Isolation and structural characterization of ...

Glycobiology vol. 7 no. 8 pp. 1099-1109, 1997

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Sevim Duvar, Jasna Peter-Katalinic'1, Franz-Georg Hanisch2 and Johannes Miithing3

action as well as receptors for blood components and macromolecules of the extracellular matrix.

Institute of Cell Culture Technology, University of BielefeldJP.O. Box 100131, 33501 Bielefeld, Germany, 'Institute for Medical Physics and Biophysics, University of Miinster, 48149 Miinster, Germany, and 2Institute of Biochemistry, University of Cologne, 50931 Koln, Germany

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

3 To whom correspondence should be addressed at: Universitat Bielefeld, Technische Fakultat, Arbeitsgruppe Zellkulturtechnik, Postfach 100131, D33501 Bielefeld, Germany

© Oxford University Press

The luminal side of blood vessels is covered with a continuous monolayer of endothelial cells. For a long time, the inner lining of the vasculature system was interpreted as a uniform layer of cells that provide a protective, nonadherent surface and transport various substances from or into the bloodstream. Many new functions of the vascular epithelium have been described meanwhile, e.g., the important role of endothelial cells in the regulation of blood coagulation, inflammation, and immune response (Zimmerman et al, 1992; Bevilacqua, 1993; Feizi, 1993; Smith, 1993; Varki, 1994; Lasky, 1995). Endothelial cells throughout the vascular system share many common structural and functional characteristics, but on the other hand they also show a wide diversity depending on the nature of the vascular bed (Fajardo, 1989). This structural heterogeneity is suggested to play a functional role in the extravasation of cancer cells mediated by specific adhesion molecules resulting in organ-preference of metastatic tumor cells and unique organ colonization patterns (Pauli et al, 1990). The active processes of endothelial cells include production of bioactive lipids such as arachidonic acid metabolites (Whatley et al, 1990), and for metabolism, drug transport, and glycoconjugate drug design, research on endothelial cells has become a growing field of clinical and pharmaceutical investigation (Audus et al, 1990; Karlsson, 1991). Glycosphingolipids (GSLs) are composed of long-chain base and fatty acid, which together make up the ceramide portion, and carbohydrate moieties. Structures (Hakomori, 1989; Yu and Saito, 1989), functions (Prokazova et al, 1988; Igarashi et al, 1989; Saito, 1989; Hakomori, 1990; Schnaar, 1991; Zeller and Marchase, 1992; Hakomori and Igarashi, 1995), and metabolism of GSLs (Ledeen, 1989; Schwarzmann and Sandhoff, 1990; van Echten and Sandhoff, 1993) have been widely reviewed. Gangliosides are characterized by the presence of one or more sialic acid units in the oligosaccharide chain. The parent compounds are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), which are known to play crucial roles in various biological functions (Varki, 1992; Schauer etal, 1995). GSLs are located primarily in the outer leaflet of the plasma membrane, and in polarized epithelial cells asymmetric distribution of GSLs on the apical surface and the basolateral membrane has been reported (Nichols et al, 1988; Spiegel et al, 1988). This indicated the existence of mechanisms which can produce different degrees of polarity for specific lipids in polarized epithelial cells. Al1099

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Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 109 primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gaipi-4(Fucal3)GlcNAcpi-R antibody indicated the presence of lipid bound Lewis" antigen, whereas the isomeric Lewis3 structure (Gaipi-3(Fuccd-4)GlcNAcpi-R) was not detectable. G M3 substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. GM3-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C 18 sphingosine was the only long chain base, whereas variation occurred due to C ^ o ^ a and C 16 fatty acids. Terminally a2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (

References Abo.T. and Balch,C.M. (1981) A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J. Immunol., 127, 1024— 1029. Audus.K.L., Bartel.R.L., Hidalgo.IJ. and Borchardt.R.T. (1990) The use of cultured epithelial cells and endothelial cells for drug transport and metabolism studies. Pharmacol. Res., 7, 435-451. Bergelson.L.D. (1995) Serum gangliosides as endogenous immunomodulators. Immunol. Today, 16, 483-486. Bethke.U., MiithingJ., Schauder.B., Conradt,P. and Miihlradt.P.F. (1986) An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms. J. Immunol. Methods, 89, 111-116. Bethke.U., Kniep,B. and Muhlradt.P.F. (1987) Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes. J. Immunol, 138, 4329-4335. Bevilacqua,M.P. (1993) Endothelial-leukocyte adhesion molecules. Annu. Rev. Immunol, 11, 767-804. Blaszczyk.M., Hansson.G.C, Karlsson,K.-A., Larson.G., Stromberg.N., Thurin,J., Herlyn,M., Steplewski.Z. and Koprowski,H. (1984) Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies. Arch. Biochem. Biophys., 233, 161-168. Bouhours.D., Pourcel,C. and Bouhours,J.-F. (1996) Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing major epitope for human xenoreactive antibodies (Galcd—3Gal), blood group H determinant and N-glycolylneuraminic acid. Glycoconjugate J., 13, 947—952. Bovin,N.V. (1997) Carbohydrate-carbohydrate interaction. In Gabius,H.J. and Gabius.S. (eds.), Glycosciences. Chapman & Hall, Weinheim, pp. 277-289. Cacic.M., Miithing.J., Krachum,I., Neumann.U. and Weber-Schiirholz.S. (1994) Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining. Glycoconjugate J., 11, 477-485. Chien,J.-L., Li,S.-C, Laine.R. and Li,Y.-T. (1978) Characterization of gangliosides from bovine erythrocyte membranes. J. Biol. Chem., 253, 40314035. Chou,D.K.H., Ilyas,A.A., Evans.J.E., Costello.C, Quarles,R.H. and Jungalwala,F.B. (1986) Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. J. Biol. Chem., 261, 11717-11725. Ciucanu.I. and Kerek,F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209-217. Duvar,S., Miithing.J., Mohr.H. and LehmannJ. (1996) Scale up cultivation of primary human umbilical vein endothelial cells on microcarriers from spinner vessels to bioreactor fermentation. Cytotechnology, 21, 61-72. Eggens,I., Fenderson.B., Toyokuni.T., Dean.B., Stroud.M. and Hakomori,S.-I. (1989) Specific interaction between Lex and Le* determinants. J. Biol. Chem., 264, 9476-9484. Fajardo.L.F. (1989) The complexity of endothelial cells. Am. J. Clin. Pathol, 92, 241-250. Feizi.T. (1993) Oligosaccharides that mediate mammalian cell-cell adhesion. Curr. Opin. Struct. Biol, 3, 701-710. Gillard,B.K., Jones.M.A. and Marcus.D.M. (1987) Glycosphingolipids of human umbilical vein endothelial cells and smooth muscle cells. Arch. Biochem. Biophys., 256, 435-445. Gillard,B.K., HeathJ.P., Thurmon,L.T. and Marcus.D.M. (1991) Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells. Exp. Cell Res., 192, 433-444. Hakomori,S.-I. (1989) Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res., 52, 257-331. Hakomori,S.-I. (1990) Bifunctional role of glycosphingolipids. J. Biol. Chem., 265, 18713-18716. Hakomori,S.-I. and Igarashi.Y. (1995) Functional role of glycosphingolipids in cell recognition and signaling. J. Biochem., 118, 1091-1103. Hanisch,F.-G. and Peter-KatalinicJ. (1992) Structural studies on fetal mucins from human amniotic fluid. Eur. J. Biochem., 205, 527-535. Hanisch,F.-G., Hanski,C. and Hasegawa,A. (1992) Sialyl Lewis" antigen as defined by monoclonal antibody AM-3 is a marker of dysplasia in the colonic adenoma-carcinoma sequence. Cancer Res., 52, 3138-3144.

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BAECs were seeded with 2 x 1 0 " cells/cm2 in fibronectin-coated (10 (xg/ml in PBS) 8 well polystyrene Chamber Slides (Tissue culture chambers, Nunc GmbH, Wiesbaden-Biebrich, Germany; no. 177445) and cultivated until subconfluence. The culture fluid was sucked off and the layer was washed three times with PBS, fixed with 7% formaldehyde, washed three times with 3% BSA in PBS and then incubated for 1 h with anti-LacCer antibody or antiGM3(Neu5Gc) antibody, each diluted 1:15 with 3% BSA in PBS. After 4-fold washing with 3%BSA in PBS, cells were incubated for 1 h with dichlorotriazinylamino fluorescein ( = DTAF) labeled secondary antibody (Dianova), diluted 1:40 in 3% BSA in PBS. The solution was removed and after 4-fold washing, the layer was incubated with 10"5% (w/v) 4',6-diamidine-2phenylindole-dihydrochloride (DAPI; Boehringer, Mannheim, Germany) in PBS, followed by embedding with 20% (w/v) Mowiol (Hoechst, Frankfurt a.M., Germany). Bound DTAF labeled antibodies as well as stained nuclei were evaluated under fluorescence microscope, equipped with filter sets adequate to the maxima of absorption/emission of DTAF (495 nm/528 nm) and of DAPI (368 nm/488 nm). For details see Cacic el al. (1994).

IV3(Neu5Ac)2,II3(Neu5Ac)2-GgOse4Cer; sialyl Lewis", sialylated lacto-Nfucopentaose II, Neu5Aca2-3Gaipi-3(Fucal-4)GlcNAcfSl-3Gal-R; sialyl Lewisx, sialylated lacto-N-fucopentaose m , Neu5Aca2-3Gaipi-4(Fucocl3)GlcNAcpi-3Gal-R; VIM-2, Neu5Aca2-3Gaipi-4GlcNAcpi-3Gaipi4(Fucal-3)GlcNAcpi-3Gal-R. Only Neu5Ac-substituted gangliosides are presented in this list of abbreviations.

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Mannoni,P., Janowska-Wieczorek,A., Turner,A.R., MacGann,L. and Turc,J.M. (1982) Monoclonal antibodies against human granulocytes and myeloid differentiation. Human Immunol, 5, 309-323. Miyatani,N., Kohriyama,T., Maeda,Y. and Yu,R.K. (1990) Sulfated glucuronyl paragloboside in rat brain microvessels. J. Neurochem., 55, 577-582. MuthingJ. (1996) High-resolution thin-layer chromatography of gangliosides. J. Chromatogr. A, 720, 3-25. MuthingJ. and Cacic.M. (1997) Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography. Glycoconjugate J., 14, 19-28. Miithing,J. and Kemminer.S.E. (1996) Nondestructive detection of neutral glycosphingolipids with lipophilic fluorochromes and their employment for preparative high performance thin-layer chromatography. Anal. Biochem., 238, 195-202. MuthingJ. and Miihlradt.P.F. (1988) Detection of gangliosides of the G M l b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment. Anal. Biochem., 173, 10-17. MuthingJ. and Neumann,U. (1993) Selective detection of terminally a2-3 and a2—6 sialylated neolacto-series gangliosides by immunostaining on thin layer chromatograms. Biomed. Chromatogr., 7, 158-161. MuthingJ., Egge,H., Kniep.B. and Miihlradt,P.F. (1987) Structural characterization of gangliosides from murine T lymphocytes. Eur. J. Biochem., 163, 407-416. MuthingJ., Portner.A. and Jager.V. (1992) Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium. Glycoconjugate J., 9, 265-273. Miithing.J., Unland.F., Heitmann,D., Orlich.M., Hanisch,F.-G., PeterKatalinic,J., Knauper.V., Tschesche,H., Kelm.S., Schauer.R. and LehmannJ. (1993) Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes. Glycoconjugate J., 10, 120-126. MuthingJ., Maurer.U., Sostaric,K., Neumann,U., Brandt.H., Duvar,S., PeterKatalinicJ. and Weber-Schiirholz,S. (1994a) Different distribution of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses. J. Biochem., 115, 248-256. MuthingJ., Steuer,R, Peter-KatalinicJ., Marx,U., Bethke.U., Neumann.U. and LehmannJ. (1994b) Expression of gangliosides G M3 (NeuAc) and GM3(NeuGc) in myelomas and hybridomas of mouse, rat, and human origin. J. Biochem., 116, 64-73. MuthingJ., Spanbroek,R., Peter-KatalinicJ., Hanisch,F.-G., Hanski,C, Hasegawa,A., Unland,F., LehmannJ., Tschesche.H. and Egge.H. (1996a) Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes. Glycobiology, 6, 147-156. MuthingJ., Duvar,S., Nerger.S., Buntemeyer.H. and LehmannJ. (1996b) Microcarrier cultivation of bovine aortic endothelial cells in spinner vessels and a membrane-stirred bioreactor. Cytotechnology, 18, 193-206. Nichols,G.E., Shiraishi,T. and Young,W.W.Jr. (1988) Polarity of neutral glycolipids, gangliosides, and sulfated lipids in MDCK epithelial cells, j . Lipid Res., 29, 1205-1213. Pauli.B.U., Augustin-Voss,H.G., El-Sabban,M.E., Johnson.R.C. and Hammer,D.A. (1990) Organ-preference of metastasis. The role of endothelial cell adhesion molecules. Cancer Metastasis Rev., 9, 175—189. Peter-KatalinicJ. and Egge,H. (1990) Desorption mass spectrometry of glycosphingolipids. Methods Enzymol, 193, 713-733. Prokazova,N.V and Bergelson,L.D. (1994) Gangliosides and atherosclerosis. Lipids, 29, 1-5. Prokazova,N.V., Orekhov,A.N., Mukhin,D.N., Mikhailenko.I.A., Kogtev.L.S., Sadovskaya.V.L., Golovanova,N.K. and Bergelson,L.D. (1987) The gangliosides of adult human aorta: intima, media and plaque. Eur. J. Biochem., 167, 349-352. Prokazova,N.V., Dyatlovitskaya,E.V. and Bergelson.L.D. (1988) Sialylated lactosylceramides. Possible inducers of non-specific immunosuppression and atherosclerotic lesions. Eur. J. Biochem., 172, 1-6. Prokazova,N.V., Mukhin.D.N., Orekhov.A.N., Gladkaya,E.M., Vasilevskaya, V.V., Mikhailenko.I.M., Sadovskaya.V.L., Bushuev,V.N. and Bergelson.L.D. (1989) Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue. Eur. J. Biochem., 180, 167-171. Reuter.G. and Schauer.R. (1988) Suggestions on the nomenclature of sialic acids. Glycoconjugate J., 5, 133-135. Saito,M. (1989) Bioactive sialoglycosphingolipids (gangliosides): potent differentiation-inducers for human myelogenous leukemia cells. Develop. Growth Differ., 31, 509-522. Saito.T. and Hakomori,S.-I. (1971) Quantitative isolation of total glycosphingolipids from animal cells. J. Lipid Res., 12, 257-259. Schauer.R., Kelm,S., Reuter.G., Roggentin,P. and Shaw.L. (1995) Biochem-


Hanski,C, Bornhoeft,G., Topf,N., Hermann,U., Stein,H. and Riecken,E.O. (1990) Detection of mucin marker for the adenoma-carcinoma sequence in human colonic mucosa by monoclonal antibody AM-3. J. Clin. Pathol, 43, 379-385. Hara,S., Takemori.Y., Yamaguchi,M., Nakamura.M. and Ohkura,Y. (1987) Fluorometric high-performance liquid chromtography of N-acetyl- and Nglycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids. Anal. Biochem., 164, 138-145. Higashi,H. (1990) N-Glycolylneuraminic acid-containing glycoconjugate as tumor-associated antigen: Hanganutziu-Deicher antigen. Trends Glycosci. Glycotechnol., 2, 7-15. Higashi.H., Hirabayashi.Y., Fukui.Y., Naiki.M., Matsumoto,M., Ueda,S. and Kato.S. (1985) Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer. Cancer Res., 45, 3796-3802. Hirabayashi.Y., Higashi.H., Kato,S., Taniguchi,M., and Matsumoto.M. (1987) Occurrence of tumor-associated ganglioside antigens with HanganutziuDeicher antigenic activity on human melanomas. Jpn. J. Cancer Res. (Gann), 78, 614-620. Igarashi.Y., Nojiri,H., Hanai,N. and Hakomori,S.-I. (1989) Gangliosides that modulate membrane protein function. Methods Enzymol, 179, 521-541. IUPAC-IUB Commission on Biochemical Nomenclature (1977) The nomenclature of lipids. Eur. J. Biochem., 79, 11-21. Jaffe.E.A. (1984) Culture and identification of large vessels endothelial cells. In Jaffe,E.A. (ed), Biology of Endothelial Cells. Martinus-Nijhoff, Boston, pp. 1-13. Jerg,K.R., Baumann.H., Keller.R. and Friedl,P. (1990) Fermentation of bovine endothelial cells for preparation of endothelial cell-surface heparan sulphate. Int. J. Biol. Macromoi, 12, 153-157. Kanda,T., Ariga,T., Yamawaki,M., and Yu.R.K. (1993) G M3 regulates protein kinase systems in cultured brain microvascular endothelial cells. /. Neurochem., 61, 1969-1972. Kanda,T., Yoshino.H., Ariga,T., Yamawaki,M. and Yu.R.K. (1994) Glycosphingolipid antigens in cultured microvascular bovine brain endothelial cells: sulfoglucuronosyl paragloboside as a target of monoclonal IgM in demyelinative neuropathy. J. Cell Biol., 126, 235-246. Karlsson,K.-A. (1991) Glycobiology: a growing field for drug design. Trends Pharmacol. Sci., 12, 265-272. Kojima,N. and Hakomori,S.-I. (1991) Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction. J. Biol. Chem., 266, 17552-17558. Kojima,N., Shiota,M., Sadahira.Y., Handa,K. and Hakomori,S.-I. (1992) Cell adhesion in a dynamic flow system as compared to static system. J. Biol. Chem., 267, 17264-17270. Lasky.L.A. (1995) Selectin-carbohydrate interactions and the initiation of the inflammatory response. Annu. Rev. Biochem., 64, 113-139. Ledeen,R.W. (1989) Biosynthesis, metabolism, and biological effects of gangliosides. In Margolis.R.U. and Margolis.R.K. (eds.), Neurobiology ofGlycoconjugates. Plenum Press, New York, pp. 43—83. Ledeen.R.W. and Yu,R.K. (1982) Gangliosides: structure, isolation and analysis. Methods Enzymol., 83, 139-191. LehmannJ., Piel.G.W. and Schulz,R. (1987) Bubble free culture aeration with porous moving membranes. Dev. Biol. Standard., 66, 227-240. LehmannJ., Vorlop,J. and Biintemeyer.H. (1988) Bubble-free reactors and their development for continuous culture with cell recycle. In Spiehr.R.E. and GriffithsJ.B. (eds.), Animal Cell Biotechnology. Vol. 3. Academic Press, London, pp. 221-237. Levery,S.B. and Hakomori,S.-I. (1987) Microscale methylation analysis of glycolipids using capillary gas chromatography-chemical ionization mass fragmentography with selected ion monitoring. Methods Enzymol, 138E, 13-25. Macher,B.A. and KlockJ.C. (1980) Isolation and chemical characterization of neutral glycosphingolipids of human neutrophils. J. Biol. Chem., 2SS, 20922096. Macher,B.A., BuehlerJ., Scudder,P., Knapp,W. and Feizi,T. (1988) A novel carbohydrate, differentiation antigen on fucogangliosidesof human myeloid cells recognized by monoclonal antibody VIM-2. J. Biol. Chem., 263, 10186-10191. MagnaniJ.L., Smith,D.F. and Ginsburg.V. (1980) Detection of gangliosides that bind cholera toxin: direct binding of h2sI-labeled toxin to thin-layer chromatograms. Anal. Biochem., 109, 399-402. MagnaniJ.L., Nilsson,B., Brockhaus.M., Zopf.D., Steplewski.Z., Koprowski.H. and Ginsburg.V. (1982) A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-W-fucopentaose H. / . Biol. Chem., 257, 14365-14369.

Glycosphingolipids of bovine aortic endothelial cells


istry and role of sialic acids. In Rosenberg.A. (ed.), Biology of the Sialic Acids. Plenum Press, New York, pp. 7-67. Schnaar.R.L. (1991) Glycosphingolipids in cell surface recognition. Glycobiology, 1, 477-485. Schwarzmann.G. and Sandhoff.K. (1990) Metabolism and intracellular transport of glycosphingolipids. Biochemistry, 29, 10865-10871. Smith,C.W. (1993) Endothelial adhesion molecules and their role in inflammation. Can. J. Physiol. Pharmacol., 71, 76-87. Spiegel.S., Matyas,G.R., Cheng.L. and Sacktor,B. (1988) Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes. Biochim. Biophys. Ada, 938, 270-278. Svennerholm,L. (1956) The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem., 1, 42-53. Svennerholm,L. (1957) Quantitative estimation of sialic acids. Biochim. Biophys. Ada, 24, 604-611. Svennerholm.L. (1963) Chromatographic separation of human brain gangliosides, J. Neurochem., 10, 613-623. Ueno,K., Ando,S. and Yu,R.K. (1978) Gangliosides of human, cat, and rabbit spinal cords and cord myelin. J. Lipid Res., 19, 863-871. Usuba.O., Fuji,Y., Miyoshi.I., Naiki,M. and Sendo,F. (1988) Establishment of a monoclonal antibody to Hanganutziu-Deicher antigen as a tumorassociated carbohydrate antigen. Jpn. J. Cancer Res. (Gann), 79, 13401348. van Echten,G. and Sandhoff.K. (1993) Ganglioside metabolism. /. Bio/. Chem., 268, 5341-5344. Varki.A. (1992) Diversity in the sialic acids. Glycobiology, 2, 25^tO. Varki,A. (1994) Selectin ligands. Proc. Natl. Acad. Sci. USA, 91, 7390-7397. Watarai.S., Kushi.Y., Shigeto,R., Misawa,N., Eishi,Y., Handa.S. and Yasuda,T. (1995) Production of monoclonal antibodies directed to HanganutziuDeicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides. J. Biochem., 117, 1062-1069. Whatley,R.E., Zimmerman,G.A., Mclntyre.T.M. and Prescott.S.M. (1990) Lipid metabolism and signal transduction in endothelial cells. Prog. Lipid Res., 29, 45-63. Wu,G. and Ledeen.R. (1988) Quantification of gangliotetraose gangliosides with cholera toxin. Anal. Biochem., 171, 368-375. Yu,R.K. and Saito.M. (1989) Structure and localization of gangliosides. In Margolis,R.U. and Margolis.R.K. (eds.), Neurobiology of Glycoconjugates. Plenum Press, New York, pp. 1-42. Zeller,C.B. and Marchase.R.B. (1992) Gangliosides as modulators of cell function. Am. J. Physiol. 262 (Cell Physiol., 31), C1341-C1355. Zimmerman,G.A., Prescott,S.M. and Mclntyre.T.M. (1992) Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol. Today, 13, 93-100. Received on February 10, 1997; revised on April 23, 1997; accepted on May 12, 1997

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