Transglycosylation and reverse hydrolysis reactions of endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EGCase: EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here transglycosylation and reverse hydrolysis reactions of EGCase from the jellyfish Cynaea nozakii. Various alkyl-GM1 oligosaccharides (alkyl-II(3)NeuAcGgOse4) were synthesized when GM1 ganglioside was treated with the EGCase in the presence of 1-alkanols. Among various 1-alkanols tested, methanol was found to be the most preferential acceptor, followed by 1-hexanol and 1-pentanol. GM1 was the best donor, followed by GD1b and GT1b, when methanol was used as an acceptor. However, neither globoside nor glucosylceramide was utilized by the enzyme as a donor substrate. The enzyme transferred oligosaccharides from various glycosphingolipids to NBD-ceramide, a fluorescent ceramide, producing NBD-labeled glycosphingolipids. In addition to the transglycosylation reaction, the enzyme catalyzed the reverse hydrolysis reaction; lactose was condensed to ceramide to generate lactosylceramide in the presence of the enzyme. These results indicate that the jellyfish enzyme will facilitate the synthesis of various neoglycoconjugates and glycosphingolipids.     

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.     

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

Conversion of endoglycoceramidase-activator II by trypsin to the 27.9 kDa polypeptide possessing full activity: purification of activator for endoglycoceramidase by trypsin treatment followed by trypsin-inhibitor agarose column application.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9 kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and, after a 1-hour pulse, with chase time. After a 1-hour pulse with 2 x 10(-6) M [3H-Sph]GM1 followed by a 4-hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/10(6) cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.     

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.     

A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide

A novel type of enzyme which hydrolyzes the linkage between the ceramide and the sugar chain in various glycosphingolipids has been found in the leech, Hirudo medicinalis. This enzyme releases the intact oligosaccharide from LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, nLcOse4Cer, GM3, GM2, GM1, GD1a and GT1 with the concurrent release of ceramides. By using tritium-labeled GM1 as substrate we found the optimum pH of this enzyme to be between pH 4 and 5. Since the enzyme cleaves the linkage between the ceramide and the sugar chain in various glycosphingolipids with no apparent preference toward the sugar chain, we propose to call this enzyme ceramide-glycanase.     

Rapid internalization of exogenous ganglioside GM3 and its metabolism to ceramide in human myelogenous leukemia HL-60 cells compared with control ganglioside GM1

Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL-60 cells were analyzed using ³H-labeled GM3 ([³H]GM3). [³H]GM3 was rapidly internalized into the cells (trypsin-resistant fraction) 8 times more than the control, ³H-labeled GM1 ([³H]GM1). In addition, not only incorporation but also metabolism of [³H]GM3 was more rapid than [³H]GM1 in HL-60 cells. Moreover, one of the metabolites was found to co-migrate with ceramide in thin-layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation-inducible GM3 in HL-60 cells rather than from non-inducing GM1.     

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.     

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.     

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.

Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Glycosphingolipid specificity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.