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Relative Glycosyltransferase Levels Determine Ganglioside Patterns1

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Relative Glycosyltransferase Levels Determine Ganglioside Patterns1 (CANCER RESEARCH 52, 5725-5731, October 15, 1992] Glycosylation Pathways in the Biosynthesis of Gangliosides in Melanoma and Neuroblastoma Cells: Relative Glycosyltransferase Levels Determine Ganglioside Patterns1 Shutian Rúanand Kenneth O. Lloyd2 Memorial Sloan-Kettering Cancer Center, New York, New York 10021 ABSTRACT The pathways leading to the biosynthesis of the various major gangliosides of mammalian cells are quite well understood. In order to elucidate some of the factors that determine the charac Specifically, three major pathways (designated a, b, and c) are teristic expression of gangliosides in malignant melanoma and neuro considered to lead to a, b, and c series gangliosides respectively blastoma the levels of ganglioside synthases (glycosyltransferases) were (Fig. 1). GM3 synthase, GD3 synthase, and possibly GT3 syn determined in a panel of cell lines from those tumors that exhibited a wide range of ganglioside composition. Sialyltransferases (GM.v (;,,,. thase play key roles in determining the prominence of each <•!,,„.andCub synthases), /V-acetylgalactosaminyltransferases (GM2 pathway (12-15). Less information is available, however, on the and G|>2 synthases), and galactosyltransferase (G|\n and GUM,syn factors that determine the overall ganglioside expression exhib thases) were analyzed in crude membrane preparations from these cells. ited by a particular cell type. In an initial attempt to analyze The results confirmed the importance of GM, and G,(, synthases in factors that control the characteristic ganglioside patterns in determining the prominence of the a (G\i.. to GI ,.,) or b (G|M to G(_,n,) melanoma and neuroblastoma tumors we have correlated gan biosynthetic pathways. The overall ganglioside composition in cells was glioside synthase levels with ganglioside expression in a number found to be dependent on the relative levels of specific enzymes acting of melanoma and neuroblastoma cell lines that exhibit a spec sequentially or in competing pathways. In general, the pattern and levels trum of different ganglioside patterns. The results show that the of transferases correlated with the actual ganglioside content of the cell line, although several important discrepancies were noted. For example, major factors in determining the ganglioside phenotype of a cell in cell lines containing high amounts of Gm ganglioside, the level of the are the relative ratios of the specific glycosyltransferases and the preceding enzyme in the pathway (<;,,, synthase) was unexpectedly low. levels of acceptors which together interact to produce the final Thus, the high (,,,,:<;,,, ratios characteristic of most neuroblastomas ganglioside profile. result from low levels of G|M synthase as well as high levels of G|>2 synthase. In other cell lines, (.,.1 synthase was completely absent, re sulting in the synthesis of <>\i>.but not (.,,> by yV-acetylgalactosami- MATERIALS AND METHODS nyltransferase I, as would be expected. It was concluded that different Cell Lines and Cell Culture. The derivation and culture conditions glycosyltransferases play key roles in determining glycolipid expression of all human melanoma cell lines used in this study have been described in different cell types. (16). Mouse melanoma cell line B78 is a subline of the B16 line (17). JB-RH was obtained from Dr. Jane Berkelhammer (University of Mis INTRODUCTION souri, Columbia, MO) and subcloned (18). Human neuroblastoma cell lines SH-SY5Y, SH-EP1, BE(2)C, and SK-N-MC were kindly provided Gangliosides (sialylated glycosphingolipids) have been found by Dr. June Biedler (Memorial Sloan-Kettering Cancer Center, New to be significant antigens in tumors of neural crest origin, i.e., York, NY); others were maintained in our laboratory. The neuroblas toma cell lines were cultured in a mixture of Eagle's minimum essential melanomas, astrocytomas, and neuroblastomas (reviewed in Refs. 1-3). For example, normal melanocytes express GM33 as medium with nonessential amino acids and Ham's nutrient mixture F-12 supplemented with 10% heat-inactivated fetal bovine serum. Cells their major gangliosides, whereas malignant melanomas syn were passaged by incubation with 0.02% EDTA and 0.12% trypsin in thesize GD.I, as well as GM1, as their major gangliosides (4, 5). phosphate-buffered saline. All cells were harvested in phosphate-buff A few melanomas also synthesize small amounts of more com ered saline and used immediately for glycolipid extraction and cell plex gangliosides, e.g., GM2 and GD2 (6). In neuroblastomas, on membrane preparation or stored at -80°C before use. the other hand, GM2 and GD2 are the major gangliosides in Chemicals and Reagents. Gangliosides (GMJ, GM2, GMI, Gma, most cell lines and tumors (7, 8). These gangliosides are recog GDib, GTibi and Lac-Cer) were purchased from Sigma Chemical Co. nized by many of the mouse (2) and human (1) monoclonal (St. Louis, MO). Gr>.iwas obtained from Calbiochem (San Diego, CA). antibodies that have been developed in recent years. Mouse Their purity was checked by TLC, as described below. Gr>2was isolated monoclonal antibodies to Gnu (9) and Gn2 (10) have been used from human neuroblastoma tissue and kindly provided by Dr. F. Hell in therapy trials in the treatment of melanoma and neuroblas ing (Memorial Sloan-Kettering Cancer Center). CMP-[4,5,6,7,8,9- toma, respectively. Moreover, some gangliosides are immuno- 14C]NeuAc (250 mCi/mmol), UDP-Ar-acetyl-n-[l-1H (N)]galac- tosamine (8.3 mCi/mmol), UDP-/V-acetyl-D-[l-14C]galactosamine (55 genic in humans and form the basis of vaccines for active im- mCi/mmol), and UDP-[4,5,-1H (N)jgalactose (43.3 Ci/mmol) were pur munotherapy in melanoma patients (11). chased from New England Nuclear (Boston, MA). Phenylmethylsulfo- nylfluoride, 2-mercaptoethanol, Triton CF-54, MgCl2, and MnCl2 were from the Sigma Chemical Co. Cacodylic acid (sodium salt), chlo Received 4/30/92; accepted 8/6/92. roform, and methanol were from Fisher Scientific (Pittsburgh, PA). All The costs of publication of this article were defrayed in part by the payment of other chemicals and reagents used were of analytical grade quality. page charges. This article must therefore be hereby marked advertisement in accord ance with 18 U.S.C. Section 1734 solely to indicate this fact. Extraction and Isolation of Glycolipids from Cells. Methods for the 1Supported by grants from the National Cancer Institute (CA08478 and extraction and isolation of acidic glycolipids were previously described CA21445). (19). Briefly, the total lipids were extracted sequentially from cell pellet 2 To whom requests for reprints should be addressed, at Sloan-Kettering Insti in 30 volumes of 2:1, 1:1, and 1:2 chloroform:methanol. A glycolipid tute, Section 6155. 1275 York Avenue, New York, NY 10021. 3 The abbreviations used are: Lac-Cer, lactosylccramide; TLC, thin-layer chro- fraction was subsequently separated from other lipids by Florosil chro- matography; SAT, sialyltransferase. Ganglioside nomenclature is according to matography of the acetylated sample (20). After deacetylation and de Svennerholm (34) salting, the acid glycolipids and neutral glycolipids were separated by 5725 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1992 American Association for Cancer Research. GANGLIOSIDE BIOSVNTHETIC PATHWAY'S LacCer tions for GD.Isynthase were based on earlier work in our laboratory (5). Assay conditions for GM, synthase were optimized for Lac-Cer and Sialyltransferase I CMP-NeuAc concentrations using membranes from SK-MEL-28 cells; (GM3 synthase) the apparent Kms for these substrates were determined to be 30 and 480 MM,respectively. Assay conditions for GM2 and GD2 synthases were Sialyltransferase optimized for GM.,, GD.i, and UDP-GalNAc concentrations using GM3 GD3 membranes from IMR32 cells; the apparent Afmswere 48 MM(GM.i)and (GD3 synthase) 71 MM(UDP-GalNAc) for GM2 synthase and 42 MM(GD3) for GD2 GalNActransferase I synthase. GM1, Gmb, GDla, and GTlb synthases were assayed under (GM2/GD2 synthase) conditions described previously (23-29). First, the indicated concentra tion of glycolipid acceptor and sugar donor were dried under a gentle GM2 GD2 stream of nitrogen. The mixture was redissolved in an aqueous cacody- late buffer solution containing the detergent and metal ion at the opti mal pH. The reaction was started at 37°Cbythe addition of 200 Mgof Galactosyltransferase membrane protein. After 1, 2, or 3 h, 2 ml of water were added, and the reaction mixture was then passed several times through C|8 Sep-Pak GM1 GDlb cartridges that had been pretreated according to the manufacturer's instruction. The cartridge was washed with 25 ml of water to remove Sialyltransferase IV nonlipid components. The bound glycolipids were sequentially eluted with 2 ml of methanol and 3 ml of chloroform:methanol (2:1) and pooled. A portion of the eluted sample was dried and counted by liquid GDla GTlb scintillation spectrometry after the addition of Liquiscint (National Diagnostics, Mannville, NJ). The remainder of the sample was ana lyzed by thin-layer chromatography. For quantitation of the products the plates were scanned with a Berthold linear analyzer (Wildbad, Ger a pathway b pathway many). Enzyme activity was calculated from the ratios of the products Fig. 1. Pathways leading to the biosynthesis of gangliosides (modified from and the total radioactivity of the samples. The radioactive products on Refs. 13 and 14). Pathway c is omitted for simplicity. the TLC plates were also detected by fluorography as described below. Thin-Layer Chromatograph}- and Fluorography. Samples were ap DEAE-Sephadex A-50 (Pharmacia, Inc.. Piscataway. NJ) chromatog- plied to silica gel 60 high-performance TLC plates (EM Science, Ger raphy (21). The ganglioside fraction was desalted with a Sep-Pak C]8 many) and chromatographed in a solvent system of chloroformtmeth- cartridge (Waters; Millipore Co., Milford, MA). The gangliosides were anol:0.2% aqueous CaCl2 (55:45:10). The plates were sprayed with identified by thin-layer chromatography and fluorography (5). resorcinol/HCl for the analysis of glycolipids extracted from cell lines.
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