Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP‐NeuAc:GM1 α2‐3‐sialyltransferase (ST‐IV) and UDP‐GalNAc:GM3 N‐acetylgalactosaminyltransferase (Gal‐NAc‐T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108‐15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST‐IV activity to 45% of that of control cells with t1/2 = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST‐IV due to the inhibition of a serine/threonine‐specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST‐IV, the activity of GalNAc‐T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc‐T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up‐regulation of GalNAc‐T in conjunction with down‐regulation of ST‐IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a‐series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108‐15 cells can be specifically regulated by both protein kinase A‐ and protein kinase C‐related phosphorylation systems.

An in vitro model of Gaucher's disease in murine neuroblastoma ϫ rat glioma NG108-15 cells was used to investigate the physiological effects of two specific inhibitors of glucosylceramide synthase, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d,l-PDMP) and N-butyldeoxynojirimycin (NB-DNJ), which have been suggested as agents for treatment of glycolipid storage disorders. Incubation of NG108-15 cells with conduritol-B-epoxide, a covalent inhibitor of glucosylceramidase, raised the intracellular concentration of glucosylceramide (GC) by more than fourfold, indicating a glycolipid composition equivalent to that of Gaucher's cells. The level of GC was decreased, and the cells were depleted of gangliosides by postincubation with d,l-PDMP or NB-DNJ. Treatment with d,l-PDMP, but not with NB-DNJ, resulted in a dose-dependent reduction of the growth rate and eventually caused cell death in NG108-15 cells on reaching confluency. An in situ detection assay using terminal nucleotidyltransferase indicated that cell degeneration was accompanied by apoptosis. Lipid analysis by high-performance TLC revealed that on incubation with d,l-PDMP, but not with NB-DNJ, the concentration of endogenous ceramide was elevated by threefold. Ceramide elevation and apoptosis were also observed when NG108-15 cells were incubated with daunorubicin, which was previously reported to induce programmed cell death by stimulation of ceramide synthesis. Structural characterization by HPLC and subsequent laser desorption mass spectrometry revealed that the endogenous ceramide contained fatty acids with chain lengths ranging from C14:0 to C24:0. The results indicate that elevation of levels of these ceramide species by incubation with d,l-PDMP or daunorubicin induces programmed cell death in NG108-15 cells. Because ceramide accumulation and cell death were not observed on incubation with NB-DNJ, its use is suggested to be less toxic than that of d,l-PDMP for treatment of Gaucher's disease and other sphingolipid storage disorders. Key Words: Apoptosis-Gaucher's disease-Neuroblastoma cells-Cell death-Glycolipids. J. Neurochem. 72, 1040Neurochem. 72, -1049Neurochem. 72, (1999.Sphingolipids are known to be involved in various biological functions, including modulation of cell proliferation, differentiation, and apoptosis (Schwarz et al., 1995;Ariga et al., 1998). The main active compounds are ceramide and its glycosylated, e.g., glycosphingolipids, or phosphorylated, e.g., sphingomyelins, derivatives (Schwarz et al., 1995;Ballou et al., 1996). Ceramide, mainly generated from signal-induced cleavage of sphingomyelin by sphingomyelinase, has been shown to arrest cell cycle and to stimulate apoptosis (Jarvis et al., 1994;Ji et al., 1995;Obeid and Hannun, 1995;Witty et al., 1996;Ariga et al., 1998). Recent studies have demonstrated that induction of apoptosis can also be achieved by activation of de novo synthesis of ceramide (Bose et al., 1995). Hydrolysis of ceramide by ceramidase releases sphingosine, which has been found to be ...

To characterize the sialyltransferase-IV activity in brain tissues, the activities of GMl b-, GD1 a-, GT1 b-, and GQl c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1 b, or a cod brain ganglioside mixture as the substrate. The GD1 a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg2~o r Mn 2' and Triton CF-54 for its full activity. The Vmax value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent Km values for CMP-sialic acid and GM1 were 172 and 78 NM, respectively . Cichlid fish and rat brains also contained GM1 b-, GT1 b-, and GQ1 c-synthase activities. The ratio of GM1 b-, GD1 a-, and GT1 b-synthase activities in fish brain was 1 .00 :0 .89:1 .13, respectively, and in rat brain 1 .00:0 .60:0 .63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [3H]CMP-sialic acid produced radiolabeled GQ1 c. It is interesting that the adult rat brain also contains an appreciable level of GQ1 c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1 a-or GQl c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1 a-synthase activity was inhibited by GA1 and GD1 b, but not by LacCer, GM3, or GD3 . In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GDla-synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1 b, whereas the endogenous GT1 b-synthase activity was suppressed by GA1 or GM1 . GA1, GM1, and GD1 b did not affect the endogenous GM3-and GD3synthase activities . These results clearly demonstrate that sialyltransferase-IV in brain tissues catalyzes the reaction for GQ1 c synthesis in the c-pathway as well as the corresponding steps in the asialo-, a-, and b-pathway in ganglioside biosynthesis . Key Words: Sialyltransferase-Gangliosides-GQ1c-Brain -Fish-c-Series gangliosides. J. Neurochem. 64, 385-393 (1995) .Gangliosides are sialic acid-containing glycosphingolipids and are enriched especially in vertebrate ner-nomenclature is according to Svennerholm (1963) .

The progeny of two emu breeder pairs, which had a history of producing offspring with gangliosidosis, were monitored for 15 mo. DNA fingerprinting revealed that individuals in each breeder pair were not related to each other. One breeder pair had 13 progeny that reached or exceeded the age of 1 mo, and six of these progeny developed gangliosidosis. The mean age at which these affected emus were euthanatized, with distinct neurologic disease, or died was 5.7 mo. The second emu pair had 13 progeny, seven of which developed gangliosidosis, with a mean age of euthanasia/death of 4.6 mo. Affected emus died or were euthanatized from 2 to 8 mo of age. The primary clinical sign in the affected emus was mild to severe ataxia. Severe hemorrhage into the body cavity or the muscles of the thigh was noted in 8 of 13 of the affected emus. Brain ganglioside levels were evaluated in six of the affected emus and six controls. Significant increases (P < 0.05) in gangliosides GM1 and GM3 were noted, with 2.3- and 4.9-fold increases in these two gangliosides, respectively, in affected emus. Furthermore, the diseased emu brains contained ganglioside GM2, whereas this monosialoganglioside was undetectable in the brains of normal controls. Total mean brain ganglioside sialic acid in affected emus was increased 3.3-fold in comparison with controls. Serum chemistries revealed elevated cholesterol and decreased uric acid levels in affected emus. Gangliosidosis in emus is an inherited disease process that, in the current study, caused 50% mortality in the progeny of two emu breeder pairs. The elimination of this lethal gene from emu breeder stock is essential for the long-term economic viability of the United States emu industry.

To investigate the role of Sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different Sialyltransferases (GM3‐, GD3‐, GT3‐, GD1a‐, and GT1a‐synthase) using total membrane preparations from cichlid fish and Sprague‐Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglloside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3‐synthase activity was lower than GD3‐synthase activity, whereas the opposite relationship was observed in rat brain. The GT3‐synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3‐synthase activity was detected in rat brain. The GD1a‐and GT1a‐synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a‐synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3‐, GD3‐, GT3‐, GD1a‐, and GT1a‐synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c‐series gangliosides including GT3 and polysialo‐species, whereas a‐and b‐se‐ries gangliosides were major components in rat brain. These results suggest that the species‐specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.

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 actosylceramide 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 myelm-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.

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