Sialic acid (N-acetyl neuraminic acid (Neu5Ac) or other N- and O-substituted neuraminic acids) is the most abundant terminal monosaccharide on the surface of eukaryotic cells. By virtue of their widespread distribution, their structural versatility, and their peripheral position on oligosaccharide chains of glycoconjugates, sialic acids are well suited as molecular determinants of specific biological processes, including the interaction of pathogenic microorganisms with sialylated cellular receptors (Varki, 1997), various types of cell-cell interactions (Yednock and Rosen, 1989) and T-cell activation (Powell et al., 1987; Varki, 1992). Also, cell surface sialylation has been implicated in the tumorigenicity and metastatic behavior of tumor cells, involving for example tumor cell-mediated platelet aggregation (Kijima-Suda et al., 1988) or invasive potential (Collard et al., 1986).
The differentiation of B and T lymphocytes is associated with changes in the glycosylation, and in particular sialylation, of surface differentiation antigens. The expression of B cell differentiation antigens CDw75, CDw76, HB-6, HB-4, and EBU-65, found predominantly on mature immunoglobulin (Ig)-positive B cells in lymphoid secondary follicles (Schwartz-Albiez et al., 1995), critically depends on the presence of [alpha]-2,6-linked sialic acids. The generation of this group of [alpha]-2,6-sialylated B cell differentiation antigens in COS-7 and LTK- cells has been shown to be dependent on the activity of the Golgi [beta]-galactoside [alpha]-2,6-sialyltransferase (ST6Gal I; Gal[beta]1-4GlcNAc-specific) (Keppler et al., 1992; Bast et al., 1992; Schwartz-Albiez et al., 1995). The B lymphocyte-specific adhesion and signaling molecule CD22 (Siglec-2) (Tedder et al., 1997), which is involved in the regulation of B cell activation, is a lectin specific for [alpha]-2,6-sialoglycans. In one report, CD22 has been suggested to interact in particular with CDw75 (Stamenkovic et al., 1991), but other [alpha]-2,6-linked B cell antigens may also serve as partners for B-B cell interactions (Powell and Varki, 1995).
The cell surface sialoglycoprotein CD95 (APO-1/Fas) is a member of the tumor necrosis factor/nerve growth factor receptor superfamily, and it transduces apoptotic signals into apoptosis-sensitive cells. Apoptosis is an important and well-controlled form of receptor-mediated cell death which occurs under a variety of physiological and pathological conditions (Cleveland and Ihle, 1995). Apoptosis represents a physiological mechanism that essentially contributes to the homeostasis of the organism. Inappropriate apoptosis appears to be involved in many disorders, including immune deficiencies and various malignancies (Carson and Ribeiri, 1993; Barr and Tomei, 1994). Thus, understanding the molecular mechanisms underlying the cellular control of apoptosis and its dysregulation are of pivotal importance. CD95 has an apparent molecular mass ranging from 48 to 59 kDa and in many cell types is highly sialylated (Oehm et al., 1992; Peter et al., 1995). Sialic acid residues on CD95 are present on N- and O-linked oligosaccharide chains and can account for up to 8 kDa of its apparent molecular mass. The possibility that CD95 sialylation is functionally important was raised by Peter et al., in which removal of terminal sialic acids by sialidase augmented sensitivity to apoptosis induction in various human B and T cell lines (Peter et al., 1995).
Sialic acid is also an essential component of functional cell surface receptors of many different viruses including the B lymphotropic papovavirus (LPV) (Keppler et al., 1995, and references therein). The host range of LPV in cultured human cells is tightly restricted to B lymphoma-derived cell lines, to a large extent Burkitt's lymphomas (zur Hausen et al., 1980; Haun et al., 1993). The LPV cell surface receptor appears to be a major factor in host range restriction (Haun et al., 1993; Herrmann et al., 1995). Although the molecular identity of the receptor is still unknown it is thought to be an [alpha]-2,6 sialylated O-linked glycoprotein (Haun et al., 1993; Keppler et al., 1994, 1995). The exceptional receptor-mediated tropism makes LPV an interesting candidate for the development of a virus-based vector for specific gene transfer into B lymphoma cells. Yet a more profound understanding of factors influencing the LPV tropism remains of crucial importance.
In this study, using subclones of the human B lymphoma cell line BJA-B, we show that differential cellular sialylation of glycoconjugates can drastically influence and regulate the functions of viral and signal transducing receptors.
BJA-B subclones differ in the cell surface expression of [alpha]-2,6-sialylated epitopes but not in the overall expression of B cell-associated antigens
In an attempt to phenotypically characterize the different subclones derived from the clonal human B lymphoma cell line BJA-B, we investigated the cell surface expression of B cell-associated antigens using flow cytometry. A panel of 38 mAb was used, which allows the characterization of a mature human B cell (Möller and Mielke, 1989). Initial experiments suggested that the subclones displayed a very similar binding pattern for most of the antibodies tested (not shown). However, on the basis of a very different expression of the CDw75 and CDw76 differentiation antigens, BJA-B subclones were subdivided into two groups: K88 and K43 in group I with concordant high expression levels, and K20, K4, K5, K6, K9, and K138 in group II with low expression levels. Data of one representative member of each group, namely subclones K88 and K20, are shown in Table I. Of 38 tested antibodies, 31 showed similar binding to both subclones; the median fluorescence intensities of K20 and K88 cells never differed more than 2.2-fold, with a mean similarity of 93.4 ± 34.2% (Table I, middle and bottom of right column).
Clear differences in the cell surface expression were only found for five B cell differentiation antigens, namely CDw75, CDw76, HB-4, HB-6, and EBU-65, the recognition of which is known to require [alpha]-2,6-linked sialic acid residues (Keppler et al., 1992; Bast et al., 1992; Schwartz-Albiez et al., 1995). K20 cells displayed a marked reduction in the expression of these sialoglycans, ranging from 5 to 23% of the level expressed on K88 cells (mean 12.1 ± 6.5%, Table I, top of right column, boldface numbers; and FigureTable I.
Antigen | mAb | BJA-B K88 | BJA-B K20 | K20/K88b (%) |
(median fluorescence intensity)a | ||||
CDw75 | HH2 | 470 | 34 | 7 |
CDw75 | LN1 | 1673 | 182 | 11 |
CDw75 | EBU-141 | 473 | 23 | 5 |
CDw76 | HD66 | 796 | 47 | 6 |
Unclustered | HB-4 | 1617 | 318 | 20 |
Unclustered | HB-6 | 251 | 58 | 23 |
Unclustered | EBU-65 | 153 | 20 | 13 |
CD10 | J5 | 27 | 25 | 93 |
CD19 | HD37 | 622 | 402 | 65 |
CD20 | MEM97 | 2702 | 1957 | 72 |
CD21 | HB-5 | 23 | 25 | 109 |
CD22 | HD239 | 191 | 113 | 59 |
CD23 | HD50 | 19 | 18 | 95 |
CD25 | Anti-Tac | 19 | 18 | 95 |
CD32 | KB61 | 25 | 27 | 108 |
CD37 | HD28 | 865 | 459 | 53 |
CD38 | T16 | 785 | 542 | 69 |
CD39 | AC2 | 18 | 29 | 161 |
CD40 | G28-5 | 291 | 210 | 72 |
CD44 | HK23 | 18 | 21 | 117 |
CD45RA | 4KB5 | 18 | 27 | 150 |
CD54 | LB2 | 112 | 96 | 86 |
CDw60 | UM4D4 | 11 | 10 | 91 |
CD71 | PA-1 | 64 | 47 | 73 |
CD72 | SHCL-2 | 69 | 55 | 80 |
CD74 | BU-45 | 26 | 22 | 85 |
CD77 | 424/3D99 | 564 | 653 | 116 |
CDw78 | Leu21 | 24 | 20 | 83 |
CD80 | BB1 | 33 | 74 | 224 |
CD86 | NU-B1 | 230 | 154 | 67 |
HLA-A,B,C | W6/32 | 1700 | 1930 | 114 |
HLA-DR | ISCR3 | 900 | 418 | 46 |
HLA-DP | B7/21 | 113 | 93 | 82 |
HLA-DQ | Tü22 | 28 | 23 | 82 |
IgM (H) | NMZ88.5 | 896 | 517 | 58 |
Unclustered | M9 | 719 | 716 | 100 |
Unclustered | BL4 | 23 | 18 | 78 |
Unclustered | MB2 | 19 | 21 | 111 |
Different cellular sialic acid levels in BJA-B subclones
Figure 1. Differential cell surface expression of various sialylated B cell differentiation antigens in BJA-B subclones. Overlays of cytofluorometric histograms of BJA-B subclones stained for the sialic acid-dependent B cell differentiation antigens CDw75 (mAb HH2), CDw76 (mAb HD66), EBU-65, and HB-6 (Schwartz-Albiez et al., 1995). Histograms for BJA-B K20 cells are hatched. K88 cells were pretreated with either PBS (open histogram, continuous line) or Vibrio cholerae sialidase (open histogram, broken line). Irrelevant mAb HD20, and mAb W6/32 which detects the HLA-A, -B, and -C antigens, served as negative and positive controls, respectively (bottom panel). We then addressed the question of whether the observed difference in the expression of several [alpha]-2,6-sialylated B cell epitopes on BJA-B subclones was possibly a result of a generalized difference in the sialic acid content of these cells. As a first step, we quantified the sialic acid concentrations of subclones by the thiobarbituric acid method and confirmed these results by HPLC analyses. The sialic acid contents of the group I cells, K88 and K43, were 3.1 and 3.7 nmol Neu5Ac/107 cells, respectively (Table II). Group II cells, K20 and K6, however, contained only 1/3 of this amount: 1.0 and 1.1 nmol Neu5Ac/107 cells, respectively. The sialylation phenotype of the parental BJA-B cell line (3.3 nmol Neu5Ac/107 cells) was in the range of highly sialylated group I cells. This is in agreement with our finding during subcloning that cells with a group II phenotype occur with a low frequency of only about 1 in 100 cells in the parental cell population (M.Pawlita, M.Oppenländer, G.Haun, and O.T.
Keppler, unpublished observations), thus contributing only to a very small extent to the parental sialylation phenotype. The different sialic acid content determined for group I and II cells (Table II) cannot be attributed to a difference in the overall glycoprotein expression of the cells, since quantifications of total protein per cell yielded almost identical results for all subclones. In addition, the expression of highly abundant cell surface glycoproteins such as the HLA-A, -B, and -C antigens was comparable (Table I, Figure
Table II.
Cells/Groupa
Neu5Acb (nmol/107 cells (%))c
MAA bindingd (%)
BJA-B
3.3 ± 0.6 (100)
100
BJA-B K43/I
3.7 ± 0.7 (112)
104
BJA-B K88/I
3.1 ± 0.5 (94)
104
BJA-B K20/II
1.0 ± 0.3 (30)
26
BJA-B K6/ II
1.1 ± 0.3 (33)
25
Reduction of [alpha]-2,3-linked sialic acids on group II cells
Next we determined whether the overall reduction in glycoconjugate-bound sialic acid affected also [alpha]-2,3-linked sialic acid residues, which are the most abundant sialic acid linkages on mammalian cells. BJA-B subclones were analyzed for their binding capacity of Maackia amurensis agglutinin (MAA), which detects sialic acid residues in [alpha]-2,3-linkage to Gal (Wang and Cummings, 1988). The MAA binding level of K20 and K6 cells was reduced by 74-75% as compared to group I cells K43 and K88 (Table II), demonstrating a strong reduction also of [alpha]-2,3-sialylation on group II glycoconjugates.
Sambucus nigra agglutinin (SNA) binds to sialic acid residues in [alpha]-2,6-linkage to d-N-acetylgalactosamine (GalNAc) or d-galactose (Gal) (Shibuya et al., 1987), but has also been shown to react with terminal Gal and GalNAc residues (Taatjes et al., 1988). The latter reactivity made a relative quantification of [alpha]-2,6-linkages on group I and II cells impossible, since even sialidase-pretreated K20 and K88 cells bound high amounts of SNA, while their binding of MAA and of Limax flavus agglutinin (LFA), which detects sialic acid residues irrespective of their linkage (Knibbs et al., 1993) were almost completely abolished (see also below).
Level of penultimate saccharide residues on cell surface glycoconjugates of BJA-B subclones
Figure 2. Expression of penultimate saccharide residues on cell surface glycoconjugates of BJA-B subclones. Overlays of cytofluorometric histograms of BJA-B subclones stained with either (A) FITC-conjugated lectins SBA, VVA, ConA, or TRITC-conjugated lectin PNA with the indicated saccharide specificity; or (B) with mAb 1B2-1B7 which detects the nonsialylated carbohydrate sequence Gal[beta]1-4GlcNAc-R on N-glycosyl type II chains (Young et al., 1991). MAb W6/32 served as a control. Analyses for BJA-B K88 cells (overlays, top panels), either PBS-treated (continuous line) or sialidase-pretreated (broken line) and untreated BJA-B K20 cells (lower panels) are shown. Numbers indicate mean fluorescence intensities.
To further define the glycosylation of cell surface glycoconjugates in BJA-B subclones, we analyzed their binding capacities for several fluorochrome-coupled lectins and one anti-carbohydrate antibody with specificities for different mono- or disaccharides. In particular the amount and type of saccharides found at the penultimate position (relative to sialic acid as the terminal monosaccharide) of oligosaccharide chains was investigated. Soybean agglutinin (SBA) specifically detects GalNAc and Gal residues (Lis et al., 1970). Vicia villosa agglutinin (VVA) detects GalNAc residues (Tollefson and Kornfeld, 1983), and peanut agglutinin (PNA) specifically binds to the Gal [beta]1->3 GalNAc disaccharide and also reacts with Gal residues (Lotan et al., 1975). The target saccharides of these three lectins are masked when a terminal sialic acid residue is present. Fluorochrome-conjugated lectins SBA, VVA, and PNA strongly stained the cell surface of K20 cells (Figure
MAb 1B2-1B7 is an anti-carbohydrate antibody which recognizes lacto-series oligosaccharide sequences (Young et al., 1991). The binding capacity for mAb 1B2-1B7, like that for lectins SBA, VVA and PNA, can be increased by sialidase pretreatment of cells (Campos et al., 1992). The staining pattern of this mAb on BJA-B K88 and K20 cells was similar to that seen for the three lectins (Figure
Taken together, these results indicate that the total quantity and type of oligosaccharide chains, in particular penultimate saccharides, are similar in group I and II subclones. In group II cells, however, cell surface glycoconjugates are largely unsialylated, whereas in group I cells they are sialylated to a much larger extent.
Altered 2D-electrophoretic mobility of cell surface sialoglycoproteins from a hyposialylated BJA-B subclone
Lysates from surface-biotinylated K88 and K20 cells were subjected to 2D-gel electrophoresis. The presence or absence of sialic acid as a negatively charged monosaccharide can significantly alter the isoelectric point and molecular mass of a glycoprotein, thus affecting its isoelectric focusing and electrophoretic mobility. Several major glycoprotein species were more basic in K20 cells as compared with K88 cells (see, for example, CD95 in Figure
Figure 3. Different CD95 sialylation status in BJA-B subclones. CD95 was immunoprecipitated from surface-biotinylated cells (with or without sialidase pretreatment) and (A) separated by 10% SDS-PAGE or (B) subjected first to isoelectric focusing and subsequently separated by 10% SDS-PAGE (2D-analysis). Biotinylated CD95 was detected on nitrocellulose membranes by peroxidase-conjugated streptavidin and an ECL reaction. The sensitivity of BJA-B subclones toward CD95-mediated apoptosis correlates with their sialylation status
We investigated the sialylation status of CD95 in BJA-B subclones. Cell surface proteins were biotinylated, and CD95 was immunoprecipitated using mAb anti-APO-1 (Trauth et al., 1989) and separated by SDS-PAGE. CD95 from group I subclones, K43 and K88, migrated markedly slower than that from group II subclones K5, K4, K9, K138, K6, and K20, with a difference in apparent molecular mass of 4-7 kDa (Figure
Next we determined whether group I and II subclones differ in their susceptibility to CD95-mediated apoptosis. Hyposialylated group II cells were far more susceptible than group I cells to apoptosis induced by either cytotoxic mAb anti-APO-1 (Figure
Figure 4. The sensitivity of BJA-B subclones towards CD95-mediated apoptosis correlates with their sialylation status. BJA-B K88 or K20 cells were cultivated for 14 h in the presence of different concentrations of cytotoxic mAb anti-APO-1 (A and C) or hCD95L (B), produced in transiently transfected 293T cells. Subsequently, the percentage of apoptotic cells was analyzed by the method of Nicoletti et al.. Additionally, sialidase-pretreated K88 cells were tested. Control cells in (B) were incubated with supernatants of untransfected 293T cells diluted 1:2 in RPMI-based culture medium. (D) binding of mAb anti-APO-1 to K88 or K20 cells at antibody concentrations ranging from 10 to 10,000 ng/ml was analyzed by flow cytometry, mAb HD20 served as a negative control. (A, B, D) values shown are the arithmetic mean ± SD of 3-6 determinations. (C) Apoptosis values are the mean of duplicates from one experiment.
To investigate the possibility that the enhanced apoptosis observed in K20 cells was simply the result of altered binding of the mAb anti-APO-1 to its extracellular epitope on hyposialylated CD95, we compared the antibody binding capacity of K20 cells with that of K88 cells. No significant differences were determined (Figure Binding of LPV to its sialylated receptor correlates with the cell surface expression of B cell differentiation sialoglycans in BJA-B subclones
When tested for their LPV binding capacity and susceptibility to infection, group I and II subclones displayed marked differences. Both the receptor binding capacity and the level of infection in hyposialylated K20 cells was more than 90% lower than that of K88 cells (Figure
The LPV susceptibility determined for seven different subclones and parental BJA-B cells was found to be correlated with their cell surface expression of the [alpha]-2,6-sialylated B cell differentiation antigens CDw75 and CDw76 (Figure
Figure 5. Sialic acid-dependent LPV binding and infection are reduced in hyposialylated K20 cells. LPV binding and infection was determined as described previously (Haun et al., 1993; Keppler et al., 1994). Pretreatment of K88 cells with sialidase was performed as outlined in experimental procedures. Values are the mean of two experiments and presented relative to values determined for untreated K88 cells (=100%).
Figure 6. Correlation of LPV susceptibility and expression of [alpha]-2,6-sialylated B cell differentiation antigens in BJA-B subclones. (A) Parental BJA-B cells and seven BJA-B subclones were analyzed for (1) their LPV susceptibility determined as ng LPV-VP1 per mg total protein in extracts of infected cells and (2) the expression of cell surface sialoglycans CDw75 and CDw76 determined as the median fluorescence intensity of 10,000 cells in a FACScan analysis. Values represent the mean of 2-3 independent experiments and are shown on logarithmic scales. (B) Parental BJA-B cells were stained for the CDw75 antigen using mAb HH2 and subjected to fluorescence-activated cell sorting. The CDw75-lowest expressing cells (7.9%) and the CDw75-highest expressing cells (7.3%) were isolated by sorting and subsequently infected with LPV for 2 h at 37°C. To allow only one viral replication cycle, neutralizing LPV antiserum was added and cells were cultivated for 48 h. The percentage of LPV-VP-positive cells was scored by indirect immunofluorescence microscopy.
Taken together, these results demonstrate that the first step of LPV infection, i.e., sialic acid-dependent virus attachment, is impaired in hyposialylated K20 cells. The correlation of either the sialylation status or the expression level of the [alpha]-2,6-sialylated cell surface antigens CDw75 and CDw76, in BJA-B subclones with their LPV susceptibility suggests that differential sialylation of the putative LPV sialoglycoprotein receptor or of an associated sialoconjugate on the cell surface represents a means of regulating virus infection in this cell line. ST6Gal I activity does not limit sialoglycan expression in BJA-B cells
In COS-7 cells the generation of the [alpha]-2,6-sialylated B cell differentiation antigens CDw75, CDw76, HB-4, HB-6, and EBU-65 has been shown to be limited by the low activity of ST6Gal I (Bast et al., 1992; Keppler et al., 1992). It has been suggested that differential expression of ST6Gal I in human B and T lymphocytes may also regulate the generation of [alpha]-2,6-sialylated differentiation antigens on the cell surface (Tedder et al., 1997). However, BJA-B K88 and K20 cells, which are heterogenous for the expression of [alpha]-2,6-sialylated B cell differentiation antigens (Table I, Figure
Figure 7. Northern blot analysis of ST6Gal I mRNA expression in BJA-B K88 cells, K20 cells, K20[alpha]2,6ST transfectants, which constitutively overexpress a recombinant ST6Gal I, hairy cell leukemia line JOK-1 and ST6Gal I-deficient B-lymphoblastoid cell line IM-9. The blot was hybridized with a [[alpha]-32P]-ST6Gal I cDNA probe (top panel). Ethidium bromide staining of the gel demonstrated that poly-A+-enriched RNA were present in similar amounts and quality for each cell line.
Despite ST6Gal I overexpression the phenotype of the K20[alpha]2,6ST cells in regard to the expression of CDw75, CDw76, LPV susceptibility (Figure Exogenous resialylation of hyposialylated BJA-B cells
We attempted to restore functionally relevant glycoconjugate sialylation in group II cells by exogenous resialylation using a combination of rat liver ST6Gal I and CMP-Neu5Ac. The low increase of binding of fluorescein-conjugated LFA, which detects sialic acid residues irrespective of their linkage (Knibbs et al., 1993; Figure
Figure 8. Exogenous resialylation of cell surfaces. BJA-B K88, either pretreated with Vibrio cholerae sialidase (+) or PBS (-), and K20 cells were incubated in resialylation buffer with (+) or without (-) the addition of rat liver ST6Gal I and analyzed. (A) Cell surface-bound sialic acid was quantified by flow cytometry using LFA-FITC. The relative means of triplicates of one of two comparable experiments are shown. (B) Cells were incubated with LPV for 3 h at 4°C, washed, and cultivated for 68 h. LPV infection was quantified as the amount of LPV-VP1 relative to the total protein content of cellular extracts. Values are the mean of triplicates. Bars indicate the SD.
The correlation between the heterogeneity of glycoconjugate sialylation in tumors and various tumor-related biological functions, including the metastatic and invasive phenotype of cells, has been investigated in detail (Yogeeswaran and Salk, 1981; Irimura and Nicolson, 1985; Collard et al., 1986). The present study for the first time addressed the question of whether differential sialylation in a clonal cell line could affect the function of a viral and a signal transducing receptor. The identical immunoglobulin heavy chain gene rearrangement of BJA-B subclones demonstrates their clonal origin (M.Pawlita, M.Oppenländer, G.Haun, and O.T.Keppler, unpublished observations), and they were indistinguishable on the basis of the cell surface expression of most B cell antigens. Furthermore, the quantity and type of penultimate saccharides on oligosaccharide chains of cell surface glycoconjugates were comparable. The major structural difference found between subclones was their level of sialylation involving both specific sialoglycoproteins, such as CD95 or the transferrin receptor, as well as their overall sialic acid content. This supports the interpretation that the marked differences between group I and II cells regarding sialic acid-dependent LPV susceptibility and CD95-mediated apoptosis are to a large extent related to the differential degree of sialylation of the cells. Also, sialidase pretreatment of highly sialylated group I cells influenced sialic acid-dependent functions in a similar fashion as seen in untreated group II cells. Sialidase-treatment of K88 cells resulted in a 5- to 12-fold reduction of binding for sialic acid-specific lectin LFA and mAbs against CDw75, CDw76, EBU-65, and HB-6, whereas in the functional assays, i.e., LPV binding and infection (3- to 5-fold reduction) and CD95-mediated apoptosis (2-fold reduction), these cells displayed an "intermediate" phenotype. This difference could be due to differential desialylation of cell surface glycoconjugates by Vibrio cholerae sialidase under the conditions used. Also, the increase of cell surface sialylation on group II cells or sialidase-pretreated group I cells by exogenous resialylation using CMP-Neu5Ac and rat liver ST6Gal I was only marginal as determined by LFA-FITC binding and yielded no increase in the functional assays. Both differences could be due to differential accessibility of sialylated acceptor sites on specific glycoconjugates to the externally applied sialidase and sialyltransferase.
Interestingly, the 3-fold reduction of the glycoconjugate-bound sialic acid concentration resulted in an impairment of virus and antibody binding by factors of 5-20. The known polyvalence of LPV particle binding to its sialylated receptor (Herrmann et al., 1997), and of various IgM-mAbs binding to their sialylated epitopes, may explain this discrepancy.
Dysregulated apoptosis can promote cancer development, both by allowing accumulation of dividing cells and by obstructing removal of genetic variants with enhanced malignant potential. Multiple mechanisms may be involved in tumor cell-resistance to CD95-mediated death (O'Connell et al., 1996; Whiteside and Rabinowich, 1998). Among them are deletion or downregulation of CD95, truncation of CD95 in its cytoplasmic domain, upregulation of negative regulatory proteins, such as the Bcl-2 family members, Fas-associated phosphatase 1 (FAP-1), FADD-like-interleukin [beta]-converting enzyme (FLICE)-inhibitory proteins (FLIP) and members of the inhibitors of apoptosis family. The metastatic and invasive potential of tumor cells has been positively correlated with the amount of expressed sialic acids (Yogeeswaran and Salk, 1981; Collard et al., 1986; Dennis et al., 1986; Sawada et al., 1994). Proposed sialic acid-dependent mechanisms explaining this observation include altered adherence of tumor cells to endothelia and leukocytes (Dennis et al., 1982; Schirrmacher et al., 1982), changes in tumor cell-mediated platelet aggregation (Bastida et al., 1987), and resistance to T cell-mediated immune destruction (Werkmeister et al., 1983).
Our data now support the concept that the sialylation status of a cell can also modulate its sensitivity towards apoptosis. In the human B lymphoma cell line BJA-B, differential sialylation appears to be a regulator of CD95-mediated apoptosis. The mechanism involved is, however, unclear. Binding of cytotoxic mAb anti-APO-1 was unaffected by differential sialylation of CD95. Since CD95 requires trimerization of death domains for signal production, an altered ability to self-aggregate is conceivably related to the number of negatively charged sialic acid residues present on CD95 (Peter et al., 1995). Currently, however, we cannot rule out the possibility that additional factors further downstream in the signaling cascade contribute to the different susceptibility of group I and II cells to CD95-mediated apoptosis. An increased cellular sialylation could be an antiapoptotic mechanism of certain tumor cells, resulting in prolonged survival of hypersialylated cells in vivo. For example, metastases from a human Burkitt's lymphoma cell line xenotransplanted into SCID mice displayed a higher degree of cell surface sialylation than the parental cells (Abe et al., 1996). Reduced apoptosis of hypersialylated cells in a tumor population could be one naturally occurring mechanism for their positive selection and more malignant phenotype in vivo.
Generally, loss of virus receptor-mediated function is thought to be due to a lack of receptor protein expression. We provide evidence in this study that differential sialylation of the putative [alpha]-2,6-sialylated LPV glycoprotein receptor may be sufficient to serve as a key regulator of LPV binding and infection in the human cell line BJA-B. Factors influencing the LPV tissue tropism are of particular importance for the future development of an LPV-based vector delivering a suicidal gene into B lymphomas in vivo. The role of differential sialylation ought to be studied for other viruses, which display a rather narrow tissue tropism and require a sialylated receptor. For example, the host range of the human polyomavirus JC (JCV), the etiological agent of the progressive multifocal leukencephalopathy, appears in vitro to be mainly restricted to glial and, to a limited extent, B cells (Major et al., 1992). JCV has recently been shown to use an [alpha]-2,6-sialylated N-linked glycoprotein for binding to human glial cells (Liu et al., 1998).
The endogenous mechanisms underlying the different sialylation levels in BJA-B subclones are still unclear. In COS-7 cells the generation of the [alpha]-2,6-sialylated B cell differentiation antigens CDw75, CDw76, HB-4, HB-6, and EBU-65, which are potential interaction partners of the I-type lectin CD22, has been shown to be limited by the low activity of an ST6Gal I (Keppler et al., 1992; Bast et al., 1992). In human BJA-B cells the activity of this enzyme does not appear to be the limiting factor, since ST6Gal I mRNA levels in group I K88 and group II K20 cells are comparable, and even constitutive overexpression of a functional, recombinant human ST6Gal I had no significant effect on the sialylation status of K20 cells. Also, the markedly reduced MAA-binding capacity of group II cells and the altered isoelectric focusing pattern of the transferrin receptor, which in human placenta is almost exclusively [alpha]-2,3-sialylated (Orberger et al., 1992), demonstrate that, besides [alpha]-2,6-sialylation, [alpha]-2,3-sialylation is also impaired in group II cells. Identifying the molecular basis of regulators of sialylation, apart from sialyltransferases, will be important for the future. It can be speculated from our data that, for example, the synthesis or Golgi uptake of the sialyltransferase substrate CMP-Neu5Ac is a limiting factor for the generation of sialoglycans in hyposialylated BJA-B cells. Evaluating the contribution of glycosylation enzymes critically involved in the multistep biosynthesis of CMP-Neu5Ac such as the glucosamine-6-phosphate deaminase (Wolosker et al., 1998), two recently cloned epimerases, GlcNAc-2-epimerase (Maru et al., 1996) and UDP-GlcNAc-2-epimerase/ManNAc-kinase (Hinderlich et al., 1997; Stäsche et al., 1997) or the CMP-Neu5Ac synthetase (Münster et al., 1998) may prove helpful in understanding the molecular mechanisms underlying differential cellular sialylation. These BJA-B subclones with stable differences in the sialylation level also will be valuable to the study of other B cell-associated, sialic acid-dependent ligand-receptor interactions. Cell line and BJA-B subclones
Human B lymphoma cell line BJA-B (Burkitt's lymphoma-like, EBV-negative; Menezes et al., 1975), BJA-B subclones (Keppler et al., 1994; M.Pawlita, M.Oppenländer, G.Haun, and O.T.Keppler, unpublished observations), hairy cell leukemia cell line JOK-1 (Kontula et al., 1982), and B lymphoblastoid cell line IM-9 (Lesniak and Roth, 1976) were propagated as suspension culture in Erlenmeyer flasks with RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per ml in a humidified 5% CO2 atmosphere. BJA-B subclones were isolated either by limiting dilution from parental BJA-B cells (Keppler et al., 1994) or from a BJA-B culture with previous persistent LPV infection (M.Pawlita, M.Oppenländer, G.Haun, and O.T.Keppler, unpublished observations). All subclones are of clonal origin since they display identical immunglobulin heavy chain gene rearrangements. Most subclones isolated from unselected parental BJA-B cells displayed the group I phenotype and were not further analyzed. Group II subclones were isolated at a low frequency of ~1%. The replication times of subclones in cell culture are identical. Human embryonal kidney cells 293T (obtained from the ATCC; Pear et al., 1993) were propagated as monolayer cultures in plastic tissue culture bottles and cultivated in DMEM with the same supplements and incubation conditions as described above. LPV infection and virus binding assay
LPV infection was performed essentially as described previously (Keppler et al., 1994). Briefly, stocks of LPV were prepared from infected BJA-B cells. To determine the susceptibility of cells to LPV infection, 1 × 106 cells were washed in cold PBS and the pellet was resuspended in 300 µl cold PBS containing LPV stock virus (~5 infectious units per cell). Cells were exposed to LPV for 3-4 h at 4°C, washed twice with PBS, and subsequently cultivated for 53 to 68 h in medium. The percentage of LPV-infected cells was scored by indirect immunofluorescence microscopy with costaining for LPV T and VP antigens (Haun et al., 1993). Additionally, the amount of viral antigen produced in infected cultures was quantified in cell extracts by LPV VP1 ELISA relative to the total protein extracted (Haun et al., 1993; Keppler et al., 1994).
The indirect, nonradioactive LPV binding assay was performed as previously described (Haun et al., 1993; Keppler et al., 1994). Washed cells were serially diluted in PBS containing 0.2% gelatin, and incubated in 96-well tissue culture plates in a final volume of 200 µl (1 × 104 to 1 × 106 cells/ml) with a constant amount of purified LPV particles equivalent to 800 pg of LPV VP1 for 30 min at 37°C. After low-speed sedimentation of cells (400 × g) the amount of unbound LPV VP1 in the supernatant was quantified by ELISA.
Quantification of cellular sialic acid content
Cells (1 × 107) were harvested, washed once in PBS, and lysed by hypotonic shock in distilled water (15 min, 4°C). The crude membrane fraction was pelleted by centrifugation at 10,000 × g for 15 min and the pellet was washed twice with distilled water. The lyophilized pellet was hydrolyzed for 1 h with 300 µl of 2 M acetic acid to release sialic acids (Varki and Diaz, 1984). Sialic acids were quantified by HPLC analysis (Keppler et al., 1995) and by the thiobarbituric acid method (Aminoff, 1961). By both methods similar values for cellular sialic acids were obtained. Antibodies, lectins, staining procedures, and flow cytometry
All mouse monoclonal antibodies (mAb), including mAb HH2, EBU-141, EBU-65, HD66, HB-4, HB-6 (all of IgM isotype), to sialylated B cell differentiation antigens were obtained from panels of the "Fourth and Fifth International Workshop and Conference on Human Leukocyte Differentiation Antigens" (Schwartz-Albiez et al., 1995). MAb HH2 and EBU-141 represent the prototype reagents defining the [alpha]-2,6-sialylated CDw75 and HD66 characterizes the [alpha]-2,6-sialylated CDw76 antigen (Schwartz-Albiez et al., 1995). MAb 1B2-1B7 is an anti-carbohydrate antibody directed to neolacto-N-glycosyl type II chains (Young et al., 1991). MAb HB-4, HB-6 and EBU-65 remain so far unclustered, but are known to recognizes [alpha]-2,6-sialylated epitopes (Schwartz-Albiez et al., 1995). MAb [alpha]APO-1 (IgG3, [Kgr]) recognizes an epitope on the extracellular part of CD95 (Trauth et al., 1989). Antibody staining procedures and fluorescence-activated cell scanning on a Becton Dickinson FACScan cytometer using Cellquest II software were carried out as described previously (Keppler et al., 1992). Sorting of cells with an EPICS-752-Coulter (Coulter, Hialiah, Florida) was performed under standard conditions.
Lyophilized FITC-conjugated lectins Vicia villosa (VVA), soybean agglutinin (SBA, Glycine max), Concanavalin A (ConA, Canavalia ensiformis), and TRITC-conjugated peanut agglutinin (PNA, Arachis hypogaea) were obtained from Sigma (Deisenhofen, Germany), dissolved (1 mg/ml), aliquoted, and stored at -20°C according to the manufacturer's instructions. FITC-conjugated Limax flavus agglutinin (LFA) was from EY Laboratories (San Mateo, CA). Biotionylated Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA) were from Vector Labs. FITC-conjugated streptavidin was from Sigma and used at a final concentration of 30 µg/ml. Optimal lectin concentrations for flow cytometry, with regard to agglutination of cells and signal intensity, were determined by testing serial lectin dilutions. Untreated or sialidase-pretreated cells (1 × 106) were washed twice in cold PBS and subsequently incubated in 100 µl PBS containing either fluorochrome-conjugated lectins VVA (50 µg/ml), SBA (5 µg/ml), PNA or ConA (10 µg/ml), LFA (20 µg/ml), biotinylated SNA or MAA (each 5 µg/ml) for 45 min on ice in the dark. After washing with PBS cells were resuspended in 400 µl PBS and analyzed by flow cytometry. Surface biotinylation, immunoprecipitation, and SDS-PAGE
Cell surface proteins were biotinylated essentially as described previously (Meier et al., 1992; Peter et al., 1995). Cells (1 × 107) were washed with PBS and resuspended in biotinylation buffer (107 cells/ml; 10 mM sodium borate, pH 8.8, 150 mM NaCl). d-Biotinyl-[epsis]-amido caproic acid N-hydroxysuccinimide ester (Boehringer Mannheim, Mannheim, Germany) was freshly dissolved in dimethyl sulfoxide (10 mg/ml) and immediately added to the cell suspension to a final concentration of 50 µg/ml. After tumbling at RT for 20 min, the reaction was terminated by addition of NH4Cl to a final concentration of 10 mM. Fifty milliliters of cold washing buffer (50 mM TRIS/HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA) was added and the mixture was centrifuged. The cell pellet was lysed on ice for 30-60 min in 500 µl lysis buffer containing 30 mM TRIS/HCl pH 7.5, 150 mM protease inhibitor cocktail (Boehringer Mannheim), and 1% NP-40. Lysates were precleared by rotation with 30 µl Protein A-Sepharose plus 30 µl Staphylococcus aureus cells (Pansorbin, Calbiochem, Bad Soden, Germany). Precleared supernatants were added to 10 µg of mAb anti-APO-1 covalently coupled to 30 µl CNBr-activated Sepharose 4B and rotated for 1-3 h at 4°C. Beads were washed five times with washing buffer (lysis buffer containing 0.1% NP-40). Samples were then incubated with Vibrio cholerae sialidase (500 mU/ml) for 60 min at 37°C where indicated. Samples were either boiled in a standard reducing sample buffer for 5 min and separated by 10% SDS-PAGE or subjected to two-dimensional (2D) gel electrophoresis. After transfer of proteins onto nitrocellulose membranes, biotinylated proteins were visualized with peroxidase-conjugated streptavidin (Dianova) and an ECL reaction (Sigma). Two-dimensional gel electrophoresis
2D-Gel electrophoresis was performed as described previously (Peter et al., 1995). Briefly, a combination of isoelectric focusing (IEF) and SDS-PAGE was used to resolve proteins in two dimensions. Samples were solubilized in 9.8 M urea, 4% (vol/vol) NP-40, containing three ampholines, (1) pH 5-7, (2) pH 3.5-10 (Pharmacia, Freiburg, Germany), and (3) Servalyt pH 5-7 (Serva), each 2% (vol/vol), and DTT to a final concentration of 100 mM. Tube gels used for the first dimension were 25 cm long with an internal diameter of 2.5 mm. IEF gels were run at 1200 V for 18 h. The pH gradient after electrophoresis was linear between pH 4.6-7.2. For the second dimension 10% SDS-PAGE gels were used. Sialidase pretreatment
Washed cells (1 × 107/ml) were incubated with Vibrio cholerae sialidase (200 mU/ml) (Behring, Marburg, Germany) in PBS for 45-60 min at 37°C, washed in PBS and subjected to the respective assays. Induction and measurement of apoptosis
Untreated or sialidase-pretreated cells (5 × 105 cells/ml) were incubated in 24-well plates (Costar, Cambridge) in culture medium for 14 h at 37°C with different concentrations of mAb anti-APO-1 and a standard concentration (10 ng/ml) of soluble protein A from Staphylococcus aureus (Sigma), or in the presence of human CD95 ligand (hCD95L). hCD95L was produced in 293T cells transiently transfected with the phCD95L expression plasmid (Peter et al., 1995). Supernatants were harvested 1 week after transfection and tested for their apoptosis-inducing activity on BJA-B cells after a dilution of either 1:2 or 1:4 in RPMI 1640-containing culture medium. Quantification of DNA fragmentation as a specific measure of apoptosis was carried out essentially as described previously (Nicoletti et al., 1991). Briefly, cells were centrifuged in a minifuge (Heraeus, Hanau, Germany) at 2000 rounds per min for 5 min and washed once with PBS. Cells were then carefully resuspended in a buffer containing 0.1% (wt/vol) sodium citrate, 0.1% (vol/vol) Triton X-100 (Serva, Heidelberg, Germany) and 50 µg/ml propidium iodide (Sigma). After incubation for at least 24 h at 4°C in the dark the percentage of apoptotic nuclei was determined by flow cytometry. Stable BJA-B subclone transfectants overexpressing human [beta]-galactoside [alpha]-2,6-sialyltransferase (ST6Gal I)
The eukaryotic expression vector pEx-[alpha]-2,6-ST-Hygro (Keppler et al., 1992) contains the cDNA sequence encoding the ST6Gal I (Stamenkovic et al., 1990) under control of the cytomegalovirus immediate early promotor/enhancer element together with the hygromycin resistance gene. BJA-B subclones were transfected by electroporation with 20 µg CsCl gradient-purified DNA of pEx-[alpha]-2,6-ST-Hygro as described previously (Döffinger et al., 1988), using a voltage of 180 V. After incubation in mass culture for 2 days, transfected cells were cultivated under limiting dilution conditions in 96-well plates (Nunc, Wiesbaden, Germany) in medium containing 200 µg/ml hygromycin B (Sigma). Hygromycin-resistant clones were expanded. Northern blot analysis
Poly (A)+ RNA was isolated from 5 × 107 cells by the guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987); 10 µg RNA was separated in a 0.8% agarose/formaldehyde gel. Ethidium bromide staining of the gel was documented. Subsequently, the RNA was transferred overnight onto Hybond-N+ filters (Amersham, Arlington, IL) and then UV-crosslinked using a Stratalinker (Stratagene, Heidelberg, Germany). cDNA probes were labeled by oligo-primed labeling (Amersham) using [[alpha]-32P]-CTP. The blot was hybridized with a [[alpha]-32P]-labeled 1.9 kb Xba1 cDNA fragment of the ST6Gal I (Keppler et al., 1992), essentially as described previously (Wolf et al., 1990). Resialylation of cell surfaces
The resialylation assay was performed essentially as described previously (Gross et al., 1996). Sialidase-treated or PBS-treated cells (1 × 106) were incubated in PBS (pH 7.0) containing 1% BSA, 500 µM CMP-Neu5Ac (kind gift of H.-J.Gross) with or without the addition of rat liver ST6Gal I (40 U/ml; Boehringer Mannheim) for 2 h at 37°C. Washed cells were then either stained with LFA-FITC, infected with LPV for 3 h at 4°C or incubated with mAb anti-APO-1 (100 ng/ml) for 13 h at 37°C and further analyzed as described above.
We are grateful to H.zur Hausen for continuous support. We are indebted to S.Funderud and T.F.Tedder for kind gifts of mAb HH2 and HB-4, HB-6, respectively. We thank H.Walczak for the phCD95L expression plasmid and H.-J.Gross for CMP-Neu5Ac. We are grateful to K.Hexel for FACSort analyses. We thank G.Haun for initial experiments and helpful discussions and M.Oppenländer for expert technical assistance. We thank T.A.Scott for improving the English of the manuscript. This work was supported in part by grants from the Bundesministerium für Bildung und Forschung, Bonn, Germany (to W.R. and M.P.) and from the Wilhelm-Sander Stiftung (to M.P).
BSA, bovine serum albumin; CD, cluster of differentiation; CD95, Fas/APO-1; ConA, concanavalin A; CMP; cytidine monophosphate; FAP-1, Fas-associated phosphatase 1; FLICE, FADD-like-interleukin [beta]-converting enzyme; FLIP, FLICE-inhibitory proteins; Gal, d-galactose; GalNAc, d-N-acetyl galactosamine; GlcNAc, d-N-acetyl glucosamine; Glc, d-glucose; hCD95L, human CD95 ligand; IEF, isoelectric focusing; Ig, immunoglobulin; JCV, human polyomavirus JC; K20[alpha]2,6ST, K20 cells overexpressing the ST6Gal I; LFA, Limax flavus agglutinin; LPV, B lymphotropic papovavirus; mAb, monoclonal antibody; MAA, Maackia amurensis agglutinin; Man, d-mannose; Neu5Ac, N-acetyl neuraminic acid; PNA, peanut agglutinin; SBA, soybean agglutinin; sialic acid, N-acetyl neuraminic acid or other N-or O-substituted neuraminic acids; SNA; Sambuccus nigra agglutinin; ST6Gal I; human [beta]-galactoside [alpha]-2,6-sialyltransferase; 2D, two-dimensional; VVA, Vicia villosa agglutinin.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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