Selective changes in gangliosides of human milk during lactation: a molecular indicator for the period of lactation

Gangliosides of human milk from women at various periods of lactation were analyzed. GD3 in colostrum, particularly in the early period of lactation, was the major ganglioside, and the molar ratio of GM3 to GD3 was 0.2-0.3 in the milk at 2-6 days postpartum. In contrast, milk from women at 60-390 days postpartum contained GM3 as the major ganglioside and the molar ratio of GM3 to GD3 was more than 3. Milk at 8-40 days postpartum represented an intermediate stage in terms of the ratio of GM3 to GD3. The selective change in the molar ratio of gangliosides was observed as a phenomenon common to all human milk from different individuals at different periods of lactation, indicating that the periods of lactation can be defined on the basis of the ratio. Since glycolipids in human milk are preferentially localized in the milk fat globule membrane, which is derived from the plasma membrane of epithelial cells in the mammary gland, the changes in the ganglioside composition reported in this communication may reflect a qualitative change of the cells in the mammary gland.     

All three promoters of the acetyl-coenzyme A-carboxylase alpha-encoding gene are expressed in mammary epithelial cells of ruminants.

The activity of the enzyme acetyl-CoA-carboxylase alpha (ACC-alpha) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-alpha-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-alpha synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.     

Content, pattern and metabolic processing of rat-liver gangliosides during liver regeneration

During rat liver regeneration, the ganglioside content and distribution undergo significant changes after partial hepatectomy; total liver gangliosides increase remarkably till the 4th day after surgery, thereafter progressively decreasing to reach the values of sham-operated controls at the 12th day. The qualitative pattern is characterized by the 95% relative increase of GD1a at the 4th day and the 40% relative decrease of GD1b. In order to investigate the processes of ganglioside penetration into cells, degradation and biosynthesis, radiolabelled GM1 ([Sph-3H] GM1) was administered. One day after hepatectomy the liver uptake and metabolism of exogenous ganglioside were significantly reduced. Three days post-surgery these parameters were restored to control values; however an increased radioactivity incorporation was found in GD1a, thus suggesting an enhancement of its biosynthesis around the 4th day. The data reported here suggest that in the first two days after partial hepatectomy, the ganglioside degradation is reduced with a consequent increase of ganglioside content; later on the catabolic routes normalize and some biosynthetic processes leading to GD1a are enhanced. GD1a seems to be a marker of a peculiar transition phase of liver regeneration.     

Gangliosides in human, cow and goat milk, and their abilities as to neutralization of cholera toxin and botulinum type A neurotoxin

To elucidate the potential of mammalian milk as to protection of infants from infections, we determined the ganglioside compositions of human, cow and goat milk in relation with cholera toxin and botulinum type A neurotoxin-receptors. Gangliosides accounted for 1 to 2 μmol of lipid-bound sialic acid (LSA) in 100 ml of milk, and GD3 comprised about 69% of LSA in all milk samples. Among the milk samples examined, goat milk was found to contain an amount of gangliosides belonging to the b-pathway representing 15.8% of the total LSA. Accordingly, botulinum neurotoxin bound to GT1b and GQ1b in goat milk, but not to any gangliosides in human or cow milk. On the other hand, GM1, the cholera toxin receptor, was found to be present in all milk samples at concentrations of 0.02% to 0.77% of the total LSA and to be maintained at a relatively constant level in human milk during the postpartum period. Gangliosides from 1 ml of pooled human milk exhibited the ability to attenuate the binding of cholera toxin (30 ng) to GM1 by 93%, and those from 500 μl of goat milk completely inhibited the binding of botulinum type A neurotoxin 1.5 μg to GT1b. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2]. PVP, polyvinylpyrrolidone; LSA, lipid-bound sialic acid.     

Gangliosides in bovine milk. Changes in content and distribution of individual ganglioside levels during lactation.

Bovine milk undergoes changes in its ganglioside contents during the different stages of lactation. These contents are higher in colostrum (7.5 mg of lipid-bound NeuAc/kg) than in transitional (2.3 mg) or mature (1.4 mg) milk. The sialic acid content of milk follows a similar profile to that of gangliosides with the highest content during the first few days post partum followed by a gradual decrease towards the end of the period studied. When the individual distribution of gangliosides was examined throughout the course of lactation, several changes were also found. GD3 is the major ganglioside (about 60-70%) found; its content decreases from the first to the fifth day, increasing towards the end of the period considered. GM3, GD3 and GT3, sialyllactosylceramide-containing gangliosides account for 80-90% of the total lipid-bound NeuAc content. The most striking change in the ganglioside pattern was the gradual increase in G3.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers.     

Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.     

Ganglioside stimulation of nerve growth factor synthesis in cultured rat Schwann cells

To investigate effects of gangliosides on nerve growth factor (NGF) synthesis/secretion by Schwann cells, we obtained Schwann cells from dorsal sensory ganglia of one-day old Wistar rats and cultured them with various concentrations of a mixed ganglioside comprising GM1, GD1a, GD1b, and GT1b. NGF synthesis was evaluated by the measurement of NGF concentration in the conditioned medium using an enzyme immunoassay. In the continuous presence of 10(-3) M gangliosides, the NGF concentration in the medium showed a four fold increase on the 4th day, and it then decreased by the 8th day. The present results indicate that gangliosides promote the production/synthesis of NGF by Schwann cells.     

Suckling effects in sows: importance for mammary development and productivity

An understanding of the mechanisms regulating milk yield in sows is crucial for producers to make the best management decisions during lactation. Suckling of mammary glands by piglets is one factor that is essential for development of these glands during lactation and for the maintenance of lactation in sows. The process of mammary development is not static as the majority of it takes place in the last third of gestation, continues during lactation, is followed by involution at weaning and starts over again in the next gestation. During involution, the mammary glands undergo a rapid and drastic regression in parenchymal tissue, and this can also occur during lactation if a gland is not suckled regularly. Indeed, the pattern of regression is similar for glands that involute at weaning or during lactation. Suckling during 12 to 14 h postpartum is insufficient to maintain lactation and the process of involution that occurs in early lactation is reversible within 1 day of farrowing but is irreversible if a gland is not used for 3 days. However, milk yield from a gland which is ‘rescued’ within the first 24 h remains lower throughout lactation. Suckling does not only affect milk yield in the ongoing lactation, but it also seems to affect that of the next lactation. Indeed, non-suckling of a mammary gland in first-parity sows decreased development and milk yield of that gland in second parity. Nursing behaviour of piglets in early lactation was also affected, where changes were indicative of piglets in second parity being hungrier when suckling glands that were not previously used. It is not known, however, if the same effects would be seen between the second and third lactation. Furthermore, the minimum suckling period required to ensure maximal milk yield from a gland in the next lactation is not known. This review provides an update on our current knowledge of the importance of suckling for mammary development and milk yield in swine.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Oncogene transgenic mice: an useful model to study in vivo the relationships between gangliosides and oncogenes

Several studies have demonstrated that transfer of oncogenes in cultured cells reproducibly induces transmissible alterations in their ganglioside profile; the transfection of the same oncogene into different cell lines and the different localization of the oncogene product result in a different ganglioside expression. In the present study the modifications of the ganglioside pattern in mammary carcinomas induced in transgenic mice by the activated form of the rat neu oncogene have been investigated. Whereas control mammary tissues contain quite exclusively GM3, all neoplastic samples show a substantial decrease of this ganglioside, an accumulation in variable amount of GM3-derived species (GM1, GD3, GD1a, GD1b, GT and GQ) and the appearance of new, not yet identified, sialic acid containing molecules. Interestingly, three out of 10 tumors analyzed, even if histologically comparable to the others but with a larger dimension, show a significative difference as regard to the GM1, GD3 and GD1a content. Our data suggest that an activated oncogene may induce also in vivo a specific and transmissible alteration in the ganglioside pattern, but this distribution could be susceptible to further modifications during the tumor progression.     

Absence of ganglioside GM1 in astroglial cells from 21-day old rat brain: Immunohistochemical,histochemical, and biochemical studies

A procedure was developed for the cultivation of cells derived from the cerebral hemispheres of the 21-day old rat. Approximately 98 percent of the cells in a 10 day culture are astrocytes that contain glial fibrillary acidic protein. Analysis of the extracted gangliosides by thin layer chromatography revealed that ganglioside GM1 was absent and that the predominant ganglioside was GM3. Very small amounts of the polysialogangliosides GD1a, GD1b, and GT1b were detected. The concentration of gangliosidic NeuNAc per mg protein in these astrocytes was only 3 percent that observed in the 5 day culture of a mixed cell preparation from newborn rat brain. Immunohistochemical and histochemical studies were performed on the mixed cell population of the minced tissue of 21-day old rat brain prior to cultivation. Astrocytes did not stain for hyaluronectin. These cells also did not provide a positive staining reaction for ganglioside GM1 utilizing the antiganglioside GM1 peroxidase-antiperoxidase procedure and the biotinylated choleragen-avidin-peroxidase procedure. These two histochemical methods for ganglioside GM1 also did not stain astrocytes that had been cultured for 5 days. Oligodendroglial cells, which were also present in the uncultured 21-day-old minced brain tissue, stained positively for ganglioside GM1 and hyaluronectin. Hyaluronectin had previously been shown to be a marker for oligodendroglia. Oligodendroglial cells which were present in the 5 day cultures of 21-day old brain tissue also provided a positive reaction for ganglioside GM1. It is concluded that ganglioside GM1 is absent in astroglia. The presence of small amounts of polysialogangliosides in the "pure" astrocyte preparation is discussed.     

Weaning-induced expression of a milk-fat globule protein, MFG-E8, in mouse mammary glands, as demonstrated by the analyses of its mRNA, protein and phosphatidylserine-binding activity

A milk membrane glycoprotein, MFG-E8 [milk fat globule-EGF (epidermal growth factor) factor 8], is expressed abundantly in lactating mammary glands in stage- and tissue-specific manners, and has been believed to be secreted in association with milk fat globules. In the present paper, we describe further up-regulation of MFG-E8 in involuting mammary glands, where the glands undergo a substantial increase in the rate of epithelial cell apoptosis, and a possible role of MFG-E8 in mediating recognition and engulfment of apoptotic cells through its specific binding to PS (phosphatidylserine). Immunoblotting and RNA blotting analyses revealed that both MFG-E8 protein and MFG-E8 mRNA were markedly increased in mammary tissue within 3 days of either natural or forced weaning (pup withdrawal) of lactating mice. Using immunohistochemical analysis of the mammary tissue cryosections, the MFG-E8 signal was detected around the epithelium of such involuting mammary glands, but was almost undetectable at early- and mid-lactation stages, although strong signals were obtained for milk fat globules stored in the alveolar lumen. Some signals double positive to a macrophage differentiation marker, CD68, and MFG-E8 were detected in the post-weaning mammary tissue, although such double-positive signals were much smaller in number than the MFG-E8 single-positive ones. Total MFG-E8 in milk was also increased in the post-weaning mammary glands and, furthermore, the free MFG-E8 content in the post-weaning milk, as measured by in vitro PS-binding and apoptotic HC11 cell-binding activities, was much higher than that of lactation. In addition, the post-weaning milk enhanced the binding of apoptotic HC11 cells to J774 macrophages. Sucrose density-gradient ultracentrifugation analyses revealed that such enhanced PS-binding activity of MFG-E8 was present in membrane vesicle fractions (density 1.05-1.13 g/ml), rather than milk fat globule fractions. The weaning-induced MFG-E8 might play an important role in the recognition and engulfment of apoptotic epithelial cells by the neighbouring phagocytic epithelial cells in involuting mammary glands.     

Ganglioside composition of liver and kidney from rat after pentazocine treatment

1. Female non-pregnant rats were intramuscularly injected with pentazocine for 3 months. Liver showed a statistically significant (P less than 0.05) increase in its ganglioside content after the pentazocine treatment; in addition, no changes were found in the kidney ganglioside content. 2. We have also found changes in the ganglioside pattern of these rats after the pentazocine injection. The GM1 and GD1b liver content was decreased (P less than 0.05) in parallel with an increase (P less than 0.05) in GD3 and GT1b content; kidney showed a decrease (P less than 0.05) in GM1, GD1a and GD1b content and an increase (P less than 0.05) in GM4, GD2, GT1b and GQ content. 3. Female pregnant rats were also injected with pentazocine from the first to the nineteenth day of the gestation period. The total ganglioside content of liver and kidneys from mothers and their newborns did not show statistically significant differences after the treatment. 4. Mothers showed a decrease (P less than 0.05) in the GM1 content of liver and an increase (P less than 0.05) in the GT1b content of liver and GM1, GD3 and GD1a content of kidney. Only the GM3 content from kidney was increased (P less than 0.001). 5. Newborns showed minor changes in their ganglioside pattern. GT1b content from liver and GD2 and GQ content from kidneys were decreased (P less than 0.05).     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.     

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.     

Glycosphingolipid Glycosyltransferases in Human Fetal Brain

The developmental pattern of gangliosides in human fetal brain should reflect the activities of the respective glycosyltransferases. LA2-synthase activity, along with that of GM3-, GD3-, GM2-, and GM1-synthases, was determined in human fetal brain at 10-22 weeks of gestation. LA2-synthase is the pivotal enzyme in lacto series ganglioside formation. LA2-synthase activity decreased during the study period, mirroring a similar temporal decline in levels of the lacto series gangliosides, particularly 3'-isoLM1. The developmental profiles of the ganglio series glycosyltransferase activities demonstrate distinct changes that correspond to the ganglioside pattern between fetal weeks 10 and 22. In particular, the marked increase in GM2-synthase activity at 20 and 22 weeks of gestation and the decline in GD3-synthase activity after 15 weeks could explain the prominent expression of the a series gangliosides in this period of rapid neuronal outgrowth. However, a similar decline (two- to 2.5-fold) in GM3-synthase activity suggests a more likely conclusion, namely, that the two sialyltransferase activities are derived mainly from astroglial cells, which show a marked proliferation during the 10-15th fetal weeks. The data do not negate the hypothesis that GM3- and GD3-synthase are the critical enzymes in the regulation of ganglioside biosynthesis but do indicate a need to reevaluate the significance of GM2-synthase in expression of the a series gangliosides.