Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells

Johannes Müthing5, Sevim Duvar, Dagmar Heitmann, Franz-Georg Hanisch1, Ulrich Neumann2, Günter Lochnit3, Rudolf Geyer3 and Jasna Peter-Katalinic4

Institute of Cell Culture Technology, University of Bielefeld, P.O. Box 100131, 33501, Bielefeld, Germany, 1Institute of Biochemistry, University of Cologne, 50931 Köln, Germany, 2Clinic for Poultry of the Hannover School of Veterinary Medicine, 30559 Hannover, Germany, 3Institute of Biochemistry, University of Giessen, 35392 Giessen, Germany and 4Institute of Medical Physics and Biophysics, University of Münster, 48149 Münster, Germany

Received on June 16, 1998; revised on September 22, 1998; accepted on October 13, 1998

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 × 109 human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally [alpha]2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.

Key words: gangliosides/neutral glycosphingolipids/antibodies/Lewisx antigen/TLC immunostaining

Introduction

Glycosphingolipids (GSLs), composed of a hydrophobic ceramide moiety and a hydrophilic carbohydrate portion, are located primarily in the outer leaflet of the plasma membrane but are also found in association with intracellular organelles (Gillard et al., 1993). Their structures and functions have been widely reviewed (Hakomori, 1990; Bergelson, 1995). GSLs act as receptors for toxins, bacteria and viruses (Karlsson, 1995) and provide biological specificity for numerous cellular functions such as cell growth regulation, development, and differentiation (Feizi, 1991; Bremer, 1994; Hakomori and Igarashi, 1995). GSL oligosaccharide chains spread out in the aqueous environment at the cell surface, and this makes them excellent candidates for cell surface and cell-cell recognition molecules (Schnaar, 1991; Crocker and Feizi, 1996).

There is abundant evidence that endothelial cells are actively involved in processes that affect human health and disease (Bevilacqua, 1993; Lasky, 1995; McEver, 1997). The luminal side of blood vessels is covered with a continuous endothelium, and, due to its unique position between the intracellular and extracellular spaces, the active processes of endothelial cells include interaction with neutrophils, platelets, and other inflammatory cells. Although the importance of GSLs as cell surface antigens and receptors has been well documented in recent years, the GSL composition of endothelial cells has generally received less attention than other membrane constituents. Human umbilical vein endothelial cells (HUVECs), like all normal human diploid cells, show a finite life span in vitro and require growth factors for their proliferation and long-term cultivation. Because GSLs are present in cells in small amounts and nontransformed cells grow slowly, it is difficult to obtain sufficient amounts of purified GSLs for their detailed structural characterization. Therefore, information on the GSL composition of human endothelial cells is limited (Gillard et al., 1987). Mass cultivation of anchorage dependent cells on microcarriers is the only way to get adequate amounts. We reported a large scale cultivation of HUVECs on microcarriers in a specifically designed bioreactor (Duvar et al., 1996) and used the methodology in this study to isolate and structurally characterize GSLs of the in vitro propagated HUVECs. To our knowledge, this is the first report about the full structural characterization of the major GSLs from HUVECs by immunochemical procedures combined with mass spectrometry analysis.

Results

Bioreactor production of HUVECs

HUVECs (4.85 × 109) at high viability of 95% were produced by three batch-fermentations with identical and constant culture parameters (microcarrier concentrations, O2 tension, etc.) (Duvar et al., 1996), rendering a bulk of homogeneous cells which were used as a source for isolation and structural characterization of GSLs from HUVECs. Subconfluent grown microcarriers showed the spread morphology of HUVECs during exponential growth and confluent covered beads the typical cobblestone pattern with strict contact inhibition, resulting in a regular cell monolayer at confluence (Duvar et al., 1996).


Figure 1. Detection of globo- and neolacto-series neutral GSLs in HUVECs. Orcinol stain (lane a) and TLC immunostains with anti-GbOse3Cer (lane b), anti-GbOse4Cer (lane c), anti-nLcOse4Cer (lane d) and anti-Forssman GSL antibodies (lane e). GSL equivalents corresponding to 3 × 107 (lane a), 2.6 ×107 (lane b), 1 × 107 (lane c), 4 × 107 (lane d), and 2.6 × 107 HUVECs (lane e) were applied and chromatographed in solvent I.

Neutral GSLs of HUVECs

Total neutral GSLs were 2200 µg, which corresponded to 0.45 µg/106 cells. GSL structures were verified by immunostaining with specific anti-neutral GSL antibodies on thin-layer chromatograms. GbOse4Cer, GbOse3Cer, and LacCer were detected as the main neutral GSLs in HUVECs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). Figure 1 shows the orcinol-stained TLC of whole neutral GSLs (lane a) and as examples the TLC immunostains of whole neutral GSL fraction with anti-GbOse3Cer (lane b), anti-GbOse4Cer (lane c), anti-nLcOse4Cer (lane d), and anti-Forssman GSL antibodies (lane e). As known from former studies, the anti-nLcOse4Cer antibody also binds to nLcOse6Cer due to the identity in their terminal disaccharides Gal[beta]1-4GlcNAc-R (Figure 1, lane d). Structures longer than GbOse4Cer of the globo-series, like the Forssman GSL, were not detected (Figure 1, lane e). Neutral GSLs of the ganglio-series (GgOse3Cer and GgOse4Cer) were not found.


Figure 2. Orcinol stained thin-layer chromatogram of individual neutral GSLs from HUVECs. GSL equivalents corresponding to 3 × 107 HUVECs (lane a) and 1 µg each of HPLC purified single neutral GSLs (lanes b-h, corresponding to compounds 1-7 in Table I) were chromatographed in solvent I.

FAB and GC mass spectrometry of major neutral GSLs from HUVECs

Individual neutral GSLs were obtained by reversed phase HPLC, followed by final purification on small silica gel 60 columns. The orcinol-stained TLC of purified single neutral GSLs is shown in Figure 2. Neutral GSLs were analyzed as native samples in negative ion FAB-MS to get data on the composition and sequence of sugar chains and the type of the ceramide moiety. Beside glucosyl- and lactosylceramide (compounds 1, 2, and 3 in Table I), neutral GSLs of the globo-series were identified according to their typical hexosyl sequence ions as well-separated triaosyl- and tetraosylceramides with long (C24:0) and short (C16:0) fatty acid chains, respectively (compounds 4-7 in Table I). As an example, the FAB mass spectrum of native GbOse3Cer (Figure 2, lane f; compound 5 in Table I) is shown in Figure 3. The FAB-MS data and the structures of main neutral GSLs of HUVECs are summarized in Table I.

The structures of HUVEC neutral GSLs were further verified by GC-EIMS of partially methylated alditol acetates. The results from methylation analysis were in agreement with the glycosidic structures expected for neutral globo-series GSLs. While the neutral GSLs in fraction 1 yielded only one derivative isographic with 1,5-di-O-acetyl glucitol (terminal unsubstituted glucose in monohexosylceramide), the compounds contained in fractions 2-7 gave rise to the formation of 1,4,5-tri-O-acetyl glucitol, the expected derivative of the 4-O-substituted core sugar of mammalian GSLs. Methylation analysis of fractions 2 and 3 yielded unsubstituted galactose (1,5-di-O-acetyl derivative) in addition to 4-O-substituted glucose and thus contained lactosylceramide. Fractions 4-7 revealed the presence of a 4-O-substituted galactose (1,4,5-tri-O-acetyl derivative), inferring the occurrence of globo-type trisaccharides in these compounds. In fractions 4 and 5 the 4-O-substituted galactose was only accompanied by a terminal galactose (1,5-di-O-actyl derivative) and should be derived from the unsubstituted globotrihexosyl moiety of GbOse3Cer. A further extension of this trisaccharide unit was indicated in fractions 6 and 7 by formation of the 1,5-di-O-acetyl N-acetylgalactosaminitol in conjunction with 1,3,5-tri-O-acetyl galactitol which are the expected derivatives of GbOse4Cer.

Table I. Nominal masses of molecular mass and fragment ions of native neutral GSLs from HUVECs obtained by negative ion FAB-MSa
No.b Cer- (m/z) HexCer- (m/z) Hex2Cer- (m/z) Hex3Cer- (m/z) Hex4Cer- (m/z) Fatty acid Structure
1 648 810 - - - 24:0 GlcCer
2 648 810 - - - 24:0 LacCer
  646 808 972 - - 24:1  
  620 - - - - 22:0  
3 536 698 860 - - 16:0 LacCer
4 648 810 972 1134 - 24:0 GbOse3Cer
5 536 698 860 1022 - 16:0 GbOse3Cer
6 648 810 972 1134 1334 24:0 GbOse4Cer
7 536 698 860 1022 1225 16:0 GbOse4Cer
aAll neutral GSLs are substituted with C18 sphingosine.
bCorresponding to Figure 2.

TLC immunodetection of minor neutral GSLs in HUVECs


Figure 3. Molecular mass and fragment ions of native GbOse3Cer (neutral GSL 5 in Table I) from HUVECs obtained by negative ion FAB-MS.

Positive CD15 bands obtained with anti-Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-R antibody below nLcOse6Cer, revealed the presence of lipid bound Lewisx antigen with poly-N-acetyllactosaminyl chains (extended fucosylated neolacto-series GSLs) in HUVECs. The Lewisx positive GSLs were expressed in relatively smaller amounts in HUVECs compared to human granulocytes (data not shown). TLC immunostaining with the C0514 monoclonal antibody revealed the isomeric Lewisa antigen only in small (less than Lewisx) quantities in HUVECs (data not shown).

Gangliosides of HUVECs

Total gangliosides (ganglioside fraction I) were 8100 µg, corresponding to 1.67 µg/106 cells. The major ganglioside (~90%) within the whole ganglioside fraction was GM3(Neu5Ac), followed by terminally [alpha]2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer (8% of the total resorcinol stain). IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (making together less than 2% of the total resorcinol stain) were found in minor quantities. Predominant GM3(Neu5Ac) was verified with the polyclonal chicken anti-GM3(NeuAc) antibody by TLC immunodetection (not shown). The TLC overlay assay with the chicken anti-GM3(Neu5Gc) antibody was negative, indicating the absence of Neu5Gc-substituted GM3, as well as the search for prolonged structures of GM3(Neu5Ac), i.e., GD3 and GM2, which were not expressed by HUVECs (data not shown).

FAB and GC mass spectrometry of GM3(Neu5Ac) specimens

The resorcinol-stained TLC of the GM3(Neu5Ac) specimens obtained by reversed phase HPLC of ganglioside fraction II is shown in Figure 4. Native gangliosides were submitted to negative ion FAB-MS to define their structural parameters, such as sugar size and sequence, attachment site of Neu5Ac, and the ceramide involved. The GM3 species present in HUVECs, separated by TLC (see Figure 4), differed in the fatty acid of their ceramide moiety. The complete list of molecular and sequence fragment ions is given in Table II. The clear-cut data of the 24:0 species (compound 1 in Table II; see also Figure 4, lane b), were the molecular [M-H+]--ion at m/z = 1263 and the sequence ions at m/z = 972, 810, and 648. The same holds for mixtures, as illustrated for compound 2, consisting of two components with fatty acids 24:1 and 22:0, respectively (see Table II and Figure 4, lane c). Their molecular ions were at m/z = 1261 and 1235, and the respective pairs of sequence ions at m/z = 970/944, 808/782, and 646/620. Further species were GM3(Neu5Ac) with 20:0, 18:0/18:1, and 16:0 fatty acid chains (compounds 3, 4, and 5; see Table II and Figure 4, lanes d, e, and f, respectively).


Figure 4. Resorcinol stained thin-layer chromatogram of GM3(Neu5Ac) specimens from HUVECs. Gangliosides (ganglioside fraction I) corresponding to 1.5 × 107 HUVECs (lane a) and amounts of 2 µg (lanes b and f), 1 µg (lanes d and e), and 0.5 µg (lane c) of individual HPLC purified GM3(Neu5Ac) specimens (compounds 1-5 of Table II) were chromatographed in solvent II. The asterisk indicates a resorcinol-negative compound.

Table II. Major nominal masses of molecular mass and fragment ions of native GM3(Neu5Ac) specimens from HUVECs obtained by negative ion FAB-MSa
No.b Cer- (m/z) HexCer- (m/z) Hex2Cer- (m/z) [M-H+]- (m/z) Fatty acid
1 648 810 972 1263 24:0
2 646 808 970 1261 24:1
  620 782 944 1235 22:0
3 592 754 916 1207 20:0
4 564 726 888 1179 18:0
  562 724 886 1177 18:1
5 536 698 860 1151 16:0
aAll GM3(Neu5Ac) gangliosides are substituted with C18 sphingosine.
bCorresponding to Figure 4.

Table III. GC-MS fatty acid analysis of GM3(Neu5Ac) specimens from HUVECsa
No.b C16:0 C18:0 C18:1 C20:0 C22:0 C24:1 C24:0
1 3.8 8.7         87.5
2 3.7 9.2     41.9 25.1 20.1
3 7.2 28.4   25.5 34.4   4.5
4 18.7 68.3 7.8 5.2      
5 64.7 35.3          
aRelative amounts are based on peak ratios of individual fatty acid derivatives normalized to 100%.
bCorresponding to Figure 4.

For fatty acid analysis, GM3(Neu5Ac) specimens were hydrolyzed, the resultant fatty acid methyl esters extracted with n-hexane, and analyzed without further purification by GC-MS. Fatty acid methyl esters were identified after electron-impact ionization according to their molecular and characteristic fragment ions. The fatty acid distribution for GM3(Neu5Ac) fractions 1-5 is summarized in Table III.

GC-mass spectrometry of partially methylated alditol acetates of all 5 GM3(Neu5Ac) specimens revealed the presence of methylated 1,3,5-tri-O-acetyl galactitol and 1,4,5-tri-O-acetyl glucitol, in accordance with the glycosidic structure of GM3. No hexosamine derivative (trace at m/z = 159) or alternatively substituted hexose (trace at m/z = 118) was detectable with the exception of trace amounts of 1,5-di-O-acetyl galactitol which may be derived from partial desialylation of the ganglioside.

Detection of neolacto-series gangliosides

The procedure for selective detection of terminally [alpha]2-3 and [alpha]2-6 sialylated neolacto-series gangliosides involves TLC immunostaining of separated gangliosides with an anti-nLcOse4Cer antibody with or without V.cholerae neuraminidase preincubation. GM3(Neu5Ac) and neolacto-series gangliosides from human granulocytes, consisting of IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer, and VI3Neu5Ac-nLcOse6Cer, have been used as references. As shown in Figure 5A (lane a), each ganglioside migrates as a double band due to substitution with C24 fatty acid (upper band) and C16 fatty acid (lower band). After incubation of human granulocyte gangliosides with V.cholerae neuraminidase on a TLC plate, IV3Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer, as well as IV6Neu5Ac-nLcOse4Cer, were visualized with the anti-nLcOse4Cer (anti-Gal[beta]1-4GlcNAc-R) antibody (Figure 5C, lane a). Without neuraminidase treatment two positive IV6Neu5Ac-nLcOse4Cer bands (Figure 5B, lane a) were detected since sialylation at position 6 of the terminal galactose does not hinder recognition (Müthing and Neumann, 1993). The presence of lactosamine containing gangliosides in HUVECs was proven by this method. The two faint resorcinol positive bands below GM3(Neu5Ac) (Figure 5A, lane b) were identified as ganglioside IV3Neu5Ac-nLcOse4Cer (8% of the total resorcinol stain) as shown in Figure 5C (lane b). This structure was confirmed by FAB-MS and GC-MS outlined in the next paragraph. Double bands chromatographing at the position of reference IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (Figure 5C, lane a), were detected in the HUVEC fraction (Figure 5C, lane b; less than 2% of the total resorcinol stain).


Figure 5. Detection of neolacto-series gangliosides in HUVECs. Resorcinol stain (A), TLC immunostain with anti-nLcOse4Cer antibody (B), and TLC immunostain with anti-nLcOse4Cer antibody after V.cholerae neuraminidase treatment (C). (A) Lane a: 15 µg of gangliosides from human granulocytes, lane b: gangliosides (ganglioside fraction I) equivalent to 1.5 × 107 HUVECs; (B and C) lanes a: 1 µg of human granulocytes gangliosides, lanes b: gangliosides (ganglioside fraction I) equivalent to 1 × 106 HUVECs. The chromatography was performed in solvent II. The asterisks indicate resorcinol-negative compounds.

FAB and GC mass spectrometry of IV3Neu5Ac-nLcOse4Cer

Two IV3Neu5Ac-nLcOse4Cer specimens were isolated by gradient Iatrobeads chromatography (=ganglioside fraction III). The resorcinol-stained chromatogram indicated one major upper and one minor lower band on the level of reference IV3Neu5Ac-nLcOse4Cer from human granulocytes (data not shown). Basic data for structural analysis of IV3Neu5Ac-nLcOse4Cer species in HUVECs were obtained from positive ion FAB-MS after permethylation. The attachment site of the Neu5Ac at the terminal Gal was documented by m/z = 580 ([Neu5Ac-Gal]+) and 825 ([Neu5Ac-Gal-GlcNAc]+), and the diversity in the ceramide portion was documented by [M+Na]+ molecular ions at m/z = 1820, 1930, and 1932 and by Cer+ ions at m/z = 548, 658, and 660 for fatty acid species 16:0, 24:1, and 24:0, respectively (Figure 6).


Figure 6. Positive ion FAB-MS and fragmentation pattern of the IV3Neu5Ac-nLcOse4Cer fraction after permethylation.

Methylation analysis of the sample revealed the presence of the four major components 1,3,5-tri-O-acetyl galactose, 1,4,5-tri-O-acetyl N-acetyl glucosaminitol, 1,4,5-tri-O-acetyl galactitol, and 1,4,5-tri-O-acetyl glucitol. The composition and pattern of monosaccharide substitution were in agreement with that of IV3Neu5Ac-nLcOse4Cer (sialylparagloboside). However, the fraction contained a contaminating compound (~30%) corresponding to a substitution isomer; this was indicated by the formation of 1,3,4,5-tetra-O-acetyl galactitol but absence of 1,5-di-O-acetyl galactitol.

TLC immunodetection of minor gangliosides in HUVECs

Positive CD15s (sialyl Lewisx) bands obtained by TLC overlay with anti-Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-R antibody after neuraminidase treatment indicated the presence of lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains whereas the isomeric sialyl Lewisa structure was undetectable. However, it should be mentioned that TLC overlay assays with the monoclonal anti-sialyl Lewisx antibodies, CSLEX1 and AM-3, were negative. TLC immunostaining also could not detect VIM-2 antigens (CDw65) and sulfoglucuronyl-neolacto series structures with HNK-1 epitope in the ganglioside fraction of endothelial cells (data not shown).

Detection of gangliosides with GM1-core

GM1 can be visualized on a TLC plate with choleragenoid, and GD1a, GD1b, GT1b, and GQ1b by conversion with V.cholerae neuraminidase to GM1 prior to treatment with choleragenoid according to Wu and Ledeen (1988). GM1 and GD1a were detected in trace quantities in HUVECs, not detectable by resorcinol staining.

Immunohistochemistry

Immunohistochemistry should be an adequate method for determining not only the presence of membrane components but also their localization in the cellular structures. The expression of dominant neutral GSLs and of ganglioside GM3(Neu5Ac) was proved by indirect immunofluorescence microscopy using carbohydrate-specific chicken polyclonal antibodies. Subconfluent grown cell layers cultivated in chamber slides showed different GSL specific plasma membrane and subcellular distribution patterns. A series of immunofluorescence micrographs revealing the distribution of the neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer (according to the biosynthesis pathway) was performed in parallel with DAPI staining of nuclear DNA in the same fields. The color reaction obtained with the anti-LacCer antibody revealed a weak plasma membrane staining, but a strong intracellular stain of LacCer positive structures around the nucleus. As an example of the series of immunofluorescence micrographs, the anti-GbOse3Cer stained HUVECs are shown in Figure 7A in parallel with the corresponding DAPI staining of nuclear DNA in the same field (Figure 7B). The anti-GbOse3Cer antibody showed increased plasma membrane staining and a network-like distribution in the cytoplasm in the vicinity of the nucleus. The anti-GbOse4Cer antibody indicated homogeneous plasma membrane distribution of GbOse4Cer and a strong subcellular expression. Positive staining was also obtained with the anti-nLcOse4Cer and the anti-Lewisx (CD15) antibodies. A control staining with an anti-GgOse4Cer antibody was negative. Ganglioside GM3(Neu5Ac) was found to be predominantly expressed on the cell surface, showing only moderate fluorescence around the nucleus. Parallel cultures stained with the secondary DTAF-labeled antibodies only were negative in all cases. To confirm the lipid nature of positive stainings by anti-GSL antibodies, control cultures were treated with methanol and chloroform/methanol (1:1, by vol.) before immunostaining. Considerable reduction of immunofluorescence intensity was always obtained but various amount of remaining fluorescence indicated in some cases cross-reactivity with protein-bound oligosaccharides.


Figure 7. Immunofluorescence staining of subconfluent grown HUVECs with anti-GbOse3Cer antibody. (A) Fluorescence micrograph, (B) DAPI stain of the same field. Scale bar, 7.5 µm.

Discussion

As environmental factors in cell cultures (pH value, hypoxia, and shear stress) alter the physiology of endothelial cells (Worthen et al., 1987; Farber and Rounds, 1990; Michiels et al., 1994) we paid particular attention to the scale-up cultivation of adherently growing endothelial cells, using a membrane stirred and bubble-free aerated 2 l bioreactor system (Duvar et al., 1996). This system accomplished the technical demands of controlled culture conditions and low stress in swirled culture flasks. This bioreactor equipment was also successfully used for large scale production and GSL characterization of bovine aortic endothelial cells (Müthing et al., 1996b; Duvar et al., 1997). In both cases we used serum from the same species: calf serum for bovine and human serum for HUVECs, reflecting to the in vivo environment of both cell types. Therefore, the GSL expression for example of HUVECs (Gillard et al., 1987) or of porcine aorta endothelial cells (Bouhours et al., 1996), cultivated in fetal calf serum supplemented medium (xenogenic for both species) and under hardly reproducible conditions in culture flasks, does not permit a detailed comparison with regard to the quantities particularly of minor GSLs due to the different culture conditions. However, we will discuss and refer to the differences of major GSL series of endothelial cells from different species and tissues, which might not be altered by using various culture conditions, in the following paragraph.

The GSL composition of endothelial cells vary greatly between different species and within the same species in regard to their origin from different vascular beds. Primary porcine endothelial cells were found to express GbOse4Cer and GbOse3Cer as the major neutral GSLs (like HUVECs) and Gal[alpha]1-3Gal- and Fuc[alpha]1-2Gal-terminated penta- and heptaglycosylceramides of the neolacto-series (Bouhours et al., 1996). The predominance of neolacto-series and the lack of globo-series neutral GSLs is characteristic for bovine aortic endothelial cells (Duvar et al., 1997), whereas HUVECs are enabled to synthesize both series as shown by us and others (Gillard et al., 1987). Bovine brain and aortic endothelial cells express almost the same major neutral and acidic GSLs (Kanda et al., 1994). However, the most prominent difference between these two cell types was the biosynthesis of sulfoglucuronyl nLcOse4Cer in brain, which was undetectable in aortic endothelial cells using the monoclonal antibody HNK-1. The presence of this sulfate and glucuronic acid-carrying GSL was also reported in rat brain microvessels (Miyatani et al., 1990). We did not find it in HUVECs, indicating its specific expression in brain vascular cells of different species. The overall feature of all endothelial cells analyzed so far is the dominance of GM3. However, GM3-expression also indicates the strongest difference between the species because cells of porcine (Bouhours et al., 1996) and bovine origin (Kanda et al., 1994; Duvar et al., 1997) synthesize GM3(Neu5Gc), whereas the N-glycolylated variant of GM3 is not synthesized by human cells. The same difference was found for the substitution of neolacto-type gangliosides with Neu5Gc in bovine and Neu5Ac in human endothelial cells (Duvar et al., 1997).

GSLs with Lex and sialyl Lewisx determinants were recognized by TLC immunostaining in the neutral and acidic GSL fraction, respectively, of unstimulated HUVECs. However, sialyl Lewisx was not detectable with the monoclonal antibody CSLEX1, but with an anti-Lewisx monoclonal antibody after neuraminidase treatment. These results are in agreement with reports from Renkonen and co-workers showing the expression of sialyl Lewisx epitope on HUVECs and on lymph node high endothelial venules, where it facilitates the L-selectin-dependent lymphocyte homing to lymph nodes (Majuri et al., 1994; Renkonen et al., 1997, and references therein).

Globotriaosylceramide and galabiosylceramide having a terminal Gal[alpha]1-4Gal disaccharide sequence are the specific receptors for verotoxins 1 and 2 (Lingwood et al., 1987; Waddell et al., 1990; and references therein). Both toxins have been found to be associated with hemorrhagic colitis and hemolytic uremic syndrome, pathologies of the endothelial cells lining the capillaries of the gastrointestinal tract and the renal glomerulus, respectively. Lingwood and coworkers have demonstrated the important role of GbOse3Cer fatty acid microheterogeneity responsible for different verotoxins binding capacities. This was shown by use of the TLC overlay and microtiter plate binding assay (Pellizzari et al., 1992) as well as in cell reconstitution experiments in vitro (Kiarash et al., 1994) indicating that the binding of verotoxin to GbOse3Cer in a lipid matrix is affected by the ceramide fatty acid chain length of the GSL. C20:0 and C22:1 fatty acid containing GbOse3Cer had the greatest capacity to bind verotoxin 1, whereas C18:0 and C18:1 homologues showed the greatest capacity for verotoxin 2c binding. In all cases an unsaturated fatty acid in GbOse3Cer increased verotoxin binding (Kiarash et al., 1994). Our study showed that GbOse3Cer with C24:0 and C16:0 fatty acid were the dominant GbOse3Cer species in HUVECs. This could at least in part explain the lower accessibility of verotoxins towards GbOse3Cer in HUVECs compared to renal endothelial cells. Also the latter cells have 50 times larger GbOse3Cer content compared to HUVECs (Obrig et al., 1993).

Regarding the presumed functional aspects of endothelial GSLs, several GSLs have been reported to be involved in biomolecular interaction via 'head-to-head" contact by interaction of their oligosaccharides belonging to different cells (for reviews, see Hakomori 1990; Hakomori and Igarashi, 1995; and references therein). GSLs with Lewisx motifs, GM3 and lactosylceramide of HUVECs are potential candidates for heterotypic cell aggregation of leukocytes and endothelial cells (Eggens et al., 1989; Kojima et al., 1991, 1992). The specific modulation of cell surface GSLs by inflammatory mediators such as interferon-[gamma], tumor necrosis factor-[alpha], and interleukin-1 suggests that GSLs may play a role in the altered adhesive and receptor activities of activated endothelial cells (Gillard et al., 1990; van de Kar et al., 1992). Using in vitro cultivated cells like HUVECs or other endothelial cells in order to elucidate the functional role of GSLs in cell-cell or cell-toxin interactions, full characterization of GSL structures, such as reported in this study for in vitro grown HUVECs, is a necessary prerequisite. Referring to the early report published two decades ago by Huang (1978), the functional role of GSLs in cell-cell adhesion events still remains to be clarified and is of outstanding importance for further investigations.

Materials and methods

Cells and culture conditions

Primary HUVECs were isolated according to the procedure described by Gimbrone et al. (1974) and the modifications of Friedl et al. (1989). HUVECs were cultivated in gelatin precoated polystyrene culture flasks (Duvar et al., 1996) in a 1:1 mixture of Iscove's MDM and Ham's F12 basal medium (Gibco BRL, Eggenstein, Germany) supplemented with 20% (v/v) human serum (German Red Cross Blood Transfusion Service, Institute Springe, Germany), 2.1 g NaHCO3, 1.25 µg/l human basic fibroblast growth factor (bFGF, Sigma GmbH, Deisenhofen, Germany), 5 mg/l sodium heparin (Serva Chemie, Heidelberg, Germany), 2 mmol/l glutamine, 12.5 µmol/l [beta]-mercaptoethanol, 5 mg/l iron saturated human transferrin (Behring Werke AG, Marburg, Germany), 65 mg/l benzylpenicillin, and 100 mg/l streptomycin sulfate (=culture medium). A cell bank of HUVECs was established with cells of the third passage, which were used for large scale production on microcarriers in bioreactors (see next paragraph).

Mass cell cultivation

Anchorage dependent HUVECs were propagated on gelatin precoated Dormacell microcarriers (Pfeifer & Langen, Dormagen, Germany) as recently reported in detail (Duvar et al., 1996). Briefly, inoculums for 2 l bioreactor cultivations, corresponding to the seventh passage, were prepared in 200 ml spinner cultures containing 3 g/l of Dormacell microcarriers (type 2.9% nitrogen content). Scale-up batch fermentations were performed in a 2 l membrane stirred bioreactor as outlined elsewhere (Duvar et al., 1996). The microcarriers covered with HUVECs were washed twice with phosphate-buffered saline (PBS) before chloroform/methanol extraction (see below).

Isolation of GSLs from HUVECs

GSLs from 4.85 × 109 cells corresponding to a volume of about 330 ml packed beads covered with HUVECs, which were obtained from 3 consecutive batch fermentations, were isolated according to standard procedures (Ledeen and Yu, 1982) as described in detail by Duvar et al. (1997). Neutral GSLs and gangliosides were separated by anion exchange chromatography on DEAE-Sepharose CL-6B (Pharmacia Fine Chemicals, Freiburg, Germany) as reported by Müthing et al. (1987). The ganglioside fraction was incubated for 1 h at 37°C in aqueous 1 N NaOH to saponify phospholipids followed by neutralization with acetic acid and dialysis. Neutral GSLs and gangliosides were further purified by adsorption chromatography on silica gel 60 (Merck) and Iatrobeads 6RS-8060 (Macherey-Nagel, Düren, Germany), respectively, as described by Ueno et al. (1978). Finally, neutral GSLs were purified by Florisil (Merck) chromatography as their peracetylated derivatives (Saito and Hakomori, 1971).

Purification of neutral GSLs

Individual neutral GSLs were obtained by reversed phase HPLC. The HPLC system used (Gilson Abimed, Langenberg, Germany) consisted of three M303 HPLC pumps, a high pressure mixer M811 and a fraction collector M202. The whole fraction of neutral GSLs was dispersed in 1 ml of methanol/water (90/10, v/v), applied to a reversed phase column (Nucleosil 7RP18 250 mm × 4 mm, Macherey-Nagel) and eluted with an one step gradient from methanol/water (90/10, v/v) to methanol/water (100/0, v/v). Final purification of single neutral GSLs was performed on a small silica gel 60 column by stepwise elution from chloroform/methanol (7/1) to (2/1), each by volume.

Ganglioside fractionation

The whole ganglioside fraction eluted from Iatrobeads with chloroform/methanol (1/2, by vol.) was designated as ganglioside fraction I. GM3(Neu5Ac) and IV3Neu5Ac-nLcOse4Cer were isolated by successive chloroform/methanol elution with 85/15, 75/25, 65/35, 60/40, 55/45, 1/3 (each by volume), and methanol. Elution with chloroform/methanol (65/35, v/v) gave ganglioside fraction II (GM3(Neu5Ac)) and the pooled (60/40, v/v) and (55/45, v/v) eluates represented ganglioside fraction III (IV3Neu5Ac-nLcOse4Cer). Individual GM3(Neu5Ac)-specimens were obtained by reversed phase HPLC as described above for the purification of individual single GSLs.

High performance thin-layer chromatography

GSLs were separated on high-performance thin-layer chromatography plates (HPTLC-plates, size 10 cm × 10 cm, thickness 0.2 mm, Merck; Art. no. 5633). Neutral GSLs were chromatographed in solvent I (chloroform/methanol/water, 120/70/17, each by vol.) and gangliosides in solvent II (chloroform/methanol/water, 120/85/20, each by vol.), the latter supplemented with 2 mM CaCl2. Neutral GSLs were visualized with orcinol (Svennerholm, 1956) and gangliosides with resorcinol (Svennerholm, 1957). Chromatograms were scanned with a CD60 scanner (Desaga, Heidelberg, Germany) equipped with an IBM compatible personal computer and densitometric software. Bands were measured in reflectance mode at 580 nm (resorcinol) and 550 nm (orcinol) with a light beam slit of 0.1 × 2 mm.

Reference GSLs

A reference neutral GSL fraction with LacCer and nLcOse4Cer as main components was prepared from human granulocytes (see Müthing et al., 1994a). A ganglioside mixture composed of GM3(Neu5Ac), IV3Neu5Ac-nLcOse4Cer (sialylparagloboside), IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer was isolated from human granulocytes as previously described (Müthing et al., 1993).

Monoclonal and polyclonal antibodies; cholera toxin B subunit

Neutral GSL specific monoclonal and polyclonal antibodies and their respective dilutions employed for TLC immunostaining have been recently reported (Duvar et al., 1997). The polyclonal rabbit anti-GgOse4Cer and chicken anti-LacCer, anti-GbOse3Cer, anti-GbOse4Cer, and anti-nLcOse4Cer antibodies have also been characterized in earlier publications (Bethke et al., 1986; Müthing and Mühlradt, 1988; Müthing and Neumann, 1993; Müthing et al., 1994a; Müthing and Kemminer, 1996; Müthing and Cacic, 1997). The specificity of the monoclonal anti-Forssman GSL antibody has been previously reported (Bethke et al., 1986, 1987). The monoclonal anti-Lewisx, anti-GgOse3Cer, and anti-Lewisa antibodies were the same as in our recent study (Duvar et al., 1997).

Ganglioside-specific monoclonal and polyclonal antibodies and their respective dilutions employed for TLC immunostaining have been recently reported (Duvar et al., 1997). The polyclonal chicken anti-GM3(Neu5Ac), anti-GM3(Neu5Gc), and anti-nLcOse4Cer (the latter used for the detection of neolacto-series gangliosides after neuraminidase treatment) have been characterized in our former reports (Müthing and Neumann, 1993; Müthing et al., 1994a,b; Müthing and Kemminer, 1996). The anti-sialyl Lewisx antigen antibody producing hybridoma (clone CSLEX1, HB 8580) was from the American Type Culture Collection (ATCC; Rockville, MD). The anti-sialyl Lewisx (AM-3), the VIM-2 specific CDw65 and the anti-HNK-1 monoclonal antibody, the latter specific for sulfoglucuronyl-neolacto series GSLs, were the same as in our previous studies (Müthing et al., 1996a; Duvar et al., 1997). Sialyl Lewisa and sialyl Lewisx were detected with antibodies from clones C0514 and 88H7 (see above), respectively, after Vibrio cholerae neuraminidase treatment (see below). The hybridoma clone producing monoclonal anti-melanoma antibody (Dippold et al., 1980) specific for ganglioside GD3 was obtained from the ATCC (HB 8445).

Cholera toxin B subunit (=choleragenoid) specific for ganglioside GM1 was from Sigma (no. C-7771) and goat anti-choleragenoid antiserum from Calbiochem (Frankfurt a.M., Germany; no. 227040).

TLC immunostaining

Secondary rabbit anti-chicken IgG, goat anti-rat IgG, goat anti-mouse IgG and IgM, and goat anti-rabbit IgG antisera, all affinity chromatography-purified and labeled with alkaline phosphatase, were purchased from Dianova and used in a 1:2000 dilution (Duvar et al., 1997). Two reviews concerning the details of the TLC immunostaining procedure have been recently published (Müthing, 1996, 1998). Anti-Lewisa, anti-Lewisx, anti-sialyl Lewisx, and anti-VIM-2 antigen (CDw65) antibodies were used in a 'microscale method" (Müthing et al., 1996a) by applying 80 µl aliquots per cut lane of chromatographed GSLs. Neuraminidase treatment of neolacto-series gangliosides with [alpha]2-3 substituted sialic acid is necessary prior to immunostaining with anti-nLcOse4Cer antibody, whereas [alpha]2-6 sialylated neolacto-type gangliosides can be detected without enzyme treatment, since sialylation at position 6 of the terminal galactose does not hinder recognition (Müthing and Neumann, 1993). Silica gel fixed plates were incubated with 2.5 mU/ml V.cholerae neuraminidase (Behring Werke AG) for 2 h at room temperature, followed by immunostaining with anti-nLcOse4Cer antibody (for details, see Müthing and Neumann, 1993; Müthing et al., 1994a).

The TLC binding assay using choleragenoid for specific detection of GM1 was developed by Magnani et al. (1980) and was used according to the modifications described by Müthing et al. (1992).

Mass spectrometric analyses of GSLs

The permethylation was carried out according to Ciucanu and Kerek (1984). Native GSLs and the derivatives were structurally characterized by negative and positive ion mode fast atom bombardment-mass spectrometry (FAB-MS), respectively, as previously reported (Peter-Katalinic and Egge, 1990).

Fatty acid analysis

For fatty acid analysis, GSLs were hydrolyzed according to Gaver and Sweeley (1965) with 200 µl of aqueous and methanolic HCl (1 M HCl, 10 M H2O) at 100°C for 16 h. The resultant fatty acid methyl esters were extracted three times with 500 µl n-hexane, analyzed by capillary GC-MS, and identified as their molecular and characteristic fragments using the instrumentation described previously (Lochnit et al., 1997). For the separation of fatty acid species a fused-silica capillary column (DB-1701, 0.25 mm ID, 30 m; ICT, Bad Homburg, Germany) was used. The column temperature was increased at 7°C/min from 80°C to a final temperature of 320°C and held isothermally for 10 min. Spectra were recorded after electron-impact ionization at an electron energy of 1.1215 × 10-17 J.

Methylation analysis

The GSLs were methylated according to Ciucanu and Kerek (1984) and subjected to sequential hydrolysis, reduction with NaBD4 and O-acetylation as described by Levery and Hakomori (1987). The samples were taken up in 20 µl of chloroform and aliquots were injected onto a 15 m DB5 capillary column which was heated from 100 to 300°C in a temperature gradient of 10°C/min. The eluted partially methylated alditol acetates were detected by 70 eV EIMS in an MD800 (interface: 250°C; ion source: 200°C) using the scan modus or single ion monitoring at m/z 118, 159, 175, 189, 190, 205, 231, 233, 234, 261, 275, 290, 305, 318, and 346.

Immunohistochemistry

HUVECs were seeded with 2 × 104 cells/cm2 in fibronectin-coated (Sigma, 10 µg/ml in PBS) 8 well polystyrene Chamber Slides (Nunc GmbH, Wiesbaden-Biebrich, Germany; no. 177445), cultivated until subconfluence, and immunostained as described for bovine aortic endothelial cells (Duvar et al., 1997).

Acknowledgments

This work was supported financially by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 549 and by a graduate fellowship of the University of Bielefeld for S.Duvar. We are grateful to Prof. Dr. H.Egge (University of Bonn), in whose laboratory the FAB-MS has been carried out and Prof. Dr.-Ing. J.Lehmann for his generous support. We thank Prof. Dr. Ana Marusic (School of Medicine, University of Zagreb, Croatia) for critical reading and correction of the manuscript.

Abbreviations

BSA, bovine serum albumin; DAPI, 4[prime],6-diamidine-2-phenylindole-dihydrochloride; DTAF, dichlorotriazinylamino fluorescein; FAB-MS, fast atom bombardment-mass spectrometry; GC-EIMS, gas chromatography-electron impact mass spectrometry; GSL(s), glycosphingolipid(s); HNK-1, monoclonal antibody recognizing HSO3-3GlcA[beta]1-3Gal[beta]1-4GlcNAc-R structures; HPLC, high-performance liquid chromatography; HPTLC, high performance thin-layer chromatography; HUVECs, human umbilical vein endothelial cells; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid (Reuter and Schauer, 1988); PBS, phosphate-buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Gal[beta]1-4Glc[beta]1-1Cer; globotriaosylceramide or GbOse3Cer, Gal[alpha]1-4Gal[beta]1-4Glc[beta]1-1Cer; gangliotriaosylceramide or GgOse3Cer, GalNAc[beta]1-4Gal[beta]1-4Glc[beta]1-1Cer; globotetraosylceramide or GbOse4Cer, GalNAc[beta]1-3Gal[alpha]1-4Gal[beta]1-4Glc[beta]1-1Cer; gangliotetraosylceramide or GgOse4Cer, Gal[beta]1-3GalNAc[beta]1-4Gal[beta]1-4Glc[beta]1-1Cer; lacto-N-neotetraosylceramide or nLcOse4Cer, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer; Forssman GSL, GalNAc[alpha]1-3GalNAc[beta]1-3Gal[alpha]1-4Gal[beta]1-4Glc[beta]1-1Cer; lacto-N-norhexaosylceramide or nLcOse6Cer, Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer; GM3, II3Neu5Ac-LacCer; GM1, II3Neu5Ac-GgOse4Cer; GD1a, IV3Neu5Ac,II3Neu5Ac-GgOse4Cer; GD1b, II3(Neu5Ac)2-GgOse4Cer; GT1b, IV3Neu5Ac,II3(Neu5Ac)2-GgOse4Cer; GQ1b, IV3(Neu5Ac)2,II3(Neu5Ac)2-GgOse4Cer; sialyl Lewisa, sialylated lacto-N-fucopentaose II, Neu5Ac[alpha]2-3Gal[beta]1-3(Fuc[alpha]1-4)GlcNAc[beta]1-3Gal-R; sialyl Lewisx, sialylated lacto-N-fucopentaose III, Neu5Ac[alpha]2-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal-R; VIM-2, Neu5Ac[alpha]2-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-3Gal-R. Only Neu5Ac-substituted gangliosides are presented in this list of abbreviations.

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5To whom correspondence should be addressed at: Universität Bielefeld, Technische Fakultät, Arbeitsgruppe Zellkulturtechnik, Postfach 100131, D33501 Bielefeld, Germany


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