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.
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
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.
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
-
±
±
±
±
±
++++
Table II.
Cells | HMST-1 | 2G10 | 1H4 |
Nalm6 | n.d. | ++ | n.d. |
Nalm12 | - | +++ | ± |
KM3 | - | ++++ | - |
Reh | n.d. | +++ | n.d. |
DLD-1 | ++++ | +++ | ++++ |
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
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
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
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.
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
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 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
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).
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
The sLex structures on B lymphoid cell lines were presented on O-linked oligosaccharides of glycoprotein(s) (Figure
There are four major O-glycan fundamental structures, core 1, core 2, core 3, and core 4 (Figure
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
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
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
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 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.
Table V.
Table VI.
Table III.
A
B
C
Discussion
Materials and methods
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
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]
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
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.
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.
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.
10To whom correspondence should be addressed at: Division of Hemopoiesis, Jichi Medical School, Minamikawachi, Tochigi 329-0498, Japan.