critically regulates the expression levels of antigenic determinant CD15s produced by FucT-VII and ST3GalIV.

Results

Cell surface expression of sLex recognized by KM93 mAb and selected differentiation markers in B lymphoid cell lines

The results of indirect immunoflowcytometry analyses using mAb KM93 are summarized in Figure 1. Pre-B leukemia cell lines NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, and Reh expressed cell surface sLex epitope more than 85% of the cells. On the other hand, lymphoblastoid cell lines, KJM-LCL and SSK-LCL, and myeloma cell line U266 exhibited only small percentages of positive cells. Burkitt's lymphoma cell lines Ramos and BJAB displayed high percentages of sLex positive cells, while EB virus positive Burkitt's lymphoma cell lines Raji and Daudi were negative or very weakly positive, respectively. We also used the other anti-sLex mAbs, CSLEX-1 and 2H5. KM93-positive pre-B lymphoid cell lines NALL1, Nalm6, KM3, Reh, and BJAB cells were negative or only weakly positive for both CSLEX-1 and 2H5 (data not shown).


Figure 1. Indirect immunoflowcytometry analyses of sLex expression on human B lymphoid cell lines. Cell surface sLex expression on various human B cell lines was analyzed using KM93 mAb. Solid and dotted lines in each panel represent the histograms of KM93-reactive and control cells, respectively. Ordinate and abscissa of each panel represent the cell numbers and the relative fluorescence intensity, respectively.

We characterized expression of several cell surface differentiation markers in B lymphoid cells using anti-CD9, CD10, CD20, CD21, and CD22 mAbs (Table I). CD10, one of the established B cell differentiation marker, was strongly expressed in NALL1, Nalm1, Nalm6, Nalm12, KM3, Reh, Ramos, BJAB, and Daudi cells, while Nalm18 and Raji were weakly positive and Nalm16, KJM-LCL, SSK-LCL, and U266 cells were negative. By contrast, another established B cell marker CD20 was negative or very weakly positive in Nalm6, Nalm12, Nalm16, Nalm18, KM3, Reh, and U266, while Nalm1 cells were moderately positive and NALL1, KJM-LCL, SSK-LCL, Ramos, BJAB, Raji, and Daudi cells were strongly positive. Mature B cell marker CD21 was strongly expressed in Raji and Daudi, while KJM-LCL and SSK-LCL were moderately positive and NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, Reh, Ramos, BJAB, and U266 were negative or only weakly positive. However, CD22 was strongly or moderately positive in all cells except for Nalm6 and U266. Moreover, CD9 was strongly or moderately positive in NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, Reh, KJM-LCL, and SSK-LCL, while Ramos, BJAB, Raji, Daudi, and U266 cells were negative. Consequently, NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, and Reh were thought to have pre-B characteristics. Judging from CD21 reactivity, KJM-LCL, SSK-LCL, Ramos, BJAB, Raji, and Daudi were thought to have mature characteristics compared with NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, Reh, and KM3 cells. Taken together with CD10 and CD21 reactivities, however, maturation stages of the CD21-positive cell lines were tentatively considered as the following order (from immature to mature); Ramos, BJAB, Daudi, Raji, KJM-LCL, and SSK-LCL.

Expression of type1 and type2 N-acetyllactosamine structures and another important selectin ligand sialyl-Lea (sLea) was also examined by flow cytometry analyses using mAbs HMST-1, 2G10, and 1H4, respectively (Table II). Neither sLea antigen nor type1 backbone structures were detected in pre-B lymphoid Nalm12 and KM3 cells, while the control human colon carcinoma cell line DLD-1 expressed both antigens. On the other hand, type2 chains were highly reactive with 2G10 mAb in pre-B cell lines Nalm6, Nalm12, KM3, and Reh.

Table I. Indirect immunoflowcytometry analyses of differentiation marker expression
Cells CD9 CD10 CD20 CD22 CD21 CD43 CD44
NALL1 ++++ ++++ ++++ ++++ ± ++++ ++++
Nalm1 ++++ ++++ +++ ++++ - ++++ ++++
Nalm6 ++++ ++++ - + ± ++++ -
Nalm12 ++++ ++++ - ++++ ± ++++ ++
Nalm16 ++++ ± - +++ - ++++ ++++
Nalm18 +++ ++ + ++++ - ++++ ++++
KM3 ++++ ++++ - ++++ ± ++++ -
Reh +++ ++++ - +++ - ++++ -
KJM-LCL +++ ± ++++ +++ ++ ++++ ++++
SSK-LCL +++ - ++++ ++++ +++ ++++ ++++
Ramos - ++++ ++++ ++++ ++ ++++ ±
BJAB - + ++++ ++++ ++ ++++ -
Raji - ++ ++++ ++++ ++++ - ±
Daudi - ++++ ++++ ++++ ++++ ± ++++
U266 - ± ± ± ± ± ++++
Cell surface expression of selected differentiation markers on various human B cell lines was analyzed using specific mAbs. The expression was shown in a semiquantitative 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%.

Table II. Indirect immunoflowcytometry analyses of differentiation marker expression on human B cell and colon carcinoma cell lines
Cells HMST-1 2G10 1H4
Nalm6 n.d. ++ n.d.
Nalm12 - +++ ±
KM3 - ++++ -
Reh n.d. +++ n.d.
DLD-1 ++++ +++ ++++
Cell surface expression of type1 and type2 N-acetyllactosamine structures and another selectin ligand sLea was analyzed using specific mAbs, HMST-1, 2G10, and 1H4, respectively. The expression was shown in a semiquantitative manner as in Table I. n.d.; not done.

Cell surface sLex is mainly expressed on O-linked oligosaccharides in B lymphoids


Figure 2. Effects of PDMP, Bz-[alpha]-GalNAc, and swainsonine on sLex expression in Nalm6. Cell surface sLex expression was analyzed by indirect immunoflowcytometry using KM93 mAb in Nalm6 cells treated with 20 µM PDMP, 4 mM Bz-[alpha]-GalNAc, and 10 µg/ml swainsonine for 3 days. From top to bottom: no treatment control, PDMP, Bz-[alpha]-GalNAc, swainsonine treatment. Solid and dotted lines in each panel represent the histograms of KM93-reactive and control cells, respectively. Ordinate and abscissa of each panel represent the cell numbers and the relative fluorescence intensity, respectively.

To locate a possible site of cell surface sLex expression recognized by KM93 mAb, Nalm6 cells were treated with inhibitors for sugar chain biosynthesis or processing and then analyzed by flowcytometry (Figure 2). Although the fluorescence intensity peak shifted slightly to the left by d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) treatment, the percentage of KM93 positive cells did not significantly change. On the other hand, KM93 reactive cells were strikingly reduced by benzyl-[alpha]-GalNAc (Bz-[alpha]-GalNAc) treatment. However, swainsonine did not alter the pattern. As control studies using HL-60 cells, it was tested that PDMP and swainsonine were effective to inhibit paragloboside expression and type 2 chain expression, respectively. Furthermore, not only in Nalm6 cells but also in NALL1, Nalm12, and KM3 cells the sLex expression was abolished by Bz-[alpha]-GalNAc treatment. These results suggest that O-linked oligosaccharides are major carriers of the cell surface sLex structures recognized by KM93 mAb in B lymphoid cells.

Cell surface sLex of B lymphoids mediates E-selectin dependent-cell adhesion under low-shear-force condition

To investigate the roles of cell surface sLex structures of B lymphoids, low-shear-stress COS cell adhesion analyses were conducted using E-selectin-transfected COS-1 cells, COS1E5 (Figure 3). First, sLex-positive Nalm6 cells bound to COS1E5 cells, while negative Raji cells did not bind to COS1E5 cells. The Nalm6 cells did not bind to the control COS-1 cells transfected pCR3 vector alone. Subsequently, cell adhesion blocking experiments using specific mAbs were conducted. While the control mAb slightly suppressed Nalm6 cell binding, it was not statistically significant. Comparing with the control, anti-E-selectin (1.2B6) and anti-sLex antibody (KM93) significantly blocked Nalm6 cell adhesion. This indicated that the binding of sLex-positive pre-B leukemia cell line was mediated by sLex on the cell surface and E-selectin expressed on the COS-1 cells. Furthermore, the Nalm6 cell binding was significantly blocked by Bz-[alpha]-GalNAc pretreatment. On the contrary, PDMP or swainsonine pretreatment did not block at all the Nalm6 binding. These demonstrated that the functional sLex structures were located on O-linked rather than N-linked chains the cell surface glycoprotein(s) in pre-B leukemia cell line.


Figure 3. Low shear force COS cell adhesion analyses. B lymphoid cells were treated with or without inhibitor, washed, labeled with BCECF-AM(De Clerck et al., 1994), pretreated with or without anti-sLex mAb (KM93) or the control mAb (mouse anti-IgG; Sigma), and resuspended in unsupplemented RPMI1640 medium. Separately, COS1E5 cells grown on a cover glass in 35 mm dishes (assay plate) were pretreated with or without anti-E-selectin mAb or the control mAb. Then, low-shear-force COS cell adhesion assay was performed (Snapp et al., 1997) as described in Materials and methods. Upper panel, analyses using the COS1E5 cells; lower panel, analysis using the mock-transfected COS1m cells. The values are expressed as averages ± SEM of three experiments.

Semiquantitative reverse transcribed polymerase chain reaction (RT-PCR) analyses of glycosyltransferase expression

Subsequently, semiquantitative RT-PCR analyses were performed to elucidate expression of glycosyltransferases involved in the synthesis of KM93-reactive sLex structure on O-linked oligosaccharides. Among several possible glycosyltransferases, Fuc-TVII, CMP-NeuAc:Gal[beta]1->4GlcNAc [alpha]2->3sialyltransferase ([alpha]2->3ST; ST3Gal IV), UDP-Gal:GlcNAc[beta]1->3Gal [beta]1->4galactosyltransferase ([beta]1->4GalT), and C2GnT were examined as shown in Figure 4. First, internal standard glutalaldehyde-phosphate dehydrogenase (GAPDH) was constitutively amplified and its radioactivity levels were utilized for normalization of glycosyltransferase expression. The signals for Fuc-TVII were varied from cells to cells in spite of the cell surface sLex expression. Namely, Fuc-TVII was strongly amplified in several KM93 reactive cell lines, NALL1, Nalm1, Nalm6, Nalm16, KM3, and Reh, while the signals were weak in the other reactive cell lines, Nalm12, Nalm18, Ramos, and BJAB. In addition, sLex negative cell lines, SSK-LCL, KJM-LCL, Daudi, and U266, exhibited high Fuc-TVII expression, although another negative Raji did not display strong signal of Fuc-TVII. On the other hand, PCR products of ST3Gal IV and [beta]1->4GalT were strongly or moderately amplified in almost all cell lines in spite of their KM93 reactivity. Only one exception was the weak amplification of [beta]1->4GalT PCR product in U266. However, it was suggested that both transferases were constitutively expressed in sLex-positive and negative cell lines. By contrast, it was striking that PCR products of C2GnT were strongly amplified in sLex-positive cell lines, NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, Reh, Ramos, and BJAB, while there were no or only trace amounts of signal in the negative cell lines, KJM-LCL, Raji, Daudi, and U266. Although SSK-LCL exhibited a certain amount of PCR product for C2GnT, the intensity was significantly weaker than those of KM93-positive cell lines.


Figure 4. Quantitative RT-PCR analyses of glycosyltransferase transcript expression in human B lymphoid cell lines. Total RNA was extracted from various cells grown in logarithmic phase. One microgram RNA was reverse-transcribed and one-twentieth volume of the reaction mixture was subjected to PCR reaction using oligonucleotide primers specific to the respective glycosyltransferase cDNAs (Fuc-TVII, [alpha]2->3ST, [beta]1->4GalT, and C2GnT) and the control GAPDH cDNA (see Tables IV and V). PAGE was conducted using one-fifth volume of the reaction mixture, and the bands were visualized by autoradiography.

For fucosyltransferase expression in Nalm12, Nalm18, Ramos, and BJAB cells, we further conducted RT-PCR analyses by increasing the PCR cycle numbers from 27 to 30 using primer pairs for Fuc-TVII. The Fuc-TVII PCR products were clearly detected by increasing the cycle numbers. On the other hand, Fuc-TIV expression was analyzed by PCR with 27 and 30 cycles, and Fuc-TIV was not detected both in 27 and 30 cycle amplification.

Activities of glycosyltransferases that are involved in sLex expression

Subsequently, enzymatic activities of glycosyltransferases involved in sLex and O-glycan core structure synthesis were examined using membranous fractions from B lymphoid cell lines, Nalm6, Reh, Raji, and U266, as summarized in Table III. The results may be rendered nonquantitative by the presence of inhibitory substances and interfering glycosyltransferases that may differ in different cell lines. However, we thought we could obtain the total feature of their activities.

These cell lines exhibitied almost the same level of GDP-Fuc:NeuAc[alpha]2->3Gal[beta]1->4GlcNAc [alpha]1->3fucosyltransferase ([alpha]1->3FucT), and [beta]1->4GalT activities, and there was no significant difference from each other. [alpha]2->3ST activities, however, were relatively low in the KM93-positive Nalm6 and Reh cells, while the negative Raji and U266 displayed relatively or significantly high activities. On the other hand, UDP-GlcNAc:Gal[beta]1->4GlcNAc [beta]1->3N-acetylglucosaminyltransferase (elongation [beta]1->3GlcNAcT) activities were significantly lower in sLex-negative U266 than in the positive Nalm6 and Reh cells. However, the difference of the [beta]1->3GlcNAcT activities between KM93-positive Nalm6 and Reh cells and the negative Raji cells was not statistically significant. By contrast, C2GnT activities in sLex-positive Nalm6 and Reh were significantly higher than those in the negative Raji and U266 cells.

Enzymatic activities of UDP-GlcNAc:GalNAc [beta]1->3N-acetylglucosaminyltransferase (core 3 GlcNAc-transferase, C3GnT) for synthesis of core 3 structure (GlcNAc[beta]1->3GalNAc[alpha]1->Ser/Thr) and UDP-GlcNAc:GlcNAc[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase (core 4 GlcNAc-transferase, C4GnT), for synthesis of core 4 structure (GlcNAc[beta]1->3(GlcNAc[beta]1->6)GalNAc[alpha]1->Ser/Thr), were also assayed in human pre-B leukemia cell lines. As shown in Table III, however, there was no detectable activities for C3GnT and C4GnT in Nalm6 and Reh cells.


Table III. Glycosyltransferase activities in human B cell lines
Activities of glycosyltransferases were measured using respective PA- or PNP-oligosaccharides as acceptors and total membranous fractions as enzyme preparations. The values are expressed as pmol/mg protein/h and as means of triplicate assays ± SD. Statistical analyses were conducted with Student's t test; * indicates p < 0.01. N.D., not detected; n.d., not done.

Comparison of C2GnT activities and C2GnT RT-PCR products in B lymphoid cell lines

The results of the enzymatic activities and semiquantitative RT-PCR analyses for C2GnT were presented comparatively in Figure 5. Nalm6 and Reh cells exhibited 8-25 times higher C2GnT enzyme activities than Raji and U266 cells (Figure 5A). In addition, the RT-PCR products for C2GnT were strongly amplified 8 to 180 times in Nalm6 and Reh cells comparing with Raji and U266 cells (Figure 5B,C). Thus, sLex-positive Nalm6 and Reh cells have more than 8 times higher C2GnT activities and message expression than the negative Raji and U266 cells.

   A

   B

   C

Figure 5. Expression of C2GnT activity and message in human B lymphoid cell lines. Lane 1, Nalm6; lane 2, Reh; lane 3, Raji; and lane 4, U266. (A) Activities of C2GnT were measured using Gal[beta]1->3GalNAc[alpha]1->PNP as acceptors and total membranous fractions as enzyme preparations. The values are representative of three independent assays and expressed as pmol/mg protein/h. (B) Quantitative RT-PCR analyses of C2GnT and PSGL-1 (see Tables IV and V). (C) Quantitative evaluation of the results obtained by BAStation Bio-Image Analyzer. Ordinate shows the relative intensity of the signal with the intensity of Raji set at 1.0. Data shown are representative of three independent analyses.

Analyses of PSGL-1, CD43, and CD44 expression in B lymphoids

In some cases, bioactive carbohydrate structures are expressed on some specific core proteins and could be dependent on expression of such core proteins. PSGL-1 was demonstrated to express sLex structures on its O-linked oligosaccharide chains (Wilkins et al., 1996) and mediate cell adhesion through the sLex structures. So we first investigated the PSGL-1 expression as shown in Figure 5B. Although PCR product for PSGL-1 was strongly amplified in U266 cells, the product was not significantly observed in the other cell lines, Raji, Nalm6, and Reh. It was true that the faint signal and weak signals were detected in Raji and Reh cells, respectively. However, the sizes were a little bit smaller than 1.2 kb and the signals could be nonspecific. Therefore, we could not convincingly conclude that not only U266 but also Nalm6, Reh, and Raji cells express PSGL-1. Second, we analyzed the cell surface expression of heavily O-glycosylated CD43 and moderately O-glycosylated CD44 by indirect immunoflowcytometry as summarized in Table I. CD43 was strongly positive in KM93-positive cell lines, NALL1, Nalm1, Nalm6, Nalm12, Nalm16, Nalm18, KM3, Reh, Ramos, and BJAB, and negative in KM93-negative Raji and Daudi cells. However, sLex-negative KJM-LCL and SSK-LCL cells were shown to express CD43 glycoprotein in their cell surface. On the other hand, CD44 expression was strong in KM93-positive NALL1, Nalm1, Nalm16, and Nalm18 cells, and in the negative KJM-LCL, SSK-LCL, Daudi, and U266 cells, while CD44 was negative or only weakly positive in KM93-positive Nalm6, Nalm12, KM3, Reh, Ramos, and BJAB cells, and in the negative Raji cells.

Detection of anti-sLex mAb-reactive glycoprotein

By Western blotting using KM93 mAb, glycoproteins bearing sLex structure-containing O-linked sugar chain(s) were detected as shown in Figure 6. The strongly positive pre-B lymphoid cell lines, NALL1, KM3, Reh, and Ramos cells exhibited at least one major KM93-reactive glycoprotein on 5% polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (lanes 1-4). The negative B lymphoid cell lines SSK-LCL, Raji, and U266, however, displayed no such protein (lanes 5-7). The molecular size of the major KM93-reactive glycoprotein was about 150 kDa, although the size seemed to vary a little bit from cell line to cell line (lanes 1-4). Due to the Bz-[alpha]-GalNAc pretreatment of NALL1 and Nalm6 cells at 4 mM concentration, the band became faint comparing with the no treatment control (lane 8 versus 9 and lane 10 versus 11). Besides the major ~150 kDa glycoprotein, we could observe several minor glycoproteins that reacted with KM93 antibody. They were ~250 kDa, ~200 kDa (lane 10), ~100 kDa (lanes 1 and 8), and others in molecular size, and disappeared by the Bz-[alpha]-GalNAc treatment. However, no other band was detected even on 10% gel (data not shown).


Figure 6. Detection of anti-sLex mAb-reactive glycoproteins. Proteins of B lymphoid cells treated with or without 4 mM Bz-[alpha]-GalNAc for 4 days were solubilized and subjected to 5% PAGE analyses in the presence of sodium dodecyl sulfate. Immunostaining of sLex antigen-bearing glycoprotein was conducted as described in Materials and methods. Lane 1, NALL1; lane 2, KM3; lane 3, Reh; lane 4, Ramos; lane 5, SSK-LCL; lane 6, Raji; and lane 7, U266 cells. Twenty micrograms of protein were loaded on lanes 1-7. Lane 8, NALL1 treated without Bz-[alpha]-GlcNAc; lane 9, NALL1 treated with Bz-[alpha]-GalNAc; lane 10, Nalm6 treated without Bz-[alpha]-GlcNAc; lane 11, Nalm6 treated with Bz-[alpha]-GalNAc. Thirty micrograms of protein were loaded on the lanes 8 and 9, and 50 µg of protein were loaded on the lanes 10 and 11. Arrows indicate the positions of calibrated and prestained molecular mass standard mixture Kaleidoscope (Bio-Rad; a, myosin 208 kDa; b, [beta]-galactosidase 144 kDa; and c, bovine serum albumin 87 kDa).

Discussion

KM93-reactive sLex antigen was expressed on immature B lymphoids, while it was negative or only weakly positive in the other mature B cell lines (Figure 1, Table I). KM93 has been introduced by Hanai et al. (Hanai et al., 1990) and used for the detection of sLex antigen on human T cells and the expression cloning of Fuc-TVII (Ohta et al., 1993; Sasaki et al., 1994) that was demonstrated as the responsible fucosyltransferase for the E-selectin ligand synthesis during human T cell activation (Knibbs et al., 1996). There are several other established mAbs against sLex including FH-6, SNH-3, 2H5, and CSLEX-1. Expression of CD15s recognized by each mAb is somewhat different from each other in human lymphocytes (Schlossman et al., 1995). In the present study, it was demonstrated that human pre-B lymphoid cell lines were positive for KM93 but negative for CSLEX-1 and 2H5. It is possible that KM93 is different in its recognition epitope from the other antibodies and preferentially reacts with sLex in O-glycans. However, further elucidation would be required.

The sLex structures on B lymphoid cell lines were presented on O-linked oligosaccharides of glycoprotein(s) (Figure 2). In addition, E-selectin-mediated cell adhesion of the pre-B cell line was significantly blocked in the low-shear force COS cell adhesion by inhibiting the O-glycan biosynthesis (Figure 3). Taken together with the Western blot results (Figure 6), sLex structures on the ~150 kDa O-glycosylated protein are suggested to play an important role on E-selectin-dependent adhesion of pre-B lymphoid cells with endothelial cells. It has been reported that O-glycans play important roles on cell adhesion, and multivalency presented by O-glycans is one of the essential characteristics to potentiate cell adhesion strength (Shimizu et al., 1993). The present results support the importance of O-glycans in E-selectin-mediated human B lymphoid cell adhesion.

There are four major O-glycan fundamental structures, core 1, core 2, core 3, and core 4 (Figure 7; Schachter et al., 1989a). They are synthesized through the actions of UDP-Gal:GalNAc[alpha]1->Ser/Thr [beta]1->3galactosyltransferase, C2GnT, C3GnT, and C4GnT. In this study, neither C3GnT nor C4GnT activity was detected in human pre-B lymphoids. However, C2GnT activities were exhibited in B lymphoid cell lines (Table III). These are in a good agreement with the previous report on human leukocytes and leukemia cells (Brockhausen et al., 1991). Moreover, C2GnT activities and transcript were not expressed in sLex-negative mature B lymphoid cell lines but highly expressed in sLex-positive pre-B lymphoids (Table III, Figures 4 and 5). Therefore, sLex antigen may be created on the termini of the N-acetyllactosamine unit repeats extended from [beta]1->6N-acetylglucosamine residue of the core 2 backbone structure(s) in human pre-B lymphoid cell lines. Although we do not exclude the other possibility that the type2 chains are extended from the [beta]1->3Gal residue of core 1 and/or [beta]1->6GlcNAc residue of I antigen structures as well, sLex epitopes on the core 2 backbones appear to play an important role before differentiation of pre-B lymphoid cells into more mature stage.


Figure 7. Schematic presentation of common O-linked oligosaccharide core biosynthesis and possible formation of sLex antigen epitope on the type2 N-acetyllactosamine unit backbone extended from [beta]1->6GlcNAc residue of core 2 sequence. Core 1 [beta]1->3GalT, UDP-Gal:GalNAc[alpha]1->Ser/Thr [beta]1->3galactosyltransferase. In human B lymphoid cell lines, C2GnT was suggested to hold a key role on the biosynthetic control of sLex epitopes recognized by KM93 mAb.

In our present study, C2GnT is suggested to play a critical role on the regulation of sLex expression in pre-B lymphoid cell lines (Figure 4, Table III). Although only weak Fuc-TVII activity was detected in several sLex-positive Nalm12, Nalm18, Ramos, and BJAB cells, Fuc-TVII PCR products were clearly amplified by increasing the PCR cycle numbers from 27 to 30. This suggests the existence and the adequacy of Fuc-TVII transcript expression for the cell surface sLex structure synthesis.

Among known [alpha]2->3sialyltransferases, ST3Gal IV was cloned as the one involved in the biosynthesis of the sLex and the levels of sLex antigens were increased by the transfection of ST3Gal IV in Namalwa KJM-1 cells (Sasaki et al., 1993). In our present study, ST3Gal IV was constitutively expressed in sLex-negative cell lines as well as in the positive cells (Figure 4). Moreover, the enzymatic activities were rather significantly higher in sLex-negative cells than the positive cell lines (Table III). As the expression levels of ST3Gal IV transcripts were not parallel to the enzyme activity levels in sLex-negative cells, these elevated activities may be due to the other [alpha]2->3sialyltransferase(s). Taken together, it appears that ST3Gal IV does not hold a key role in the regulation of expression levels of KM93-reactive sLex antigens in B lymphoid cells.

Expression levels of [beta]1->4GalT transcript and enzyme activity were constitutive throughout B lymphoid cell lines in spite of positive or negative sLex expression (Figure 4, Table III). In addition, enzymatic activities of elongation [beta]1->3GlcNAcT, another glycosyltransferase involved in the synthesis of type2 N-acetyllactosamine units, did not necessarily differ between sLex positive and negative B cell lines. However, the GlcNAc-transferase activities were significantly lower in sLex-negative U266 than the positive Nalm6 and Reh cells, while the KM93-negative Raji cells showed the relatively low activities compared with the positive cell lines (Table III). Therefore, elongation [beta]1->3GlcNAcT may be assisting C2GnT in the control of KM93-reactive sLex expression levels, and further clarification of the role of the elongation [beta]1->3GlcNAcT would be required.

By contrast, C2GnT expression in both transcript levels and enzyme activity correlated well with sLex expression. The expression levels of C2GnT transcript and activity in sLex-positive cells were more than 8 times higher than those in the negative cells (Figures 4 and 5; Table III). These findings taken together with the results of fucosyltransferase and sialyltransferase activities suggest that C2GnT plays a critical role on the control of KM93-reactive sLex expression (Figure 7). C2GnT has been reported to play an important role for PSGL-1 binding to P-selectin in collaboration with the determinative role of Fuc-TVII (Kumar et al., 1996; Li et al., 1996). In addition, sLex structures are present at the termini of O-glycans extended from core 2 backbone structures of leukosialin, CD43 (Maemura et al., 1992). However, sLex expression on PSGL-1 during T cell activation needs simultaneous Fuc-TVII upregulation as well and the molecular size of our sLex antigen determinant-carrier O-glycoprotein (~150 kDa; Figure 6, lanes 1-4 and 8-10) is distinct from the ones of PSGL-1 (110 kDa) and CD43 (135 kDa). Therefore, sLex structures may not be significantly presented on the PSGL-1 on the pre-B cells investigated, and PSGL-1 may not play a significant role in our system. This is in good agreement with a recent report that PSGL-1 is essential for adhesion to P-selectin but not E-selectin (Snapp et al., 1997). Likewise, CD43 may not play a significant role in the E-selectin mediated adhesion of B lymphoid cells. This is supported by the finding that only minor proportions of O-linked oligosaccharides of CD43 glycoprotein have poly-N-acetyllactosaminyl extensions and sLex structures on their termini (Maemura et al., 1992). As the expression of moderately O-glycosylated CD44 did not correlate with cell surface KM93-reactive sLex expression (Figure 1, Table I), all heavily and moderately O-glycosylated glycoproteins may not necessarily bear significant sLex structures in B lymphoids. Our ~150 kDa glycoprotein was distinct from L-selectin, ~76 kDa, and lysosomal membrane glycoproteins (lamp-1 and lamp-2), ~115 kDa in molecular size (Lee et al., 1990; Pilarski et al., 1991). Although we have not yet performed characterization including the accessibility to the E-selectin molecule, the ~150 kDa O-glycoprotein could be a novel E-selectin glycoprotein ligand. The equivalent of mouse E-selectin ligand-1 (ESL-1; Steegmaier et al., 1995) in human hematopoietic cells has not yet been identified. Our ~150 kDa glycoprotein might be one of the ESL-1 equivalents. However, as ESL-1 was reported to be an N-glycosylated protein, extensive identification and elucidation of our ~150 kDa O-glycosylated glycoprotein should be required.

Materials and methods

Cells and cell cultures

Human pre-B leukemia cell line NALL1 was kindly provided by Prof. Isao Miyoshi and Dr. Ichiro Kubonishi (Kochi Medical School, Nankoku, Japan). Human pre-B leukemia cell line Reh was kindly provided by Dr. Naoya Nakamura (Fukushima Medical College, Fukushima, Japan). Human pre-B leukemia cell lines, Nalm1, Nalm12, Nalm18, and KM3 were kindly supplied by Dr. Jun Minowada (Fujisaki Cell Center, Hayashibara Biochemical Research Institute, Fujisaki, Japan) through Drs. Masaki Saito and Masatsugu Ohta (Hokkaido University School of Medicine, Sapporo, Japan). Lymphoblastoid cell lines (LCL) KJM-LCL and SSK-LCL were obtained from Cancer Cell Repository (Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan). Nalm6 cells were kindly provided by Dr. Jun Minowada through Prof. N. Sakaguchi (Kumamoto University, Kumamoto, Japan). Human Burkitt's lymphoma cell lines Ramos, Raji, and Daudi, were from Human Science Research Resource Bank (HSRRB, Osaka, Japan). Human myeloma cell line U266 was kindly supplied by Dr. Y. Yanagihara (National Sagamihara Hospital, Sagamihara, Japan). Daudi was cultured in RPMI-1640 medium supplemented with 20% fetal calf serum. The other B lymphoid cell lines and human colon carcinoma cell line DLD-1 cells (from HSRRB) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum.

Chemicals

CMP-[sialic-4,5,6,7,8,9-14C]NeuAc (300.9 mCi/mmol), GDP-[fucose-(U)-14C]Fuc (273 mCi/mmol), and UDP-[glucosamine-1-14C]GlcNAc (60 mCi/mmol) were obtained from New England Nuclear (Boston, MA, USA). Unlabeled GDP-fucose (GDP-Fuc) was from Wako Pure Chemicals (Osaka, Japan). Unlabeled CMP-NeuAc, UDP-Gal, and UDP-GlcNAc were from Sigma (St. Louis, MO). Pyridylaminated (PA) oligosaccharides, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc-PA (nLcOse4-PA) and NeuAc[alpha]2->3Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc-PA (IV3NeuAc-nLcOse4-PA), were prepared as described previously (Kondo et al., 1990) using nLcOse4 and IV3NeuAc-nLcOse4 that were prepared by endoglycoceramidase (Ito et al., 1989) from nLcOse4Cer and IV3NeuAc-nLcOse4Cer, respectively. nLcOse4Cer and IV3NeuAc-nLcOse4Cer were purified from human erythrocytes. GlcNAc[beta]1->3Gal[beta]1->4Glc-PA was prepared from nLcOse4-PA by [beta]-galactosidase digestion as described previously (Shigeta et al., 1987). p-Nitrophenyl oligosaccharides, Gal[beta]1->3GalNAc[alpha]1->PNP, Gal[beta]1->3(GlcNAc[beta]1->6)GalNAc[alpha]1->PNP, GalNAc[alpha]1->PNP, GlcNAc[beta]1->3GalNAc[alpha]1->PNP, were obtained from Toronto Research Chemicals (Toronto, Canada). PDMP was prepared as described previously (Inokuchi and Radin, 1987). Guanidinium thiocyanate was purchased from Fluka (Buchs, Switzerland) and cesium chloride was from Nakarai Tesque (Kyoto, Japan). All other reagents were of the highest grade commercially available.

Indirect immunoflowcytometry analyses

Indirect immunofluorescence analyses of cell surface differentiation antigen expression were carried out by FACScan (Becton-Dickinson) as described previously (Nakamura et al., 1992). mAbs used against differentiation antigens were DU-ALL-1 (CD9; Sigma), OKBcALLa (CD10; Ortho Diagnostics Systems, Tokyo, Japan), KM93 (CD15s; Seikagaku, Tokyo, Japan), CSLEX-1 (CD15s; ATCC HB8580), 2H5 (CD15s; Pharmingen, San Diego, CA), B9E9 (CD20; Sigma), WEHI-B2 (CD21; Japan Turner, Suita, Japan), 4KB128 (CD22; Dako Japan, Kyoto, Japan), DF-T1 (CD43; Sigma), A3D8 (CD44; Sigma), and 1H4 (sLea; Seikagaku, Tokyo, Japan). Anti-paragloboside mAb 1B2 was obtained from the hybridoma (ATCC TIB189). Anti-Gal[beta]1->4GlcNAc mAb 2G10 and anti-Gal[beta]1->3GlcNAc mAb HMST-1 were kindly supplied by Prof. J. Hata (Keio University, Tokyo, Japan) and Prof. S. Nozawa (Keio Univ., Tokyo, Japan), respectively. The second antibody was FITC-conjugated goat F(ab[prime])2 anti-mouse IgG plus IgM (Tago, Inc., Burlingame, CA). Mouse anti-IgG mAb (IgM) was obtained from Sigma (St. Louis, MO) and used as a control first antibody.

Inhibition of sugar chain biosynthesis

Biosynthesis of glycolipid sugar chains was inhibited by culturing the cells in the presence of 20 µM PDMP for 3 days. For inhibition of O-linked sugar chain biosynthesis, the cells were cultured with 4 mM Bz-[alpha]-GalNAc (Sigma, St. Louis, MO) for 3 days (Kuan et al., 1989). Inhibition of N-linked oligosaccharide processing was conducted by culturing the cells with 10 µg/ml swainsonine (Genzyme, Cambridge, MA) for 3 days (Elbein et al., 1982).

Preparation of E-selectin-transfected COS-1 cells

E-Selectin-transfected COS-1 cells were prepared by introducing human full length E-selectin cDNA using electroporation technique followed by G418 selection and limited dilution as described previously (Tsunoda et al., 1995). The cDNA was cloned by RT-PCR method using total RNA from human umbilical vain endothelial cells activated with recombinant IL-1[beta] at 10 U/ml concentration for 4 h. In the reverse transcription step, SuperScript II reverse transcriptase (Life Technologies Inc., Gaithersburg, MD) and oligo(dT) primer (Pharmacia, Upsala, Sweden) were used. The sequences of the specific primers for human E-selectin cDNA (Bevilacqua et al., 1989) and the PCR conditions were summarized in Tables V and V. The amplified cDNA was directly subcloned to pCR3 mammalian expression vector (Invitrogen, Carlsbad, CA) and a sense oriented clone was chosen and designated as pCR3-E-selectin. E-Selectin expression in the monoclonal transfectants was confirmed by indirect immunoflowcytometry and RNA blot analyses, and the clone with the highest E-selectin expression was designated as COS1E5 cells.

Low-shear-force COS cell adhesion assay

E-Selectin-dependent cell adhesion of B lymphoid cell lines were evaluated by low-shear-force COS cell adhesion assay with modification (Snapp et al., 1997). Lymphoid cells were labeled with BCECF-AM as described previously (De Clerck et al., 1994) and contacted on a constantly rocking platform for 15 min at 4°C with COS1-E5 cells grown on a cover glass in 35 mm dishes (assay plate). After washing five times and fixation, the cover glass was removed from the plate and placed on a slide glass. The fluorescence-labeled cells were counted on a fluorescence microscope system (BX-60/BX-FLA; Olympus, Tokyo, Japan) and the mean number of cells bound to COS1E5 cells per 1 cm2 was determined. Before the assay, B lymphoid cells were grown in the presence or absence of 4 mM Bz-[alpha]-GalNAc for 3 days and/or pretreated with or without anti-sLex mAb (KM93; 50 µg/ml at final concentration) or the control mAb (mouse anti-IgG; 50 µg/ml at final concentration; Sigma). Separately, COS1E5 cells were pretreated with or without anti-E-selectin mAb (1.2B6; 50 µg/ml; T Cell Diagnostics, Cambridge, MA) or the control mAb (mouse anti-IgG; 50 µg/ml; Sigma) before the assay.

Semiquantitative reverse transcribed-PCR analysis

Semiquantitative RT-PCR analysis was conducted as described previously with slight modification (Furukawa et al., 1996). Briefly, 1 µg RNA, which was extracted by guanidinium-cesium chloride method from various B lymphoid cells grown in logarithmic phase, was reverse-transcribed by SuperScript II using oligo(dT) primer. One-twentieth volume of the reaction mixture was subjected to PCR reaction (total 50 µl). Conditions of the reactions and the nucleotide sequences of the primers were summarized in Tables IV and IV. PAGE (5%) was carried out in Tris borate-EDTA buffer at 50 V using one-fifth volume (10 µl) of the PCR reaction mixtures, and the signals were visualized by autoradiography. The results were quantitated with a BAStation Bio-Image Analyzer (Fuji Film, Tokyo, Japan). The PCR cycle numbers were determined by the extensive control experiments for the respective primer pairs so that the amplification efficiency remained constant and the amplified PCR product was directly proportional to the quantity of the used RNA.

Table IV. Summarized data of quantitative PCR analysis conditions
PCR conditions E-Selectin Fuc-TVII ST3GalIV [beta]1->4GalT C2GnT PSGL-1 GAPDH
Tris-HCl buffer 60 mM ->a -> -> -> -> ->
pH 9.0 9.0 10.0 9.0 10.0 9.0 8.5
MgCl2 (mM) 1.5 2.0 2.0 1.5 2.0 1.5 1.5
(NH4)2SO4 15 mM ->a -> -> -> -> ->
dATP 250 µM ->a -> -> -> -> ->
dGTP 250 µM ->a -> -> -> -> ->
dTTP 250 µM ->a -> -> -> -> ->
dCTP 250 µM None ->a -> -> -> ->
[[alpha]-32P]dCTP None 2 µCi ->a -> -> -> ->
Primers (µM each) 10 ->a -> -> -> -> ->
Cycle numbers 32 27 27 25 26 26 20
a->, same as left.

Table V. Nucleotide sequences of the primer pairs used in this study
cDNA Forward primer Reverse primer
E-Selectina 5[prime]-aag-tca-tga-ttg-ctt-cac-agt-tt-3[prime] 5[prime]-aac-tta-aag-gat-gta-aga-agg-c-3[prime]
Fuc-TVIIb 5[prime]-atg-tct-ttg-gcc-gtg-cca-atg-gac-3[prime] 5[prime]-agc-gga-tct-cag-gcc-tga-aac-caa-3[prime]
ST3Gal IVc 5[prime]-aca-cac-tcc-tcg-tcc-tgg-tag-ct-3[prime] 5[prime]-cta-cag-ctc-ttg-ccc-agg-tca-gaa-3[prime]
[beta]1->4GalTd,e 5[prime]-caa-gaa-gcc-ttg-aag-gac-tat-g-3[prime] 5[prime]-aaa-acg-cta-gct-cgg-tgt-ccc-gat-3[prime]
C2GnTf 5[prime]-gca-atg-agt-gca-aac-tgg-aag-t-3[prime] 5[prime]-aat-tgc-ccg-taa-tgg-tca-gtg-tt-3[prime]
PSGL-1g 5[prime]-tgg-tgc-cat-gcc-tct-gca-act-cct-3[prime] 5[prime]-tga-gct-aag-gga-gga-agc-tgt-gca-3[prime]
GAPDHh 5[prime]-cca-ccc-atg-gca-aat-tcc-atg-gca-3[prime] 5[prime]-tct-aga-cgg-cag-gtc-agg-tcc-acc-3[prime]
aBevilacqua et al., 1989.
bSasaki et al., 1994.
cSasaki et al., 1993.
dMasri et al., 1988.
eNakazawa et al., 1993.
fBierhuizen et al., 1992.
gSako et al., 1993.
hMaier et al., 1990.

Table VI. Summary of assay condition for glycosyltransferases
Conditions Donor Radiolabel (nmol) Acceptor (nmol) Buffer (µmol, pH) Divalent cation (µmol) Detergent (µg) Enzyme prep. (µg) Others (µmol) HPLC column
[alpha]1->3FucT GDP-Fuc +, 1.88 PA-OLSa (20) Sod. cacodylate (3.75, 6.8) MnCl2 (0.63) Triton CF-54 (75) 100-300   ODS-80TMe
[alpha]2->3ST CMP-NeuAc +, 5.0 PA-OLSa (20) Sod. cacodylate (3.75, 6.5) None Triton CF-54 (75) 100-300   ODS-80TMe
[beta]1->4GalT UDP-Gal -, 7.5 PA-OLSa (20) Sod. cacodylate (3.75, 6.8) MnCl2 (0.25) Sod. deoxych. (2.5) 100-200   ODS-80TMe
Elongation [beta]1->3GlcNAcT UDP-GlcNAc -, 0.5 PA-OLSa (20) MOPSc (5.0, 7.5) MnCl2 (0.5) Triton X-100 (125) 100-200   ODS-80TMe
C2GnT UDP-GlcNA +, 25 PNP-OLSb (25) MESd (3.75, 7.0) None Triton X-100 (25) 100-300 GlcNAc (2.5)Sod. EDTA (0.25) PALPAK(N)f,g
C3GnT UDP-GlcNA +, 25 PNP-OLSb (25) MESd (3.75, 7.0) None Triton X-100 (25) 100-300 GlcNAc (2.5)Sod. EDTA (0.25) PALPAK(N)h
C4GnT UDP-GlcNA +, 25 PNP-OLSb (25) MESd (3.75, 7.0) None Triton X-100 (25) 100-300 GlcNAc (2.5)Sod. EDTA (0.25) PALPAK(N)h
aPA-OLS, Pyridylaminated oligosaccharide.
bPNP-OLS, p-Nitrophenyl oligosaccharide.
cMOPS, 3-(N-Morpholino)propanesulfonic acid buffer.
dMES, 2-(N-Morpholino)ethanesulfonic acid buffer.
eNakamura et al., 1992.
fBierhuizen et al., 1992.
gSchachter et al., 1989b.
hBrockhausen et al., 1991.

Glycosyltransferase assays

The total membranous fractions were prepared, aliquoted, and stored at -80°C until use for glycosyltransferase assays as described previously (Nakamura et al., 1991). The assay conditions for each glycosyltransferase activity are summarized in Table VI.

[alpha]1->3FucT activities were assayed essentially by the method described for nLcOse4 [beta]1->3GlcNAc-transferase assay (Nakamura et al., 1992) with modification. GDP-[fucose-(U)-C14]Fuc (5.4 µl) was added in a microtube and then dried. GDP-Fuc, acceptor substrate NeuAc[alpha]2->3Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc-PA, sodium cacodylate buffer, MnCl2, Triton CF-54, and the enzyme preparation were mixed and incubated at 37°C for 1-3 h in a total volume of 25 µl. The reaction was stopped by heating at 100°C for 2 min. The samples were then passed through a 0.22 µm Millipore filter, and an aliquot of the each filtrate was applied to a TSK gel ODS-80TM column (4.6 × 150 mm, TOSOH, Tokyo, Japan) and separated by HPLC. Elution was performed at 50°C with a 0.1 M acetate buffer (pH 4.0) containing 0.15% n-butanol at a flow rate of 1.0 ml/min. The product was separated with fraction collector, and the transfer of the radioactive Fuc to the acceptor substrate was determined by liquid scintillation spectrometry.

[alpha]2->3ST activities were determined as follows. CMP-[sialic-4,5,6,7,8,9-14C]NeuAc (2.5 µl) was added in a microtube and then dried. Cold CMP-NeuAc, acceptor substrate Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc-PA, sodium cacodylate buffer, Triton CF-54, and the enzyme preparation were mixed and incubated at 37°C for 1-3 h in a total volume of 25 µl. The reaction products were processed as [alpha]1->3FucT assays, and separated by HPLC system with fraction collector. The transfer of the radioactive NeuAc to the acceptor was determined by liquid scintillation spectrometry.

[beta]1->4GalT activities were assayed as follows. Cold UDP-Gal, acceptor substrate GlcNAc-Gal[beta]1->4Glc-PA, sodium cacodylate buffer, sodium deoxycholate, MnCl2, and the enzyme preparation were mixed and incubated at 37°C for 1-3 h in a total volume of 25 µl. The reaction products were processed to HPLC analyses as [alpha]1->3FucT assays. The eluates were subjected to quantitation by fluorescence intensity using pyridylaminated Gal as a standard.

Elongation [beta]1->3GlcNAcT activities were measured as described earlier (Nakamura et al., 1992).

C2GnT activities were determined essentially as described previously (Bierhuizen et al., 1992). Briefly, UDP-[glucosamine-1-14C]GlcNAc (0.4 nmol), cold UDP-GlcNAc (24.6 nmol), Gal[beta]1->3GalNAc[alpha]1->PNP, and GlcNAc were added in a microtube and then dried. 2-(N-Morpholino)-ethanesulfonic acid buffer, sodium EDTA, Triton X-100, and the enzyme preparation were mixed and incubated in a total 25 µl at 37°C for 1-3 h. The reaction product was purified by C18 Sep-Pak (Waters, Milford, MA) column chromatography and analyzed by HPLC on a column (0.46 × 25 cm) of PALPACK (type N, TAKARA, Kyoto, Japan) taking Gal[beta]1->3(GlcNAc[beta]1->6)GalNAc[alpha]1->PNP as a standard. The column was developed isocratically with acetonitrile/water, 83:17 (vol/vol) under the conditions described previously (Schachter et al., 1989b).

C3GnT and C4GnT activities were determined as described for C2GnT except for the acceptor substrates, GalNAc[alpha]1->PNP and GlcNAc[beta]1->3GalNAc[alpha]1->PNP, respectively. The retention time of GlcNAc[beta]1->3(GlcNAc[beta]1->6)GalNAc[alpha]1->PNP was determined using the enzymatic reaction product that was generated from the assay using GlcNAc[beta]1->3GalNAc[alpha]1->PNP as an acceptor substrate and porcine colonic mucosal tissue as an enzyme source (Brockhausen et al., 1985).

Western blotting analysis

The cells were solubilized in 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethansulfonic acid buffer (pH 7.2) containing 2% Triton X-100 by brief sonication. The solubilized protein was finally suspended in Laemmli's sample buffer, then 30 µg of the protein was heated at 100°C for 5 min and subjected to 5% or 10% PAGE analysis in the presence of sodium dodecyl sulfate. After transfer to an Immobilon-Psq polyvinylidene fluoride membrane (Millipore, Bedford, MA) by Transblot SD cell (Bio-Rad, Richmond, CA), the membrane was blocked with phosphate-buffered saline without Ca2+ (PBS) containing 0.01% Tween 20 (T-PBS) and 1% bovine serum albumin at 4°C overnight, incubated with KM-93 (anti-sLex) mAb, washed three times with T-PBS, and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgM in T-PBS. Detection of HRP was carried out with the ECL Western blotting reagents (Amersham, UK).

Protein assay

Protein was determined by an Amido-Schwarz dye-binding method (Schaffner and Weissmann, 1973) with bovine serum albumin as a standard.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas No. 05274105 and No. 09240229, and by a Grant for General Scientific Research No. 09670161 from the Ministry of Education, Science and Culture, Japan, and by Research Grant 95KI033 from Ichiro Kanehara Research Foundation. We are indebted to Drs. I. Miyoshi, I. Kubonishi, N. Nakamura, J. Minowada, M. Saito, M. Ohta, N. Sakaguchi, Y. Yanagihara, J. Hata, and S. Nozawa for generous gift of materials. We thank Dr. Akira Makita (Honorary Prof. of Hokkaido University School of Medicine and Prof. of Hokkaido Bunkyo Junior College) and Dr. Masaki Saito for their continuous encouragement, Dr. Hiroshi Nakada (Prof. of Kyoto Sangyo University) for his valuable comments, and Ms. Yukiko Fukuda and Taeko Inageta for their technical assistance.

Abbreviations

sLex, sialyl-Lex (sialylated Lewis antigen with Gal[beta]1->4GlcNAc backbone), sLea, sialyl-Lea (sialylated Lewis antigen with Gal[beta]1->3GlcNAc backbone); PA, pyridylamine; PNP, p-nitrophenyl; PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; Bz-[alpha]-GalNAc, benzyl-[alpha]-GalNAc; mAb, monoclonal antibody; RT-PCR, reverse transcribed-PCR; PSGL-1, P-selectin glycoprotein ligand-1; GAPDH, glutalaldehyde-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; T-PBS, PBS containing 0.1% Tween 20; HRP, horseradish peroxidase. Fucosyltransferases and sialyltransferases are designated according to the recommendations (Breton et al., 1996; Tsuji et al., 1996), respectively. Sugar sequences and glycosphingolipids are designated according to the Nomenclature Committee of the IUPAC (Recommendations of IUPAC-IUB Commission on Biochemical Nomenclature, 1977). Paragloboside, nLcOse4Cer, Gal[beta]1->4GlcNAc[beta]1->3Gal[beta]1->4Glc[beta]1->Cer; Core2GnT or C2GnT, UDP-GlcNAc:Gal[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase; C3GnT, UDP-GlcNAc:GalNAc [beta]1->3N-acetylglucosaminyltransferase; C4GnT, UDP-GlcNAc:GlcNAc[beta]1->3GalNAc (GlcNAc to GalNAc) [beta]1->6N-acetylglucosaminyltransferase; [beta]1->4GalT, UDP-Gal:GlcNAc[beta]1->3Gal [beta]1->4galactosyltransferase; [alpha]1->3FucT, GDP-Fuc:NeuAc[alpha]2->3Gal[beta]1->4GlcNAc [alpha]1->3fucosyltransferase; [alpha]2->3ST, CMP-NeuAc:lactoneotetraose [alpha]2->3sialyltransferase; elongation [beta]1->3GlcNAcT, UDP-GlcNAc:nLcOse4 [beta]1->3N-acetylglucosaminyltransferase.

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10To whom correspondence should be addressed at: Division of Hemopoiesis, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan.


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