Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4270-4274, September 1979 Biochemistry Differential activities of glycolipid glycosyltransferases in Tay- Sachs disease: Studies in cultured cells from cerebrum (gangliosides/blood group-related glycosphingolipids/galactosyltransferases/sialyltransferases/fucosyltransferases) MANJU BASU*, KATHLEEN A. PRESPER*, SUBHASH BASU*, LINDA M. HOFFMANt, AND STEVEN E. BROOKSt *Department of Chemistry, Biochemistry and Biophysics Program, University of Notre Dame, Notre Dame, Indiana 46556; and tNeuroscience Center of the Kingsbrook Jewish Medical Center and the Downstate Medical Center of the State University of New York, Brooklyn, New York 11203 Communicated by Edwin T. Mertz, May 29, 1979 ABSTRACT Four different glycolipid:glycosyltransferase Table 1. The present report is concerned with the biosynthesis activities involved in the biosynthesis in vitro of gangliosides of sialylneolactotetraosylceramide (AcNeu-nLcOse4Cer) (15, and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from 16) in TSD transformed cells and the lack of synthesis of either the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD GM1 ganglioside (11, 17) or blood group-active glyco- cultured brain cells contained little activity of either UDP- sphingolipid HI (17, 18). Gal:GM2 (81-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GMla from GM2 (Tay-Sachs) gan- MATERIALS AND METHODS glioside, or GDP-Fuc:nLcOse4Cer (al-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of HI gly- Cell Culture. TSD cell cultures were established at Kings- colipid from nLcOse4Cer. These cells contained a potent in- brook Jewish Medical Center (12). The cells were passaged hibitor of the second reaction (catalyzed by a Golgi-rich mem- serially as diploid strains from the cerebrum of a 20-week-old brane fraction from bovine spleen), whereas no inhibition of TSD fetus. Cultures established from TSD brain displayed the first reaction (catalyzed by a membrane fraction from 14- typical glia-like morphology. Cultures were transformed with day-old embryonic chicken brain) was observed. The activity the DNA virus SV40 in order to establish a permanent cell line of UDP-al:LcOse3Cer (f1-4)galactosyltransferase (GaIT4; EC 2.4.1.86) was 30- to 80fold higher than the activity of GalT-3. The (Fig. la). Cultures were grown in 250-ml Falcon plastic flasks presence of CMP-AcNeu:nLcOse4Cer sialyltransferase activity containing 15 ml of Eagle's minimal essential medium sup- and the absence of either GaIT-3 or FucT-2 suggested a probable plemented with antibiotics (penicillin/streptomycin/Fungi- pathway for the synthesis of sialylneolactotetraosylceramide zone; per ml, 100 units/100,g/1.25 ug), 10% fetal bovine [GM1b(GlcNAc)] in addition to a specific blockage of GMla serum, and nonessential amino acids. The cells were incubated ganglioside synthesis from GM2 in these TSD transformed in a humidified atmosphere of 95% air/5% CO2 and harvested cells. with Hanks' balanced salt solution (Ca2+- and Mg2+-free) Tay-Sachs disease (TSD) is one of several ganglioside storage containing 0.1% EDTA and 0.1% trypsin. A strain of these cells diseases. It is classified as GM2-gangliosidosis type I because of showing a reduced anchorage dependence (Fig. lb) was isolated its neuronal accumulation of GM2 ganglioside (1-3) and its according to the method described by Risser and Pollack (19). asialo derivative (4), gangliotriaosylceramide (GgOsesCer). The cells were washed three times with phosphate-buffered Since the publication by Klenk in 1939, it has been reported saline and centrifuged; the packed cells were homogenized in repeatedly that TSD is a clinically (5), morphologically (6, 7), 0.32 M sucrose containing 0.1% 2-mercaptoethanol and the and genetically (8) well-defined entity, but its exact biochemical homogenate was used as enzyme source. cause is evidently very complex. It has been observed that Materials. UDP-['4C]galactose (274 mCi/mmol), CMP- cultured skin fibroblast cells from TSD patients lack 3-D-hex- [4-'4C]AcNeu (1.68 mCi/mmol), and GDP-L-[14C]fucose (174 osaminidase A (9), but these cultured cells do not accumulate mCi/mmol) were purchased from New England Nuclear (1 GM2 ganglioside. In addition to the absence of lysosomal hex- Ci = 3.7 X 1010 becquerels). Unlabeled UDP-galactose was osaminidase A (10), the accumulation of GM2 could be the purchased from Sigma. Unlabeled CMP-AcNeu and GDP-L- result of a severe deficiency of a synthetic enzyme such as fucose were prepared according to the methods of Kean and UDP-galactose:GM2 (f1-3)galactosyltransferase (11), which Roseman (20) and Schachter et al. (21), respectively. The blood catalyzes the synthesis of GM1. group B-active pentaglycosylceramide Gal(al-3)Gal(f31-4)- In an attempt to study TSD in cultured cells, Hoffman et al. GlcNAc(f1-3)Gal(31-4)Glc-Cer (nLcOse5Cer) was isolated (12) have investigated the gangliosides in a glial cell strain that from rabbit (22) and bovine (23, 24) erythrocytes. Both neo- was derived from the cerebellum of a TSD fetus. They have lactotetraosylceramide (nLcOse4Cer) [Gal(/31-4)GlcNAc(31- shown that there is a 5-fold increase in GM2 content in TSD cer- Abbreviations: TSD, Tay-Sachs disease; SV40, simian virus 40; ECBM, cerebellar cells compared with cultures derived from the embryonic chicken brain membrane; BSGM, bovine spleen Golgi-rich ebellum of a normal age-matched control. Retently Schneck membrane; LacCer, lactosylceramide; LcOse3Cer, lactotriaosylcer- and his coworkers (13) have established a permanent line of glial amide [GlcNAc(f1-3)Gal(f31-4)Glc-Cer]; nLcOse4Cer, neolacto- cells from fetal TSD cerebrum for the study of this disease. In tetraosylceramide [Gal(fll-4)GlcNAc(j1-3)Gal(f31-4)Glc-Cerl; addition to GM3 and GM2 gangliosides, Hoffman et al. (14) nLcOse5Cer, neolactopentaosylceramide [Gal(a1-3)Gal(f1-4)- have found a high level of N-acetylglucosamine-containing GlcNAc(f1-3)Gal(11-4)Glc-CerI; GgOse4Cer, gangliotetraosylcera- in these simian virus 40 TSD mide [Gal(l31-3)GalNAc(f3l-4)Gal(f3l-4)Glc-Cerl; GM3, hlematoside ganglioside (SV40)-transformed [AcNeu(a2-3)Gal(f31-4)Glc-Cer]; GM2, Tay-Sachs ganglioside [Gal- cells. This led us to investigate the four key reactions shown in NAc(f31-4)Gal(3-2 AcNeu)(31-4)Glc-Cer]; GalT-3, UDP-galac- tose:GM2 (31-3)galactosyltransferase (EC 2.4.1.62); GalT-4, UDP- The publication costs of this article were defrayed in part by page galactose:nLcOse4Cer (31-4)galactosyltransferase (EC 2.4.1.86); charge payment. This article must therefore be hereby marked "ad- FucT-2, GDP-fucose:nLcOse4Cer (al-2)fucosyltransferase (EC vertisement" in accordance with 18 U. S. C. §1734 solely to indicate 2.4.1.89); SAT-3, CMP-AcNeu:nLcOse4Cer (a2-3)sialyltransferase. this fact. Lipid abbreviations are according to ref. 38. 4270 Downloaded by guest on October 2, 2021 Biochemistry: Basu et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4271 Table 1. Enzymatic reactions studied in TSD cultures Activity Donor Acceptor Product (p1-3)Galactosyltransferase UDP-[14C]Gal GalNAc-Gal-Glc-Cer (GM2) GM1a (GalT-3; EC 2.4.1.62) AcNeu (f1-4)Galactosyltransferase UDP-[14C]Gal GlcNAc-Gal-Glc-Cer nLcOse4Cer (GalT-4; EC 2.4.1.86) (LcOse3Cer) (a2-3)Sialyltransferase (SAT-3) CMP-[14C]AcNeu Gal-GlcNAc-Gal-Glc-Cer GMlb(GlcNAc) (nLcOse4Cer) (al -2)Fucosyltransferase GDP-[14C]Fuc nLcOse4Cer HI (FucT-2; EC 2.4.1.89) (al-3)Galactosyltransferase (EC UDP-[14C]Gal nLcOse4Cer nLcOse5Cer (B) 2.4.1.87) 3)Gal(31-4)Glc-Cerj and lactotriaosylceramide (LcOse3Cer) were interpreted according to Bjorndal et al. (28). Fertilized [GlcNAc(31-3)Gal(31-4)Glc-Cer] were prepared by sequential eggs were obtained from Rose Hatchery (South Bend, IN). removal of terminal galactose units with purified fig a-galac- Purification of Membrane-Bound Glycosyltransferases. tosidase (25) and a combination of fig a-galactosidase and A Golgi-rich membrane fraction was prepared from fresh bo- papaya 3-galactosidase (16, 26), respectively. GM2 ganglioside vine spleen tissue according to a published method (18) and was isolated from TSD brains according to a modification of contained high activity of GDP-L-fucose:nLcOse4Cer (al- the method of Svennerholm (2, 27). The purified glyco- 2)fucosyltransferase (FucT-2). The P3 membrane fraction sphingolipids were analyzed by gas/liquid chromatography which contained high UDP-galactose:GM2 (31-3)galactosyl- and gas chromatography/mass spectrometry. Mass spectral data transferase (GalT-3) activity was prepared (29) from 14-day-old embryonic chicken brain. Glycolipid:Glycosyltransferase Assays. Complete incu- bation mixtures for the galactosyltransferase assay contained the following components (in Amol) in final volumes of 0.1 ml: acceptor glycosphingolipids, 0.05; Triton CF-54/Tween 80 (2:1), 0.3 mg; MnCl2, 0.25; sodium cacodylate/HCl buffer (pH 7.2), 10.0; UDP-[14C]galactose, 0.04 (1.85 X 106 cpm/,umol), and enzyme fractions consisting of homogenates of TSD cells, 0.15-0.3 mg, or membranes from embryonic chicken brain and bovine spleen, 0.14-0.2 mg of protein [estimated by the method of Lowry et al. (30)]. The mixtures were incubated for 2 hr at 37°C, and the reaction was stopped by the addition of 2.5 ,umol of EDTA and 10ll of chloroform/methanol (2:1, vol/vol). The whole liquid content and a 100-Al chloroform/methanol (2:1) wash of the tube were spotted on Whatman 3MM paper and assayed by a double chromatographic method described pre- viously (16, 26, 31). The radioactivities of appropriate areas of each chromatogram were determined quantitatively by liquid scintillation techniques with a Beckman scintillation counter (model LS-3133T). Incubation conditions for glycolipid sialyltransferase were the same as described above, except that the incubation mixture contained the following (in ,umol): cacodylate/HCl buffer (pH Table 2.