The biosynthesis of gangliosides. Labelling of rat brain gangliosides in vivo

1. After injection of [6-(3)H]glucosamine into 8-day-old rats it was found that all the major brain gangliosides and their sialyl groups were labelled at essentially the same rate, except the hematoside, which was the least labelled. In 18-day-old rats it was found that the two major gangliosides with the sialyl (2-->8)-sialyl linkage, and their sialyl groups were more labelled than the hematoside, the Tay-Sachs ganglioside, the other two major gangliosides and their respective sialyl groups. 2. No difference was found in any of the cases studied between the specific radioactivities of the neuraminidase-resistant and -labile sialyl groups belonging to the same ganglioside. The same was found for the specific radioactivities of the galactosyl groups proximal and distal to the ceramide moiety of total brain gangliosides from rats injected with [U-(14)C]glucose. From this it was concluded that partial turnover of the ganglioside molecule does not occur. 3. A model for the synthesis of gangliosides is presented that accounts for results from previous experiments in vitro and the lack of precursor-product relationships observed in experiments in vivo.     

Cellular gangliosides promote growth factor-induced proliferation of fibroblasts

Cell surface gangliosides have been proposed as modulators of transmembrane signaling. In this study, we used two complementary approaches to investigate the function of cellular gangliosides in the response of mammalian fibroblasts to growth factors. First, inhibition of glucosyl ceramide synthase by a new specific inhibitor of d-l-threo-1-phenyl-2-hexadecanoylamino-3 -pyrrolidino-1-propanol-HC l (glucosylceramide synthase), which depletes cellular gangliosides at a concentration of 1 microm without causing an increase in ceramide levels, blocked epidermal growth factor-stimulated proliferation of fibroblasts. Similarly, responses to several other growth factors that activate receptor tyrosine kinases, including fibroblast growth factor, insulin-like growth factor-I, and platelet-derived growth factor, were inhibited by 50-100%. Conversely, enrichment of cellular gangliosides by preincubation of the mouse and human fibroblasts with exogenously added gangliosides enhanced growth factor-elicited cell proliferation. Novel findings of this study, distinguishing it from previous similar studies, include differential effects of preincubation versus continuous incubation of cells with gangliosides on growth factor-dependent cell proliferation and the growth factor-like action of NeuNAc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuNAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer when cells are pretreated with the ganglioside.     

Characterization of the lacto series of gangliosides, especially of disialo-lacto-N-norhexaosyl ceramide isolated from adult bovine nasal cartilage

A disialosylganglioside was isolated from adult bovine nasal cartilage, and its structure was determined by analysis of sugar composition, permethylation analysis, exoglycosidase treatment, and mild acid hydrolysis. The structure of this ganglioside was identified as disialo-lacto-N-norhexaosyl ceramide, NeuNAc(alpha 2-8)NeuNAc-(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(1-4)Glc(1-1)Cer. Furthermore, we also isolated from this cartilage gangliosides whose structures were presumed to be monosialo-lacto-N-norhexaosyl ceramide, and mono- and disialo-lacto-N-neotetraosyl ceramide. The major fatty acids of the four gangliosides isolated were palmitic, stearic, behenic and lignoceric acids. The predominant long chain bases were sphingenine, heptadecasphingenine and hexadecasphingenine.     

N-Acetylneuraminic acid molecules as possible serotonin binding sites.

5-Hydroxytryptamine (5-HT), but not acetylcholine, carbamylcholine or L-D-noradrenaline, binds to ox brain ganglioside micelles, to phosphatidylcholine smectic mesophases (liposomes) containing gangliosides and to the glycoprotein fetuin, through the negatively charged N-acctylneuraminic acid (NeuNAc) residues. The 5-HT binding to NeuNAc is reversible, saturable, prodeeds in a 1: 1 fashion and can be specifically blocked by 7-methyltryptamine. Thc affinity constant at equilibrium for the reaction is of the order of 10² 1. mol-¹. No special ganglioside was identified as specifically associating with the amine. A terminal NeuNAc in the gangliosides is not a prerequisite for binding, although it seems important for binding 5-HT in entire mcmbrane preparations (MARCHBANKS, 1966) or for bringing about the 5-HT induced contraction of smooth muscle cells (WOOLLCY & GOMML 1964a). It is proposed that in 5-HT target cells, NeuNAc residues, probably attached to membrane surface glycoprotein(s) are involved in thc mechanism of action of the drug.     

Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.     

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a-similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. G _{d 3} and G _{d 1b} decreased rapidly in per cent; correspondingly, G _{d 1a} increased during embryonic life and became the major ganglioside in place of G _{d 3}. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.     

Neuraminidase in Calf Retinal Outer Segment Membranes

Abstract: An enzyme catalyzing the hydrolysis of sialic acid ( N -acetylneuraminic acid: NeuNAc)-containing glycoconjugates has been found in bovine retinal rod outer segment (ROS) membranes. The enzymatic activity is optimal at pH 4.0 and is stimulated by 0.15% Triton X-100. Total activity was determined by the release of NeuNAc from endogenous and exogenous substrates (GDla). The ROS enzyme preferentially hydrolyses the ROS gangliosides, possibly because they are more accessible than the glycoproteins as substrates for the neuraminidase. Release of NeuNAc from gangliosides leads to important changes in the ganglioside patterns; whereas the amounts of GM1 increased throughout the incubation, the levels of polysialogangliosides GTlb and GD3 diminished owing to their rapid hydrolysis. The finding that gangliosides are hydrolysed more extensively than glycoproteins suggests that endogenous ROS gangliosides may be the principal source of metabolically available sialic acid in ROS. It was also observed that the activity of ROS neuraminidase is not affected by illumination of the membranes.     

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.     

A new chemical procedure for the preparation of gangliosides carrying fluorescent or paramagnetic probes on the lipid moiety

A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic acid. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%. The labelled deacetylated GM1 molecular species are then N-acetylated by means of acetic anhydride with quantitative yield. The chemical process of insertion of labelled fatty acid and reconstitution of GM1 ganglioside has been confirmed by GLC-MS and NMR analyses. Fluorescence and electron spin resonance experiments indicate that the labelled gangliosides behave similarly to natural GM1, in both the aggregation properties and the capability to be transferred from micelles to vesicular dispersions of phospholipids.     

GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3-/GM2-Synthase Activity Ratio Throughout Development

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a ganglioside of the lactosylceramide series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N-acetylneuraminic acid (NeuNAc)] to adult (30% of total gangliosidic NeuNAc). GD3-synthase specific activity increased about 1.5-fold from birth to day 7 and essentially plateaued thereafter. The GD3-/GM2-synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GD1a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other "b" pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3-/GM2-synthase activity ratio throughout development of the tissue.     

GLYCOPROTEINS AND GLYCOLIPIDS OF SUBCELLULAR FRACTIONS FROM BOVINE RETINA. INCORPORATION OF MANNOSE AND SIALIC ACID1

Abstract— Endogenous lipids and proteins of bovine retina subcellular fractions were labelled from CMP-[³H]NeuNAc and GDP-[¹⁴C]mannose. The bulk of NeuNAc and mannose transfer activity was in membranes other than those from the rod outer segment (ROS). Lighter and heavier membranes, obtained from ROS free membranes by density gradient centrifugation, were the most active for the incorporation of NeuNAc and mannose, respectively. NeuNAc bound to a lipid indistinguishable from gangliosides, and a lipid that contains mannose (mannolipid-I) were found in the fraction extractable with chloroform-methanol (2:1, v/v). Mannose was also incorporated into a lipid fraction extractable with chloroform-methanol-water (1:1:0.3, by vol) (mannolipid-II). Mannolipid-I and mannolipid-II were labile to mild acid hydrolysis. In the presence of ROS free membranes, radioactivity of mannoli-pid-I was transferred to mannolipid-II and from this to proteins. Analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the proteins labelled from GDP-mannose migrated as a broad peak covering the range of molecular weights 20,000–30,000 and including the zone of rhodopsin migration. The proteins labelled from CMP-NeuNAc showed four radioactive peaks that were coincident with three out of four periodic acid-Schiff (PAS) positive bands.     

Periodate-induced transformation of human peripheral blood lymphocytes and the resultant oxidation of membrane sialyl, galactosyl and fucosyl residues

Oxidation of human peripheral mononuclear cells with sodium periodate results in lymphocyte activation. Period-date, at optimal mitogenic concentrations, oxidizes membrane sialyl residues (NeuNAc) essentially into the 7 carbon analogue (C7-NeuNAc). Fucosyl and galactosyl residues are also oxidized by periodate, since propane 1,2-diol and glycerol are isolated in acid hydrolysates of lymphocytes oxidized by periodate and reduced by tritiated borohydride. The neuraminidase pretreatment of lymphocytes induces a 40-50% decrease of their response to periodate. Neuraminidase treatment of 108 human peripheral lymphocytes liberated 9.6 microgram NeuNAc (31 nmol), representing 68.5% of the total content. The neuraminidase treatment dramatically enhances the recovery of glycerol in hydrolysates of lymphocytes treated successively with periodate and tritiated borohydride.     

Ganglioside binding pattern of CD33-related siglecs

Our study deals with the interaction of CD33 related-siglecs-5,-7,-8,-9,-10 with gangliosides GT1b, GQ1b, GD3, GM2, GM3 and GD1a. Siglec-5 bound preferentially to GQ1b, but weakly to GT1b, whereas siglec-10 interacted only with GT1b ganglioside. Siglec-7 and siglec-9 displayed binding to gangliosides GD3, GQ1b and GT1b bearing a disialoside motif, though siglec-7 was more potent; besides, siglec-9 interacted also with GM3. Siglec-8 demonstrated low affinity to the gangliosides tested compared with other siglecs. Despite high structural similarity of CD33 related siglecs, they demonstrated different ganglioside selectivity, in particular to the Neu5Acalpha2-8Neu5Ac motif.     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.     

The lipid composition of axons from bovine brain

Abstract— Bovine axons derived from myelinated CNS axons were found to have 13.5% lipid. This lipid was composed of 20.1% cholesterol, 20.1% galactolipid, 14.6% ethanolarhine phosphatides (56.4% in the plasmalogen form), 18.3% choline phosphatides (10.0% in the plasmalogen form), 9.3% sphingomyelin, 5.6% phosphatidyl serine and 3.4% phosphatidyl inositol. The bovine axons had 0.33 μg ganglioside NeuNAc/mg dry weight. The axon gangliosides were found to contain the four major types of bovine gangliosides, as well as gangliosides G_M2 and G_D3. The latter two amounted to 20.9 and 15.8 mole per cent respectively, of total gangliosides. On a molar basis, about one half of the gangliosides were monosialogangliosides, with a decreased contribution by gangliosides G_T1 and GD_1b relative to ganglioside distributions which have been reported for most other CNS components. The relationship of the bovine axonal lipid composition to bovine white matter and its constituents, as well as to other CNS and PNS axonal preparations is discussed.     

Theoretical studies on the conformation of monosialogangliosides and disialogangliosides

The preferred conformation of gangliosides GM3, GM2, GM1, GD1a and GD1b have been studied by computing their potential energies. The conformation of NeuNAc in GM3 differs from that expected for the same residue in GM2 and GM1. The NeuNAc residues in GM2 and GM1 exhibit identical conformations. Theory predicts that the terminal NeuNAc of GD1a is conformationally similar to that of GM3 and that the internal one is similar in conformation to those present in GM2 and GM1 in agreement with NMR studies. The differences in chemical shifts of the C2 and C3 carbons of the internal and terminal NeuNAc of GD1a have been attributed to differences in orientation. The present studies suggest that the binding site of cholera toxin is much smaller than that of tetanus toxin. The preferred shape of these gangliosides correlate well with their biological properties.     

Selective shedding of gangliosides by cells of mouse ascitic sarcoma-37

The profile and content of gangliosides associated with tumour cells of mouse sarcoma-37 and exfoliated from the cell surface were determined. It was shown that 20% of tumour gangliosides shed from the cell surface and only about 7% of all the shed gangliosides were contained in plasma membrane fragments. When incubated in vitro at 37 degrees C for 1 hour, the tumour cells were found to release less than 2% of cellular gangliosides. The main components of cellular gangliosides corresponded in their chromatographic behaviour to GM2 (31-32% of the total ganglioside content), GD3 (17-18%), GD1a (9-11%), GD2 (16-17%) and hematosides (19-21%). The profiles of in vivo and in vitro shed gangliosides were similar but strongly differed from those of cellular gangliosides: the relative content of GM2 was 70-75% and the other gangliosides were found in negligible amounts. The data show that GM2 accumulation in extracellular spaces is rather the result of its selective shedding than the shedding of products of "incomplete" cellular ganglioside synthesis.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Possible Role of Cyclic AMP in the Receptor-Mediated Regulation of Glycosyltransferase Activities in Neurotumor Cell Lines

Exposure of mouse neuroblastoma cell line N4TGl to opiates or [D-Ala2,D-Leu5] enkephalin produced a naloxone-reversible inhibition of cyclic AMP synthesis and prevented, in a concentration-dependent manner, the formation of both ganglioside GM2 (GalNAc-[NeuNAc]-Gal-Glc-ceramide) from GM3 (NeuNAc-Gal-Glc-ceramide) and ganglioside GM1 (Gal-GalNAc-[NeuNAc]-Gal-Glc-ceramide) from GM2 in cell-free extracts. In contrast, the receptor-mediated elevation of intracellular cyclic AMP levels by agents such as prostaglandin E1 (in the presence of isobutylmethylxanthine) or the addition of the cyclic AMP derivatives (dibutyryl cyclic AMP) markedly stimulated the activities of UDP-GalNAc:GM3,N-acetylgalactosaminyltransferase and UDP-Gal:GM2,galactosyltransferase. An overall increase in the synthesis of gangliosides more complex than GM3 was also observed in the mouse neuroblastoma x hamster brain explant hybrid cell line NCB-20 following elevation of cyclic AMP levels by treatment with serotonin and pargyline. The data presented support the hypothesis that cyclic AMP may have a role in the regulation of sialoglycosphingolipid biosynthesis.