Differential expression of [alpha]2-6 sialylated polylactosamine structures by human B and T cells

Bernhard Kniep6, Knut Schäkel, Manfred Nimtz1, Reinhard Schwartz-Albiez2, Marc Schmitz, Hinnak Northoff3, Ramon Vilella4, Martin Gramatzki5 and E. Peter Rieber

Institut für Immunologie der Technischen Universität Dresden, Postfach 88115, D-01101 Dresden, Germany, 1Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany, 2Deutsches Krebsforschungszentrum, Forschungschwerpunkt Tumorimmunologie 710, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany, 3Universität Tübingen, Abteilung Transfusionsmedizin, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany, 4Servei d'Immunologia Hospital Clinic Villaroel 170 08036 Barcelona, Spain and 5Medizinische Klinik III, Universität Erlangen-Nürnberg, Krankenhausstrasse 12, D-91054, Germany

Received on July 6, 1998; revised on September 12, 1998; accepted on September 14, 1998

We found that human peripheral B and T cells differed in the surface expression of [alpha]2-6 sialylated type 2 chain glycans. In contrast to B cells, T cells expressed only sialoglycans with repeated N-acetyllactosamine (Galß1-4GlcNAc) disaccharides. This finding was based on the specificity of the monoclonal antibodies HB6, HB9 (CD24), HD66 (CDw76), FB21, and CRIS4 (CDw76) with the [alpha]2-6 sialylated model gangliosides IV6NeuAcnLc4Cer (2-6 SPG), VI6NeuAcnLc6Cer (2-6 SnHC), VIII6NeuAcnLc8Cer (2-6 SnOC), and X6NeuAcnLc10Cer (2-6 SnDC). We found that, in addition to their common requirement of an [alpha]2-6 bound terminal sialic acid for binding, the antibodies displayed preferences for the length of the carbohydrate backbones. Some of them bound mainly to 2-6 SPG with one N-acetyllactosamine (LacNAc) unit (HB9, HD66); others preferentially to 2-6 SnHC and 2-6 SnOC, with two and three LacNAc units, respectively (HB6 and FB21); and one of them exclusively to very polar [alpha]2-6 sialylated type 2 chain antigens (CRIS4) such as to 2-6 SnOC and even more polar gangliosides with three and more LacNAc units. These specificities could be correlated with the cellular binding of the antibodies as follows: whereas all antibodies bound to human CD 19 positive peripheral B cells, their reactivity with CD3 positive T cells was either nearly lacking (HD66, HB9), intermediate (about 65%: HB6, FB21) or strongly positive (CRIS4, 95%). Thus, the binding of the antibodies to 2-6 sialylated glycans with multiple lactosamine units appeared to determine their binding to T-cells.

Key words: 2-6 sialylation/gangliosides/polylactosamines/T cell antigens

Introduction

[alpha]2-6 sialylated type 2 chain carbohydrates are quite common constituents of several cell surface molecules such as glycolipids (Fukuda et al., 1985), and N- (Lee et al., 1990) as well as O-linked glycoproteins (Maemura et al., 1992). In spite of the widespread distribution of these structural components, [alpha]2-6 sialylated glycan specific monoclonal antibodies (mAbs) show rather specific staining profiles on leukocyte populations (Bast et al., 1992). This led to a different classification of the [alpha]2-6 glycan specific antibodies during previous Workshops on Human Leukocyte Differentiation Antigens. To explain this different cellular binding, we asked to what extent other parts of the glycan chain were recognized in addition to the common NeuAc[alpha]2-6Galß1-4GlcNAc sequence. It is known that the [alpha]2-6 sialyl group in longchain glycolipids as well as in glycoproteins is frequently linked to more than one lactosamine unit, e.g., in leukosialin (CD43) (Maemura et al., 1992) or in the tyrosine phosphatase CD45 (Furukawa et al., 1998). To analyze the influence of these additional structural elements on antibody binding, we purified and synthesized enzymatically gangliosides with an extended core structure comprising molecules with one to four lactosamine units. A considerable number of mAbs directed to terminally [alpha]2-6 sialylated glycans have been described to date (Hakomori et al., 1983; Kniep et al., 1990; Larkin et al., 1991; Bast et al., 1992; Schwartz-Albiez et al., 1995). MAbs with this reactivity bind mainly to human B cells but also, in a variable degree, to other leukocyte populations such as T-cells, NK cells, and granulocytes. No comprehensive attempts have been made to elucidate their antigen fine specificities in order to explain their different cellular reactivities. We show here that differences in the cellular specificity of the antibodies can be attributed to their specific recognition of [alpha]2-6 sialylated glycans with different numbers of LacNAc units. This partial core region specificity is in contrast to CD22ß, an [alpha]2-6 sialic acid recognizing lectin, whose binding affinity seems to be determined only by the number and spatial arrangement of the [alpha]2-6 sialylated groups (Powell et al., 1995). As far as we know, no other carbohydrate antigen has been described for which such a highly diverse set of antibodies exists. We report in this study a correlation between the ability of the mAbs to bind to long [alpha]-2-6 sialylated type 2 glycan structures and their binding to T-cells. From this observation, it can be concluded that T-cells contain at their cell surface preferentially [alpha]2-6 sialylated glycans with more than one LacNAc unit.

Results

Flow cytometric analysis of the binding of [alpha]2-6 sialylated glycan specific monoclonal antibodies to human peripheralT cells

The binding to human T cells of the five carbohydrate-specific, B-cell related antibodies HB9 (CD24) (Ling et al., 1987), HD66 (Möller et al., 1989), HB6 (Bast et al., 1992), FB21 (Nozawa et al., 1994), and CRIS4 (Möller et al., 1989), each recognizing a terminal [alpha]2-6 bound sialic acid together with a distinct part of a type 2 carbohydrate chain, is shown in Figure 1A. In spite of their common recognition of an [alpha]2-6 sialylated glycan chain, their reactivity with peripheral T cells, in contrast to their B cell reactivity (Figure 1B), varied from a few percent to 95%. Therefore, we wondered whether this diverse binding might be due to the fine specificity of the antibodies.


Figure 1. Differential binding of [alpha]2-6 sialylated glycan specific antibodiesto T and B cells. Flow cytometrical analysis was done after double immunofluorescent staining of PBMC with mAbs specific for [alpha]2-6 sialylated glycans and CD3 (A) or CD19 (B). Histograms show the fluorescence intensity of cells stained for different [alpha]2-6 sialylated glycan specific mAbs (IgM) on gated CD3+ or CD19+ cells (black). The given percentage of positively stained cells reflects the relative number of cells stained for [alpha]2-6 sialylated glycan specific mAbs having a fluorescence intensity above the highest fluorescence intensity obtained with an isotype matched control antibody (gray). Results obtained from one representative donor are shown. Among six donors studied ±SEM values were between 0.9% (HD66) and 5.7% (HB6). No significant differences in the staining patterns of CD4+ and CD8+ cells were observed.

Preparation and characterization of [alpha]2-6 sialylated gangliosides of the neolacto-series

Large differences in their pattern of immunostaining were observed when the binding of the five antibodies to the monosialoganglioside fraction of unseparated human leukocytes was compared (Figure 2). A wide range of antigens was detected either in the unpolar (HD66 and HB9, the middle polar (HB6 and FB21), or the polar region of the chromatogram (CRIS4). MAb J3-89 (Figure 2, right side) and mAb EBU-65 (not shown) served as a control since they bound to nearly all of the antigens, except to the very polar ones. We selected these five antibodies because they reflected the whole range of patterns which were also produced by several other [alpha]2-6 sialoglycan specific antibodies (not shown).


Figure 2. Binding of [alpha]2-6 sialylated glycan specific mAbs to the monosialoganglioside fraction of unseparated human leukocytes. Each lane contained the equivalent of monosialogangliosides originating from 17.5 ml blood, prepared as described in Materials and methods. The gangliosides were separated on a HPTLC silica plate. The running solvent was C/M/W (50:40:10) (V/V/V) containing 0.05% calcium chloride and the running time was 40 min. TLC immunostaining was performed as described in Materials and methods. The designations A, B, and C refer to the unpolar, middle polar, and polar [alpha]2-6 sialylated antigens, respectively.

Table I. Methylation analyses of the glycolipids with 1-4 N-acetyllactosamine (LacNAc) repeats
Peracetylated alditol [alpha]2-6 Sialylated ganglioside (no. of LacNAc repeats)
SPG (1) SnHC (2) SnOC (3) SnDC (4)
Fucitol
2,3,4-Tri-O-methyl- <0.1 0.2 <0.1 0.9
Galacitol
2,4,6-Tri-O-methyl- 1.0 2.1 3.0 4.0
2,3,4-Tri-O-methyl- 1.0 1.0 1.0 1.0
2,4-Di-O-methyl- - - - -
Glucitol
2,3,6-Tri-O-methyl- 0.3 0.3 0.7 1.0
2-N-Methylacetamido-2-deoxyglucitol
3,6-Di-O-methyl- 1.0 1.5 2.8 3.1
4,6-Di-O-methyl- - - - -
6-O-Methyl- - 0.3 <0.1 0.9

Since we aimed to correlate quantitatively the ganglioside binding of the 2-6 sialoglycan specific mAbs with their cellular reactivity, the [alpha]2-6 sialylated gangliosides were isolated from the lipid extract of unseparated leukocytes as described in Materials and methods. The final HPLC separation of the [alpha]2-6 sialoglycans was monitored in parallel by nonspecific staining with digoxigenin (DIG) (Kniep and Mühlradt, 1990) and TLC immunostaining using mAb J3-89 (origin: Pesando, Seattle; Schwartz-Albiez et al., 1995) (Figure 3). The gangliosides in the HPLC fractions 92 and 100 were identified as 2-6 SPG and 2-6 SnHC, respectively, by tandem electrospray mass spectrometry (ESI-MS) and methylation analysis (Table I) as will be described below in detail for the analogous compound 2-6 SnOC. Briefly, doubly deprotonated molecular ions at m/z 813.3 and 995.7 for 2-6 SPG and 2-6 SnHC indicating a molecular mass of 1628.6 and 1993.4 Da, respectively, were detected as the dominant components by negative ion mode ESI-MS. Upon collision induced dissociation (CID), a linear arrangement of the monosaccharide residues could be deduced from the fragment ions. The ceramide moiety was shown to consist predominantly of a C18 sphingosin acylated by a C24:1 fatty acid in the case of 2-6 SPG or a C16 fatty acid in the case of 2-6 SnHC. These results were confirmed by the identity of 2-6 SPG and 2-6 SnHC with nLc4Cer and nLc6Cer, respectively, after desialylation (not shown) and, as shown below, by their formation upon enzymatic sialylation of the neutral glycolipids of unseparated leukocytes using [alpha]2-6 sialyltransferase.


Figure 3. HPLC separation of the monosialogangliosides from human leukocytes. The gangliosides were separated as described in Materials and methods; 0.05% of each fraction were first separated on silica HPTLC plates and stained with resorcinol (not shown). Selected fractions (0.2 %) were then stained in parallel nonspecifically with DIG (left panel) and specifically with the [alpha]2-6 sialylated glycan specific mAb J3-89 (right panel). Running conditions and designations as in Figure 2.

There was a general problem concerning the separation of the polar [alpha]2-6 sialylated gangliosides from human leukocytes: because these often comigrated with their 2-3 sialylated and fucosylated counterparts, their purification from natural sources was not successful as demonstrated by their deoxyhexose (fucose) content after ESI-MS of HPLC fraction 107 (not shown). Instead, as described in detail in Materials and methods, they were prepared enzymatically from the polar neutral glycolipid (GSL) fraction using [alpha]2-6 sialyltransferase and [alpha]-l-fucosidase, purified by DEAE Sepharose chromatography and desalted on a SeP-Pak C-18 cartridge. Three antigen double bands were detected in the methanol/water 9:1 (V/V) eluate of the C-18 cartridge as shown by TLC immunostaining of an aliquot with mAb J3-89 (Figure 4). The antigens were separated by HPLC on a small silica column. Whereas the HPLC fractions 32 and 37+38 were identical to 2-6 SPG and 2-6 SnHC, respectively, the fraction 42+43 and 50+51 represented more polar antigens (not shown). Their identification was performed by mass spectrometry and methylation analysis. Fraction 43 contained pure 2-6 SnOC. Its electrospray mass spectrum is shown in Figure 5 (upper panel). Doubly deprotonated molecular ions (M-2H)2- at m/z 1123.4, 1165.4, and 1178.5 were detected indicating a homogenous oligosaccharide moiety [NeuAcHexNAc3Hex5] linked to a C18 sphingosin acylated by a C16, C22, or C24:1 fatty acid. Additional signals at m/z 1142.4 and 1169.9 can be assigned to chloride adducts of the major components (M+Cl-H)2-. The carbohydrate sequence of the glycolipid was then determined by ESI tandem mass spectrometry. The dominant molecular ion at m/z 1123.4 was selected by the first mass analyzer and subjected to CID in the collision cell of the mass spectrometer. The resulting daughter ions were then separated by the second mass analyzer (Figure 5, lower panel). An almost complete series of sequence specific carbohydrate fragments was detected, as is shown in the fragmentation scheme of Figure 5, clearly demonstrating a linear arrangement of the carbohydrate part of the molecule. Only the fragments incorporating the lipid moiety showed the expected mass shift, when a species with a different molecular mass was selected as parent ion excluding inhomogeneities of the carbohydrate part. Methylation analysis (Table I) allowed the determination of the carbohydrate linkages present in the glycolipid. The detection of 6- and 3-monosubstituted galactose and 4-monosubstituted GlcNAc in a ratio of 1:3:3 is in complete agreement with the presence of one [alpha]2-6 sialylated galactose and three LacNAc repeats. The detection of only traces of 3,4-disubstituted GlcNAc and terminal fucose indicates the absence of significant amounts of fucose linked to O-3 of the GlcNAc residue.


Figure 4. TLC immunostaining of the enzymatically prepared [alpha]2-6 sialylated gangliosides. Polar neutral glycolipids were sialylated, desalted and purified by RP18- and DEAE Sepharose chromatography, respectively, as described in Materials and methods. The DEAE Sepharose 0.05 M ammonium acetate eluate was adsorbed onto a second RP18 column and eluted with M/W 90:10 (V/V), 100% M and C/M 2:1 (V/V). Aliquots of these fractions containing the sialylated polar neutral glycolipid fraction from 1.4 × 108 human unseparated leukocytes, were separated on an HPTLC plate as described in the Figure 2 caption and immunostained with mAb J3-89. lane a, 90% M eluate; lane b, M eluate; lane c, C/M 2:1 eluate; lane d, standard containing the unseparated monosialoganglioside fraction. The designations were the same as in Figure 2.


Figure 5. Upper panel: negative ion mode ESI mass spectrum of the glycolipid fraction with three N-acetyllactosamine repeats. The doubly deprotonated molecular ion [M-2H]2- at m/z 1123.4 corresponds to a molecular mass of 2248.8 Da and is compatible with the composition NeuAc Hex5 HexNAc3 Cer. Additional signals can be assigned to the chloride adduct of the same molecule [M-H+Cl]2- at m/z 1142.4 and derivatives with larger lipid moieties at m/z 1165.4 and 1178.5 plus the respective chloride adducts. In the lower panel, the daughter ion spectrum of the dominant component obtained after CID is depicted. The assignment of the fragments is explained in the inset fragmentation scheme and allows the determination of the monosaccharide sequence and composition of the ceramide part of the molecule. Additional intense fragment ions not explained in the scheme at m/z 572, 937, and 1302 and the doubly charged species at m/z 438 and 621 are generated by unerring cleavages of the GlcNAc residues (0,2A3,0,2A5,0,2A7,2,4A5, and 2,4A7 according the nomenclature of Domon et al., 1988).

Fraction 50 yielded doubly charged molecular ions at m/z 1306.4 (10%) and 1379.1 (90%), when subjected to ESI-MS, compatible with the presence of X6NeuAc-nLc10Cer (2-6 SnDC) and large amounts of a monofucosylated derivative of 2-6 SnDC with a C18 sphingosin moiety acylated by a C24:1 fatty acid. These findings were confirmed by methylation analysis (Table I). The attachment position of the fucose residue was shown to be at the inner GlcNAc residues by a MS/MS experiment (not shown). The concentration of 2-6 SnDC was too low for ganglioside quantitation and therefore could not be included in our quantitative binding assay.

Binding of the [alpha]2-6 sialylated type 2 chain glycan specific antibodies to the gangliosides 2-6 SPG, 2-6 SnHC, and 2-6 SnOC

With the exception of the mAbs HD66/HB9, which showed a very similar immunostaining pattern, the [alpha]2-6 sialylated type 2 chain glycan specific antibodies recognized gangliosides of different polarity (Figure 2). Since the concentration of the [alpha]2-6 sialylated molecules decreased rapidly with increasing polarity, the fine specificities of the antibodies could only be determined when their reactivity with equal amounts of the three antigens was compared. Therefore, we quantitated the three gangliosides and analyzed the binding of the five antibodies (2 µg/ml) to increasing amounts of each of the three ganglioside antigens (Figure 6). The unpolar ganglioside 2-6 SPG was detected best by HD66 and HB9 and to a lesser extent by HB6 (Figure 6, upper panel). CRIS4 and FB21 did not recognize 2-6 SPG. The medium polar ganglioside 2-6 SnHC was mainly recognized by HB6, whereas the other bound weakly (HD66, CRIS4) or negligibly (FB21, HB9) (Figure 6, middle panel).


Figure 6. Binding of the mAbs HB9, HD66, HB6, FB21 and CRIS4 to 2-6 SPG (upper panel), 2-6 SnHC (middle panel) and 2-6 SnHC (bottom panel) as revealed by immunostaining. The indicated amounts of the three gangliosides were separated on five TLC plates as described in the legend of Figure 2. subjected to TLC immunostaining and the stained areas were measured as described in Materials and methods. All mAbs were used at a concentration of 2 µg/ ml.

HD66 and HB9 failed to detect the polar antigen 2-6 SnOC (Figure 6, bottom). FB21 and HB6 bound with intermediate intensity and CRIS4 very strongly. From Figure 6 it was evident that the threshold for binding was lower for antibodies detecting the polar antigens. More important, the antibody binding to 2-6 SnOC coincided with their binding to human T cells. This led us to conclude that the structure NeuAc[alpha]2-6Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4GlcNAc (NeuAc[alpha]2-6(LacNAc)3) is a part of a cell surface component of human T-cells. In spite of several trials, we failed to detect the presence of any [alpha]2-6 sialylated glycolipids in human B and T cells. Therefore, the NeuAc[alpha]2-6(LacNAc)3 component appears to be part of (a) cell surface glycoprotein(s) of human T cells.

Discussion

Our study of the antigen specificities of five representative [alpha]2-6 sialylated glycan specific mAbs served two purposes. First, to explain the largely different cellular reactivities of these mAbs, and second, to compare the surface expression of the [alpha]2-6 sialylated glycans on human B and T cells. In an extension of our previous findings (Kniep et al., 1990), we prepared [alpha]2-6 sialylated glycolipids differing in their number of LacNAc units and analyzed quantitatively the binding of five antibodies to these structures. We found that the terminal [alpha]2-6 bound sialic acid residues were not only decisive determinants but also distinct parts of the carbohydrate core chain. This is in contrast to the binding of the [alpha]2-6 sialylated glycan specific lectin CD22ß (Powell et al., 1995), for which the minimal structural recognition motif was found to be an [alpha]2-6 bound sialic acid linked to either Gal, GlcNAc, or GalNAc. The five antibodies showed preferences for [alpha]2-6 sialylated structures with distinct numbers of LacNAc disaccharide units in their core region and it appeared that their binding to 2-6 SnOC containing three LacNAc units determined their T-cell reactivity: only those antibodies which recognized this ganglioside (e.g., HB6, FB21, and, above all, CRIS4) bound also to T cells. As a conclusion, in T cells the surface expression of [alpha]2-6 sialylated glycans seems to be confined to structures containing repeated LacNAc units.

It is well known that carbohydrate specific mAbs often need a certain length of the core region for epitope recognition. As an example, antibodies directed to the Lex carbohydrate determinant fail to recognize this component on short glycolipids e.g. on the pentaglycosylceramide III3FucnLc4Cer but bind very strongly to the same determinant located on the heptaglycosylceramide V3FucnLc6Cer. The situation with the [alpha]2-6 sialylated glycan specific antibodies seems to be quite different, inasmuch as CRIS4 indeed bound to longchain gangliosides and not to short ones but HD66 and HB9 recognized only short chain gangliosides and not their longchain counterparts. We constrained ourselves to analyzing those antibodies which were more or less specific for gangliosides of a certain length of their carbohydrate chain. Other [alpha]2-6 sialylated glycan specific mAbs (e.g., J3-89 or EBU-65) showed a rather broad ganglioside specificity as they reacted with short and long chain [alpha]2-6 sialylated gangliosides (not shown). Thus, in this case, their common epitope seems to be included in the sequence NeuAc[alpha]2-6Galß1-4GlcNAc, whereas the epitopes of the five mAbs could be quite different. Since mAb J3-89 strongly bound to all [alpha]2-6 sialylated gangliosides, including 2-6 SnOC, we expected its binding to T-cells. This could be confirmed since it bound to 62% of the peripheral T-cells (not shown).

Which molecule might be their natural target on T cells? [alpha]2-6 bound sialic acid is a necessary but not sufficient determinant for epitope recognition. Since we were not able to detect 2-6 sialylated glycolipids in the lipid extracts from B- or T-cells, we suppose that the determinants of these carbohydrate specific antibodies are located on the carbohydrate chain of glycoproteins. [alpha]2-6 sialylated LacNAc repeats have been reported in N-linked glycoproteins such as in band 3 from human umbilical cord erythrocytes (Fukuda et al., 1984) as well as in the glycoproteins of the mouse lymphoma cell line BW 5147 (Merkle et al., 1987). Interestingly, it has been observed that the LacNAc repeats are preferentially found on complex-type tri- and tetraantennary N-glycans.

A differentiation dependent expression of carbohydrate structures has been described for several leukocyte glycoproteins(e.g., for leukosialin (CD43); Piller et al., 1991) and for the tyrosine phosphatase CD45 (Furukawa et al., 1998). CD22ß, an [alpha]2-6 sialylated glycan specific lectin binds to several T cell glycoproteins, especially to CD45RO (Stamenkovic et al., 1991). Recently, it has been suggested that the CD45RA isoform may be a preferred substrate for the Galß1,4GlcNAc[alpha]2,6-sialyltransferase (Baum et al., 1996). It seems likely that the epitopes recognized by the mAbs HB6, FB21, and CRIS4 are also located on these glycoproteins.

Materials and methods

Antibodies

All mAbs were of the IgM isotype. The mAbs EBU-65 (Gramatzki et al., 1991), HD66 (CDw76) (Möller et al., 1989) and CRIS4 (CDw76) (Möller et al., 1989) were prepared as described. The mAbs FB21 (Nozawa et al., 1994), J3-89 (Schwartz-Albiez et al., 1995), and HB6 (Bast et al., 1992) were obtained from the B Cell Section of the VIth Workshop and Conference on Human Leukocyte Differentiation antigens. MAb HB9 (CD24; Ling et al., 1987) was a kind gift from Dr. T. F. Tedder (Dana Farber Cancer Institute, Boston). All antibodies were quantitated using a capture ELISA and adjusted with phosphate-buffered saline, 1% bovine serum albumin (PBS/BSA) to a concentration of 2 µg/ml.

Purification and preparation of the [alpha]2-6 sialylated neolacto glycolipids

Purification of IV6NeuAcnLc4Cer (2-6 SPG) and VI6NeuAc-nLc6Cer (2-6 SnHC). Unseparated leukocytes (1 × 1012) were prepared from pooled buffy coat layers as described previously (Kniep et al., 1993). The lipids were extracted under sonication (Branson sonifier B12) for 30 min under stirring and ice-cooling successively with 5 l each of methanol (M), 2-propanol (i-P)/n-hexane(H)/water (W) 55:25:10 (W) (V/V/V) and chloroform/methanol (C/M) 2:1 (V/V). The extracts were combined and evaporated at 30°C under reducing pressure. Foaming was avoided by adding 5% 1-butanol/1-propanol 1:9 (V/V).

Folch partition. The lipid extract was taken up in 3.5 l C/M 2:1 (V/V). After addition of 700 ml W, the suspension was stirred for 30 min and allowed to separate in two phases at ambient temperature. The upper phase was withdrawn, and the lower phase was partitioned again with the same volume of theoretical upper phase. Both upper phases were combined, dried, taken up in W, and dialyzed for 3 days at 4°C.

DEAE-Sepharose chromatography. The dialyzed upper phases were lyophilized, taken up in C/M/W 30:60:8 (V/V/V) and loaded onto a DEAE Sepharose column (acetate form, 3 × 20 cm). Elution was performed with 2 l C/M/W 30:60:8, 1 l M and successively with 2 l each of 10, 20, 30, 50, 80, 150, and 300 mM ammonium acetate in M. The column was finally washed with2 l 0.8 M sodium acetate in M. Monosialogangliosides were contained in the 10, 20, and 30 mM ammonium acetate eluates. These were pooled, evaporated to dryness and desalted on a 2 × 10 cm column filled with silica RP-18 40 µm particles and preequilibrated with 20% i-P/100 mM NaCl. After washing with W, elution was performed with i-P/W 40:60, 50:50, 60:40 (all V/V), and finally with 100% i-P until UV absorbance at 215 nm was constant after each step. The i-P/W 50:50 eluate was chosen for further purification since most of the gangliosides migrated as single bands.

High performance liquid chromatography. The i-P/W 50:50 eluate from the RP-18 column was evaporated to dryness under reduced pressure, taken up in 15 ml C/M/W 82.6:16.4:1 (V/V/V) and pumped slowly (0.5 ml/min) onto 16 mm × 60 cm column filled with Lichrosorb Si 60 5 µm silica particles Merck, Darmstadt, Germany). Elution was performed with a linear gradient from C/M/W 82.6:16.4:1 to 40:50:10 in 200 min and to 20:60:20 in 100 min. Elution of the latter solvent was continued for 70 min. The flow rate was 2 ml/min. Fractions were collected every 2 min. The fractions were evaporated to dryness under a gentle stream of nitrogen. Aliquots were spotted on HPTLC plates and stained with the pan anti [alpha]2-6 sialylated glycan antibody EBU-65. [alpha]2-6 sialylated glycans were detected in the fractions 88-94, 100, 105, and 107. According to chromatographical analysis and mass spectrometry, fractions 88-94 and 100 consisted of pure 2-6 SPG and 2-6 SnHC, respectively, whereas the fractions C and D contained mainly fucosylated gangliosides.

Preparation and purification of XIII6NeuAcnLc8Cer (2-6 SnOC) and X6NeuAcnLc10Cer (2-6 SnDC). Unseparated leukocytes(3.5 × 1011) were prepared from pooled buffy coat layers and extracted with chloroform/methanol (C/M) as described previously (Kniep et al., 1993). The combined extracts were dried under reduced pressure, taken up in W and dialyzed for 3 days at 4°C. To the retentate (270 ml) 1350 ml (C/M) 2:1 were added. The mixture was stirred gently for 30 min, and the two phases were allowed to separate overnight at ambient temperature. The upper phase was separated. The lower phase was washed with 500 ml M/W 1:1 (V/V). Both upper phases were combined, adjusted to C/M/W 30:60:8 (V/V/V), and passed through a DEAE Sepharose column (acetate form, 3.8 × 8 cm). The column was then washed successively with 2 l M and then eluted with 2 l 20 mM ammonium acetate in M, 1 l 80 mM ammonium acetate in M and finally with 300 mM ammonium acetate in M. The C/M/W 30:60:8 and the M washes were dried in vacuo and taken up in 5 ml C/M 2:1. Twenty percent of the combined upper phase was used for the [alpha]2-6 sialylation.

Enzymatic sialylation. The incubation mixture for sialylation contained in 1.037 ml HEPES pH 6.5, 0.5% Triton X-100, 0.1% bovine serum albumin: upper phase neutral glycolipids from 7 × 1010 unseparated leukocytes (sonified for 15 min in HEPES-Triton), 6.12 µmol CMP-NANA (Sigma, Deisenhofen, Germany), 20 mU 2-6 sialyltransferase from rat liver (Boehringer Mannheim, Mannheim, Germany), 2 U alkaline phosphatase (Boehringer), 0.1 U [alpha]-l-fucosidase from bovine kidney (Boehringer), and 1 µmol MgCl2. After incubation for 96 h at 37°C, additional 8.14 µmol of CMP-NANA were added in 0.5 ml HEPES-Triton buffer together with 10 mU of the 2-6 sialyltransferase. A third portion of 8.14 µmol CMP-NANA was added 48 h later. The incubation was stopped after 8 days by freezing.

First desalting step. The incubation mixture was taken up in 5 ml 0.88% aqueous KCl, sonicated and passed through a Sep-Pak C-18 cartridge as described (Williams and McCluer, 1980). The desalted lipids were obtained in the M phase (30 ml).

DEAE-Sepharose chromatography. The desalted methanolic solution containing the lipid fraction was passed through a small DEAE Sepharose column (1 × 2.5 cm) in order to bind the 2-6 sialylated glycolipids. Nonsialylated glycolipids and detergent were washed out with pure M. The ganglioside fraction was eluted with 20 ml 50 mM ammonium acetate in M.

Second desalting step. The ganglioside containing fraction was taken to dryness under reduced pressure, sonicated in 5 ml aqueous 0.88% KCl and desalted on a Sep-Pak C-18 column as described above.

High performance liquid chromatography. The desalted 0.05 M ammonium acetate eluate after DEAE Sepharose chromato-graphy was dissolved in 1 ml C/M 85:15 (V/V) and injected onto a HPLC column (4 × 250 mm) filled with LiChrosorb Si 60 5 µm particles (Merck, Darmstadt, Germany). Elution was performed using a linear gradient from C/M/W 82.6:16.4:1 (V/V/V) to C/M/W 40:60:10 in 100 min at a flow rate of 1 ml/min. Fractions were collected every min. Each fraction was evaporated under a gentle stream of nitrogen and assayed for the presence of [alpha]2-6 sialylated glycolipids by thin layer chromatogram immunostaining using the pan [alpha]2-6 sialylated glycolipid specific antibody EBU-65. Immunoreactive bands were detected in fraction 32, fractions 37 and 38, fractions 42 and 43, and fractions 50 and 51, respectively. Identification of these gangliosides was performed by mass spectrometry and methylation analysis as described below. Fraction 43 contained pure 2-6 SnOC. Fraction 50 contained 2-6 SnDC (10%) and an internal fucosylated form of 2-6 SnDC (90%).

Electrospray mass spectrometry. A Finnigan MAT TSQ 700 triple quadrupole mass spectrometer (Finnigan MAT Corp., San Jose, CA) equipped with nanospray ion source (Protana, Odense, Denmark) was used for ESI-MS. The purified gangliosides were dissolved in methanol (~10 pmol/µl) and ~3 µl of solution was filled into gold coated nanospray glass capillaries (Protana, Odense, Denmark). The tip of the capillary was placed directly in front of the entrance hole of the heated transfer line of the mass spectrometer and a voltage of -800 V applied (negative ion mode). For collision induced dissociation (CID) experiments, the doubly charged parent ions were selectively transmitted by the first mass analyzer and directed into the collision cell (argon was used as collision gas) with a kinetic energy set around +40 eV.

Methylation analysis of glycolipids. For methylation analysis, glycolipids were permethylated according to Hakomori (Hakomori, 1964), purified on a Sephadex LH20 column, hydrolyzed, reduced, and peracetylated as described previously (Nimtz et al. 1990). Separation and identification of partially methylated alditol acetates was performed on a Finnigan gas chromatograph equipped with a 30 m DB5 capillary column connected to a GCQ ion trap mass spectrometer (Finnigan, San Jose, CA) running in the EI-mode.

Thin layer chromatography (TLC). TLC analysis was carried out on high performance TLC (HPTLC) Silica Gel 60 plates (Merck, Darmstadt, Germany). The running solvent was C/M/W (50:40:10) (V/V/V) containing 0.05% calcium chloride, and the running time was 40 min.

Ganglioside quantitation. For quantitation of TLC-separated gangliosides, the HPTLC plates were sprayed with resorcinol/HCl, then covered with a glass plate and heated at 95°C for 30 min. Densitometric measurements were made in transmission mode at 580 nm using a Shimadzu dual wavelength TLC Scanner CS9001 PC (Shimadzu, Düsseldorf, Germany). 0.5-3.0 µg GD3 were used for calibration.

TLC-immunostaining. Ganglioside antigens separated on HPTLC plates were detected by immunostaining using the method of Bethke (Bethke et al., 1986) with modifications (Kniep and Mühlradt, 1987).

Nonspecific immunochemical detection of gangliosides on HPTLC plates by digoxigenin-succinyl-[epsis]-aminocaproic acid hydrazide (DIG) labeling. DIG labeling was performed as described (Kniep and Mühlradt, 1990).

Flow cytometric analysis of cell surface antigens. Peripheral blood mononuclear cells (PBMC) were prepared from whole heparinized blood by Ficoll-Hypaque (Pharmacia, Freiburg, Germany) density centrifugation. Prior to immunofluorescence staining of PBMC, antibodies were subjected to centrifugation at 60,000 × g for 30 min to remove antibody-complexes and optimal antibody concentration was determined by serial dilution. For two-color fluorescence analysis PBMC were incubated with the respective carbohydrate specific antibody or the isotype matched control antibody (G155-228, IgM, Pharmingen, Hamburg, Germany), followed by PE-conjugated goat F(ab[prime])2 anti-mouse IgM (Coulter-Immunotec, Hamburg, Germany). After saturation of uncomplexed binding sites of the secondary antibody with normal mouse serum diluted 1/10 the cells were incubated with anti-CD3-FITC (UCHT1, IgG1)(Pharmingen) or anti CD19-FITC (B43, IgG1)(Pharmingen). After each incubation, cells were washed two times with cell-wash (Becton Dickinson, Heidelberg, Germany). Stained cells were analyzed on a FACScan (Becton Dickinson) using the Lysis II program.

Acknowledgments

We thank D.Y.Mason (Oxford) and T.F.Tedder (Boston) for generous gifts of antibodies

Abbreviations

2-6 SnDC, [alpha]2-6 sialosylneolactodecaosylceramide, X6Neu-AcnLc10Cer; 2-6 SnHC, [alpha]2-6 sialosylneolactohexaosylceramide, VI6NeuAcnLc6Cer; 2-6 SnOC, [alpha]2-6 sialosylneolactooctaosylceramide, VIII6NeuAcnLc8Cer; 2-6 SPG, [alpha]2-6 sialosylneolacto-tetraosylceramide, IV6NeuAcnLc4Cer; BCIP, 5-bromo-4-chloro-indolyl-3-phosphate; C, chloroform; CD, cluster of differentiation; CDw, provisional CD; CID, collision induced dissociation; CMP NANA, cytidine-5[prime]-monophospho-N-acetyl neuraminic acid; DEAE, diethylaminoethyl; DIG, digoxigenin-succinyl-[epsis]-aminocaproic acid hydrazide; ELISA, enzyme-linked immunosorbent assay; ESI-MS, electrospray mass spectrometry; FITC, fluorescein isothiocyanate; Gal, galactose; GalNAc, N-acetyl-galactosamine; GlcNAc, N-acetyl-glucosamine; GSL, glycosphingolipid; H, n-hexane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; i-P, 2-propanol; LacNAc, N-acetyllactosamine; M, methanol; MAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; TLC, thin layer chromatography; W, water.

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6To whom correspondence should be addressed at: Institut für Immunologie, Medizinische Fakultät Carl Gustav Carus, Karl-Marx-Strasse 3,D-01109 Dresden, Germany


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