Characterization, distribution and biosynthesis of the major ganglioside of rat intestinal mucosa.

The major sialic acid containing glycolipid has been isolated from rat intestinal mucosa. Characterization of this ganglioside by thin layer and gas chromatographic analysis indicates that it is an hematoside (GM3) with the major portion of the sialic acid in the N-glycolyl form. The distribution of this ganglioside was determined in villus and crypt cells isolated from rat intestine. The hematoside content of crypt cells was found to be significantly decreased when compared to villus cells. CMP-sialic acid:lactosylceramide sialyltransferase, responsible for the sialylation of lactosylceramide, was measured in differentiated villus and undifferentiated crypt cells and found to be greatly reduced in the crypt cell fraction. The present study demonstrates that marked differences in ganglioside content and biosynthesis occur in contiguous populations of cells in varying states of differentiation when isolated from normal rat intestine.     

The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 micrograms/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5'-monophosphate-N-acetylneuraminic acid: lactosylceramide sialyltransferase, and UDPgalactose: lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.     

Inhibition of ganglioside sialyltransferase activity and stimulation of neutral glycolipid exocytosis by heparin

Heparin treatment of human teratocarcinoma cells in culture has several manifestations. Accumulation of gangliosides is greatly decreased while the content of cellular neutral glycolipids is relatively unaffected. However, synthesis of neutral glycolipids is increased and large amounts of these glycolipids are exported out of the cells into the medium. In addition, sialyltransferase activity of heparin-treated teratocarcinoma cells is significantly inhibited, accounting for the decreased cellular content of gangliosides. These studies are not intended to infer any physiological role of heparin in the regulation of glycolipid biosynthesis. However, results do show that beta-D-galactoside 2,3-sialyltransferase is a heparin-binding protein and that inhibition of the enzyme activity by heparin is linked to an alteration in the secretion of most neutral glycolipid precursors and sialyltransferase acceptors. These data suggest that in addition to its biosynthetic function, this ganglioside transferase may play a regulatory role in glycolipid secretion.     

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.     

Special considerations in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1): solubilization of SAT-1 with lauryldimethylamine oxide

Lauryldimethylamine oxide (LDAO) was employed in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide alpha 2-3 sialyltransferase (SAT-1) (4). This detergent has advantages over the typically employed Triton detergents in the solubilization and stabilization of this sialyltransferase. Crude protein fractions solubilized from rat liver Golgi by several such detergents are very similar in composition as determined by two-dimensional gel electrophoresis. However, LDAO appears to activate and stabilize SAT-1 activity. It is possible that SAT-1 activation involves the structurally similar hydrophobic moieties and quaternary amino groups of LDAO and phosphatidylcholine.     

Combinatorial PCR approach to homology-based cloning: Cloning and expression of mouse and human GM3-synthase

GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.     

Phorbol ester-associated changes in ganglioside metabolism

The effect of phorbol esters on ganglioside metabolism in contact-inhibited Chinese hamster V79 cells was examined. Three phorbol esters of varying structure and tumor-promoting activity were used. Treatment of cells with tumor-promoting phorbol esters resulted in accumulation of gangliosides and increased incorporation of [1-¹⁴C]palmitate and [9-³H]sialic acid into gangliosides. Moreover, the phorbol esters were found to increase the activity of CMP-sialic acid: lactosylceramide sialyltransferase, the enzyme catalysing the first step in ganglioside biosynthesis. The magnitude of phorbol ester effects on V79 cell ganglioside metabolism correlated with the in vivo phorbol ester tumor-promoting activity.     

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.     

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells

Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.     

Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.     

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.     

Cyclic modulation of enzymes of pyrimidine nucleotide biosynthesis precedes sialoglycoconjugate changes during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat

Three enzymatic activities associated with pyrimidine nucleotide biosynthesis were monitored at weekly or bi-weekly intervals during 2-acetylaminofluorene- (0.025% in a Farber Basal Carcinogenic diet) induced hepatocarcinogenesis in the rat. Dihydroorotate dehydrogenase, the fourth of six enzymes in de novo pyrimidine biosynthesis, declined in activity while UDP kinase and CTP synthetase showed sequential increases in activity. The alterations in activity appeared to be cyclic, followed by a full or partial return to control values. Three full cycles were monitored. The first cycle preceded nodule formation. The second cycle accompanied nodule formation and preceded sialoglycoconjugate changes reported previously. The third cycle accompanied the early glycoconjugate changes. The cyclic pattern was reproducible in three separate experiments. In each cycle, the order of events was as follows: decrease in dihydroorotate dehydrogenase, sequential increases in UDP kinase, CTP synthetase and CMPsialic acid synthase, and finally increases in the enzyme lactosylceramide: CMPsialic acid sialyltransferase, lipid-soluble sialic acid and total sialic acid. In livers of animals fed 1.87% of the hepatotoxin, 4-acetamidophenol, no biochemical alterations resembling those induced by 2-acetylaminofluorene were obtained, despite acute centrilobular necrosis of the livers. The findings point to a biochemical cascade beginning with administration of carcinogen and continuing through the development of hyperplastic nodules and of frank carcinomas resulting not from hepatotoxicity but as events associated with the hepatocarcinogenic progression.     

Glycosphingolipids from bovine milk and milk fat globule membranes: a comparative study. Adhesion to enterotoxigenic Escherichia coli strains

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.     

Unusual Gangliosidosis in Emu (Dromaius novaehollandiae)

Abstract: A previous study has demonstrated an unusual gangliosidosis in emu that is characterized by the accumulation of gangliosides in the brain tissues with GM3 and GM1 predominating. To provide insight into this unique disorder of emu gangliosidosis, the current study focused on analysis of neutral glycosphingolipids and gangliosides from brain and liver tissues of affected birds and healthy controls. We found not only that the total lipid-bound sialic acid content was increased three- and fourfold in the affected brain and liver, respectively, but also that the ganglioside pattern was rather complex as compared with the control. The absolute ganglioside sialic acid content was significantly increased in the diseased tissues, with the highest elevation levels of GM3 (14-fold) and GM1 (ninefold) in the affected brain. Relative increases in content of these monosialogangliosides were also significant. GM2 was only detected in the affected brain, but not in normal controls. The neutral glycosphingolipid fraction showed accumulation of many oligosylceramides, with six- and 5.5-fold increases in lactosylceramide levels for brain and liver, respectively. The level of myelin-associated galactosylceramide (GalCer) in the brain was decreased to only 41% of that in the healthy control, whereas no difference was found in liver tissues from both groups. Besides GalCer, the brain content of sulfatide (cerebroside-sulfate esters), another myelin-associated glycolipid, decreased to only 16% of the control. The loss of myelin-associated GalCer and sulfatide strongly suggests demyelination in the affected emu brain. Our overall data are consistent with the presence of a unique form of sphingolipidosis in the affected emus, perhaps with secondary demyelination, and suggest a metabolic disorder related to total sphingolipid activator deficiency.     

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.     

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.     

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro

Summary Glycosphingolipid biosynthesis was examined using [³H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p < 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p < 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse₄Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.Abbreviations GM1 II³NeuAc-GgOse₄Cer - GM3 II³NeuAc-GgOse²Cer - MG4 IV³NeuAc-nLcOse₄Cer - MG6 VI³NeuAc V⁴Gal-IV³GlcNAc-nLcOse⁴Cer - TLC Thin-Layer Chromatography - DMEM Dulbecco's Modified Eagles' Medium     

Characterization of a CMPNeuAc: lactosylceramide alpha 2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells

1. The rainbow trout (Oncorhynchus mykiss) CMPNeuAc:lactosylceramide alpha 2----3sialytransferase enzyme from RTH-149 cells has been characterized. 2. Transfer of sialic acid to lactosylceramide was optimal at a pH of 5.9, temperature of 25 degrees C, and in the pressure of 0.3% CF-54, 10 mM Mn2+, 0.1 M sodium cacodylate, and 2 mM ATP. 3. Golgi-rich membrane fractions of RTH-149 cells were found to be enriched in sialidase activity and as such the addition of 40 microM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was necessary to assay alpha 2----3sialyltransferase activity optimally. 4. Apparent Km for donor (CMPNeuAc) and acceptor (lactosylceramide) were found to be 243 microM and 34 microM, respectively. 5. The alpha 2----3sialyltransferase characterized was found to be primarily specific for lactosylceramide though minor activity with other glycolipid acceptors was observed. 6. The presence of another sialyltransferase with differing substrate specificity was noted. 7. Properties of this enzyme, compared to analogous mammalian enzymes, are discussed.     

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.