Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.     

Fractionation of Primary Cultured Cerebellar Neurons: Distribution of Sialyltransferases Involved in Ganglioside Biosynthesis

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.     

Ganglioside biosynthesis in rat liver: effect of UDP-amino sugars on individual transfer reactions

Several glycosyltransferases participating in ganglioside biosynthesis were measured in Golgi-rich fractions from rat liver. Addition of those UDP-amino sugars to the enzyme assays which accumulate in liver after treatment of rats with D-galactosamine inhibited the transferases to different degrees. The simultaneous presence of UDP-GalN, UDP-GalNAc, UDP-GlcN, and UDP-GlcNAc in concentrations resembling their overall content in livers 6 h after D-galactosamine administration led to an inhibition of the glycolipid galactosyltransferases, GL2 and GM1 synthases of 44 and 64%, respectively. GM2 synthase was moderately inhibited whereas the sialyltransferases (GM3, GD3, and GD1a synthases) were almost unimpaired. Induction of liver cell damage by D-galactosamine did not cause any change of glycosyltransferase activities as determined in rat liver homogenates and Golgi-rich fractions. These results indicate a possible role for UDP-amino sugars in the depression of ganglioside biosynthesis observed in vivo after GalN administration.     

Therapeutic Effects of Stem Cells and Substrate Reduction in Juvenile Sandhoff Mice

Sandhoff Disease (SD) involves the CNS accumulation of ganglioside GM2 and asialo-GM2 (GA2) due to inherited defects in the β-subunit gene of β-hexosaminidase A and B (Hexb gene). Substrate reduction therapy, utilizing imino sugar N-butyldeoxygalactonojirimycin (NB-DGJ), reduces ganglioside biosynthesis and levels of stored GM2 in SD mice. Intracranial transplantation of Neural Stem Cells (NSCs) can provide enzymatic cross correction, to help reduce ganglioside storage and extend life. Here we tested the effect of NSCs and NB-DGJ, alone and together, on brain β-hexosaminidase activity, GM2, and GA2 content in juvenile SD mice. The SD mice received either cerebral NSC transplantation at post-natal day 0 (p-0), intraperitoneal injection of NB-DGJ (500 mg/kg/day) from p-9 to p-15, or received dual treatments. The brains were analyzed at p-15. β-galactosidase staining confirmed engraftment of lacZ-expressing NSCs in the cerebral cortex. Compared to untreated and sham-treated SD controls, NSC treatment alone provided a slight increase in Hex activity and significantly decreased GA2 content. However, NSCs had no effect on GM2 content when analyzed at p-15. NB-DGJ alone had no effect on Hex activity, but significantly reduced GM2 and GA2 content. Hex activity was slightly elevated in the NSC + drug-treated mice. GM2 and GA2 content in the dual treated mice were similar to that of the NB-DGJ treated mice. These data indicate that NB-DGJ alone was more effective in targeting storage in juvenile SD mice than were NSCs alone. No additive or synergistic effect between NSC and drug was found in these juvenile SD mice.     

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.     

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I)

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform–methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.     

The anti-oligosaccharide antibodies present in sera from patients with motor neuron disease and neuropathy recognize theN-glycolylneuraminic acid containing gangliotetrahexosyl oligosaccharide

We found that serum antibodies present in the serum of patients with motor neuron disease and neuropathy, which were previously shown to react with the oligosaccharide chain of ganglioside GM1(Neu5Ac), can be recognized and titred using theN-glycolylneuraminic acid containing monosialo-gangliotetrahexosylceramide, GM1(Neu5Gc), which is not a component of normal human cells. The antibody-antigen reaction was abolished by immunoabsorption with the free oligosaccharide chain. This result, together with the knowledge that these antibodies recognize several glycoconjugates, supports the conviction that these antibodies are non-specific for a gangliosidic structure.     

Gangliosides in normal human serum. Concentration, pattern and transport by lipoproteins

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.     

High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

A rapid method for the quantitation of cholesterol in each lipoprotein fraction has been developed which utilizes high-performance aqueous gel permeation chromatography followed by enzymatic reaction using reaction-type high-performance chromatography.Cholesterol in serum lipoproteins eluted from the column could be sensitively and selectively detected by the absorbance at 550 nm following the enzymatic reaction. The sensitivity of the detection for cholesterol measured by A₅₅₀ was compared with that for protein measured by A₂₅₀ using the standard lipoprotein fractions: low-density lipoprotein (LDL) and high-density lipoproteins (HDL₂ and HDL₃). The effects of changing the flow-rate and lengthening the column on the resolution of LDL and HDL were examined. Analyses of serum protein and cholesterol were performed with this method for human and animal subjects.     

Gangliosides are essential in the protection of inflammation and neurodegeneration via maintenance of lipid rafts: elucidation by a series of ganglioside-deficient mutant mice

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported by us. In this study, we compared complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among various mutant mice of ganglioside synthases, i.e. GM2/GD2 synthase knockout (KO), GD3 synthase KO, double KO (DKO) of these two enzymes and wild type. Up-regulation of complement-related genes, deposits of C1q, proliferation of astrocytes and infiltration of microglia also showed similar gradual severity depending on the defects in ganglioside compositions. In the expression of inflammatory cytokines such as IL-1β and tumor necrosis factor α, only DKO showed definite up-regulation. Immunoblotting of fractions from sucrose density gradient ultracentrifugation revealed that lipid raft markers such as caveolin-1 and flotillin-1 tended to disperse from the raft fractions with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > wild type. Decay-accelerating factor and neural cell adhesion molecule tended to disappear from the raft fraction. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. These results indicate that destruction of GEM/rafts is caused by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions.     

Identification and genetic control of two rabbit high-density lipoprotein allotypes

Rabbits were immunized with high-density lipoprotein (HDL) isolated from the serum of other rabbits by ultracentrifugation and gel filtration. Two different precipitating antibodies were elicited which distinguished two antigenically different genetic variants, i.e., allotypes, of HDL. The allotypes were identified as HDL based on the observation that on immunoelectrophoresis the antigen-antibody precipitate formed by the reaction of each of the antiallotype antisera with electrophoresed rabbit serum appeared electrophoretically in the α₁ region and reacted with Sudan black and with β-naphthyl acetate. In addition, the precipitin bands formed by the reaction of each antiallotype antiserum with a normal rabbit serum coalesced with the precipitin band formed by the reaction of goat anti-HDL with the same normal rabbit serum. The inheritance of the two allotypes is controlled by a pair of allelic genes, as shown by genetic studies of 312 progeny from 83 rabbit families. This HDL locus, designatedLhj, was shown not to be linked to theLpq locus of low-density lipoprotein nor to any of five other loci controlling allotypic specificities of different rabbit serum proteins. The use of these allotypes for studying the structure and biosynthesis of HDL is discussed. This study was supported by Research Grant PHS AI-07043 (Dr. S. Dray) and PHS AI-09241 from the National Institutes of Allergy and Infectious Diseases. One of us (K. L. K.) is the recipient of National Institutes of Health Career Development Award (AI-28687).     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside GM1 with human and fetal calf sera were studied, the following main results being obtained: (a) GM1, upon incubation with both sera gave origin to two GM1-protein complexes, which also occurred after interaction of GM1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore GM1 appeared to specifically bind serum albumin and to form GM1-albumin complexes. (b) GM1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10(-6) M), was time and concentration dependent, and resulted in a relevant degree of GM1 complexation (up to 80% of total GM1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 . 10(-4) M GM1, the ratio between bound and total GM1 increased linearly with increasing GM1 concentration; in the second phase, occurring above 2 . 10(-4) M, the ratio remained practically constant. After these findings it should be expected that GM1, when present in serum containing systems, forms complexes with albumin. This should be approximately considered when studying the effects of exogeneous GM1 in in vivo and in vitro (tissue cultures) systems.     

Metabolomic analysis of polar metabolites in lipoprotein fractions identifies lipoprotein-specific metabolic profiles and their association with insulin resistance

While the molecular lipid composition of lipoproteins has been investigated in detail, little is known about associations of small polar metabolites with specific lipoproteins. The aim of the present study was to investigate the profiles of polar metabolites in different lipoprotein fractions, i.e., very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and two sub-fractions of the high-density lipoprotein (HDL). The VLDL, IDL, LDL, HDL(2), and HDL(3) fractions were isolated from serum of sixteen individuals having a broad range of insulin sensitivity and characterized using comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOFMS). The lipoprotein fractions had clearly different metabolite profiles, which correlated with the particle size and surface charge. Lipoprotein-specific associations of individual metabolites with insulin resistance were identified, particularly in VLDL and IDL fractions, even in the absence of such associations in serum. The results indicate that the polar molecules are strongly attached to the surface of the lipoproteins. Furthermore, strong lipoprotein-specific associations of metabolites with insulin resistance, as compared to their serum profiles, indicate that lipoproteins may be a rich source of tissue-specific metabolic biomarkers.     

Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator?

Sphingosine 1-phosphate (S1P) concentration in plasma and serum has been estimated to be within 200-900 nM. Among plasma and serum components, S1P is concentrated in lipoprotein fractions with a rank order of high-density lipoprotein (HDL)>low-density lipoprotein (LDL)>very low-density lipoprotein (VLDL)>lipoprotein-deficient plasma (LPDP) when expressed as the per unit amount of protein. It is well known that LDL, especially oxidized LDL, is closely correlated and HDL is inversely correlated, with the risk of cardiovascular disease, such as atherosclerosis. Evidence was presented that a part of HDL-induced actions previously reported are mediated by the lipoprotein-associated S1P. Furthermore, S1P content in LDL was markedly decreased during its oxidation. This paper will discuss whether S1P is an atherogenic mediator or an anti-atherogenic mediator.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing <Emphasis Type="Italic">in vitro</Emphasis>

We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 μg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 7, pp. 948–954.     

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis

Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated a role of ganglioside GM3 in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by their incubation in the medium containing this ganglioside increased adhesion of these monocytes to HUVEC up to the level typical for monocytes from atherosclerotic patients. In addition, an increase in CD11b integrin expression comparable to that seen in lipopolysaccharide-activated monocytes was observed. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.     

Differential effects of three inhibitors of glycosphingolipid biosynthesis on neuronal differentiation of embryonal carcinoma stem cells

Gangliosides have been implicated in having important roles in neural development. It has been shown that disruption of ganglioside biosynthesis inhibits neurite outgrowth. However, many contradictory results have been reported. The inconsistency of these reports may result from the differential use of neuronal cell lines and inhibitors for ganglioside biosynthesis. In order to clarify the inconsistency in these studies, we utilized an in vitro neuronal differentiation model using an embryonic caricinoma (EC) stem cell line to elucidate the relationship between ganglioside expression and neural development. These cells were exposed to three different inhibitors of glucosylceramide synthase, the first enzyme committed for the biosynthesis of most of the brain gangliosides. All three inhibitors, d-threo-1-phenyl-2-decanoylamino-3-morphlino-1-propanol (D-PDMP), d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-PPPP), and N-butydeoxynojirimycin (NB-DNJ) can inhibit greater than 90% of ganglioside biosynthesis at certain concentrations, respectively. D-PDMP significantly slowed down cellular proliferation in undifferentiated P19 EC cells, inhibited neurite outgrowth, and eventually caused cell death in differentiated cells. However, no retardation in cell growth, neuronal differentiation, and neurite outgrowth was observed in cultures treated with D-PPPP or NB-DNJ despite the depletion of gangliosides. These results indicate that the effect of D-PDMP on cellular proliferation, neurite outgrowth, and survival of differentiated cells is independent of the inhibition of ganglioside biosynthesis.