Ganglioside-bound Sialic Acid Research Articles

Dietary Isomers of Sialyllactose Increase Ganglioside Sialic Acid Concentrations in the Corpus Callosum and Cerebellum and Modulate the Colonic Microbiota of Formula-Fed Piglets

Background: Sialyllactose is a key human milk oligosaccharide and consists of sialic acid (SA) bound to a lactose molecule. Breastfed infants have increased accumulation of ganglioside-bound SA compared with formula-fed infants.Objective: This study aimed to determine whether different isomers of sialyllactose enrich brain SA and modulate the microbiome of developing neonatal piglets.Methods: Day-old pigs were randomly allocated to 6 diets (control, 2 or 4 g 3′-sialyllactose/L, 2 or 4 g 6′-sialyllactose/L, or 2 g polydextrose/L + 2 g galacto-oligosaccharides/L; n = 9) and fed 3 times/d for 21 d. Pigs were killed, and the left hemisphere of the brain was dissected into cerebrum, cerebellum, corpus callosum, and hippocampus regions. SA was determined by using a modified periodic acid–resorcinol reaction. Microbial composition of the intestinal digesta was analyzed with the use of 16S ribosomal DNA Illumina sequencing.Results: Dietary sialyllactose did not affect feed intake, growth, or fecal consistency. Ganglioside-bound SA in the corpus callosum of pigs fed 2 g 3′-sialyllactose or 6′-sialyllactose/L increased by 15% in comparison with control pigs. Similarly, ganglioside-bound SA in the cerebellum of pigs fed 4 g 3′-sialyllactose/L increased by 10% in comparison with control pigs. Significant (P < 0.05, Adonis Test) microbiome differences were observed in the proximal and distal colons of piglets fed control compared with 4-g 6′-sialyllactose/L formulas. Differences were attributed to an increase in bacterial taxa belonging to species Collinsella aerofaciens (phylum Actinobacteria), genera Ruminococcus and Faecalibacterium (phylum Firmicutes), and genus Prevotella (phylum Bacteroidetes) (Wald test, P < 0.05, DeSeq2) compared with piglets fed the control diet. Taxa belonging to families Enterobacteriaceae and Enterococcaceae (phylum Proteobacteria), as well as taxa belonging to family Lachnospiraceae and order Lactobacillales (phylum Firmicutes), were 2.3- and 4-fold lower, respectively, in 6′-sialyllactose–fed piglets than in controls.Conclusions: Supplementation of formula with 3′- or 6′-sialyllactose can enrich ganglioside SA in the brain and modulate gut-associated microbiota in neonatal pigs. We propose 2 potential routes by which sialyllactose may positively affect the neonate: serving as a source of SA for neurologic development and promoting beneficial microbiota.

Dietary Isomers of Sialyllactose Increase Ganglioside Sialic Acid Concentrations in the Corpus Callosum and Cerebellum of Formula‐fed Piglets (LB320)

Sialic acid (SA) is a key component of human milk oligosaccharides and neural tissues. SA accumulates in the brain rapidly during neonatal development and is thought to play an important role in brain development . This study aimed to determine if different isomers of sialyllactose (SL) enrich brain SA of developing neonatal piglets. Day‐old pigs were randomized among 6 diets (control, 2g/L 3’‐SL, 4g/L 3’‐SL, 2g/L 6’‐SL, or 4g/L 6’‐SL, or 2g/L polydextrose + 2g/L galacto‐oligosaccharide; n=9) and fed three times per day for 21 d. Pigs were euthanized and the left hemisphere of the brain was dissected into cerebrum, cerebellum, corpus callosum, and hippocampus regions. Total and lipid‐bound (ganglioside) SA were assayed following extraction with chloroform:methanol (2:1), and free SA was calculated by difference. Protein‐bound SA was measured in the insoluble residue following suspension in PBS containing 1% Triton X‐100. SA was determined using a modified periodic acid‐resorcinol reaction. Dietary SL did not affect feed intake, growth or fecal consistency. Ganglioside‐bound SA in the corpus callosum of pigs fed 2g/L of 3’‐SL (359±16 μg SA/g wet tissue) or 6’‐SL (361±16 μg SA/g) was increased by 15% over control pigs (314±16 μg SA/g; P&lt;0.05). Similarly, ganglioside‐bound SA in the cerebellum of pigs fed 4g/L of 3’‐SL (416±14 μg SA/g) was increased by 10% over control pigs (377±14 μg SA/g; P &lt; 0.05). In conclusion, supplementation of formula with 3’‐ or 6’‐SL can enrich ganglioside SA in the brain of suckling piglets.Grant Funding Source: Mead Johnson Nutrition

GM1-gangliosidosis in American black bears: Clinical, pathological, biochemical and molecular genetic characterization

GM1-gangliosidosis is a rare progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of lysosomal β-galactosidase. We have identified seven American black bears (Ursus americanus) found in the Northeast United States suffering from GM1-gangliosidosis. This report describes the clinical features, brain MRI, and morphologic, biochemical and molecular genetic findings in the affected bears. Brain lipids were compared with those in the brain of a GM1-mouse. The bears presented at ages 10–14months in poor clinical condition, lethargic, tremulous and ataxic. They continued to decline and were humanely euthanized. The T2-weighted MR images of the brain of one bear disclosed white matter hyperintensity. Morphological studies of the brain from five of the bears revealed enlarged neurons with foamy cytoplasm containing granules. Axonal spheroids were present in white matter. Electron microscopic examination revealed lamellated membrane structures within neurons. Cytoplasmic vacuoles were found in the liver, kidneys and chondrocytes and foamy macrophages within the lungs. Acid β-galactosidase activity in cultured skin fibroblasts was only 1–2% of control values. In the brain, ganglioside-bound sialic acid was increased more than 2-fold with GM1-ganglioside predominating. GA1 content was also increased whereas cerebrosides and sulfatides were markedly decreased. The distribution of gangliosides was similar to that in the GM1-mouse brain, but the loss of myelin lipids was greater in the brain of the affected bear than in the brain of the GM1 mouse. Isolated full-length cDNA of the black bear GLB1 gene revealed 86% homology to its human counterpart in nucleotide sequence and 82% in amino acid sequence. GLB1 cDNA from liver tissue of an affected bear contained a homozygous recessive T1042 to C transition inducing a Tyr348 to His mutation (Y348H) within a highly conserved region of the GLB1 gene. The coincidence of several black bears with GM1-gangliosidosis in the same geographic area suggests increased frequency of a founder mutation in this animal population.

Brain ganglioside and glycoprotein sialic acid in breastfed compared with formula-fed infants

The concentration of sialic acid in brain gangliosides and glycoproteins has been linked to learning ability in animal studies. Human milk is a rich source of sialic acid-containing oligosaccharides and is a potential source of exogenous sialic acid. The aim of the study was to compare the sialic acid concentration in the brain frontal cortex of breastfed and formula-fed infants. Twenty-five samples of frontal cortex derived from infants who died of sudden infant death syndrome were analyzed. Twelve infants were breastfed, 10 infants were formula-fed, and 1 infant was mixed-fed; the feeding status of the remaining 2 infants was unknown. Ganglioside-bound and protein-bound sialic acid were determined by HPLC. Ganglioside ceramide fatty acids were also analyzed to determine the relation between sialic acid and long-chain polyunsaturated fatty acids. After adjustment for sex with age at death as a covariate, ganglioside-bound and protein-bound sialic acid concentrations were 32% and 22% higher, respectively, in the frontal cortex gray matter of breastfed infants than in that of formula-fed infants (P < 0.01). Protein-bound sialic acid increased with age in both groups (P = 0.02). In breastfed but not in formula-fed infants, ganglioside-bound sialic acid correlated significantly with ganglioside ceramide docosahexaenoic acid and total n-3 fatty acids. Higher brain ganglioside and glycoprotein sialic acid concentrations in infants fed human milk suggests increased synaptogenesis and differences in neurodevelopment.

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12 h light–dark cycles at 3 and 10°C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3°C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23°C; the differences were less pronounced in rats exposed to 10°C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3°C and to a lesser extent to 10°C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.

Gangliosides protect human melanoma cells from ionizing radiation-induced clonogenic cell death.

With an experimental model of spontaneous lung metastases of melanoma developed in this laboratory, a range of sublines (variants and clones) with different metastatic potential and ganglioside expression was established from a single human melanoma cell line M4Be. Using an in vitro clonogenic assay and provided that cells were cultured for no more than five passages, variations in cellular radioresistance of M4Be and seven sublines derived from M4Be were detected. This study shows a positive correlation between the cell intrinsic radioresistance of M4Be and its seven sublines and their total ganglioside content. More precisely, the proportion of radioresistant cells in M4Be and the seven sublines correlated with the number of cells determined by flow cytometry that were positively labelled with a monoclonal antibody directed to GD3 disialoganglioside. Blocking the cellular biosynthesis of gangliosides with the inhibitor Fumonisin B1 or cleaving with Vibrio cholerae neuraminidase the cell surface ganglioside-bound sialic acid in a radioresistant poorly metastatic subline increased its radiosensitivity in vitro. In contrast, enrichment of a radiosensitive metastatic subline with exogenous bovine brain GM1 increased its radioresistance in vitro. These results suggest that, in the radiation dose range important for radioprotection (0-1 Gy), membrane gangliosides radioprotect human melanoma cells in vitro.

Effect of pure bovine brain-derived gangliosides on normal human B cell proliferation in vitro

Pure gangliosides obtained from bovine brain including GM1, GD1b, GT1b and asialo-GM1 (GA1) did not induce normal human B cell proliferation in vitro. No B cell proliferation was observed either when GM1 was tested in the presence of interleukin-2 (IL-2), IL-4 or IL-6. Furthermore, the proliferative responses of human B cells induced by Staphylococcus aureus Cowan I (SAC), a T cell-independent B cell mitogen, were inhibited by these gangliosides. The degree of inhibition was influenced by ganglioside-bound sialic acid, although sialic acid per se was not inhibitory. Strongest inhibition was observed in the presence of GT1b (EC 50 8.8 μM/10 5 B cells) and lowest in the presence of GA1 (EC 50 129.5 μM/10 5 B cells) with intermediate values for GM1 and GD1b. GM1 inhibition of SAC-induced B cell proliferation did not represent cytotoxic effects and was still evident when GM1 was added 24–48 h after the beginning of the cultures. GM1 inhibition of SAC-induced proliferation was not reversed by the addition of IL-2, IL-4, IL-6 or their combination. In addition, GM1 inhibited the ILs-driven proliferative responses of SAC-induced B cell blasts. However, no inhibition of Epstein-Barr virus-induced B cell proliferative responses was observed. In conclusion, these results show that bovine brain-derived gangliosides do not induce proliferative responses of normal human B cells but, on the contrary, inhibit B cell responses induced by SAC.

Developmental profiles of gangliosides in trisomy 19 mice

The ganglioside composition of the cerebrum, cerebellum, brainstem, liver, heart, and spleen was analyzed quantitatively in trisomy 19 (Ts19) mice aged 4 to 12 days postpartum. The developmental profiles of cerebral gangliosides were similar in Ts19 mice and control littermates: Total ganglioside-sialic acid as well as the proportions of the individual gangliosides GD1a and GM1 increased with age, while the percentages of GQ1b and GT1b decreased during development. Both the accretion of the total ganglioside content and the development of the individual ganglioside fractions were delayed by 2–3 days in the Ts19 telencephalon. Likewise, the shift from the b- to the a-pathway of ganglioside synthesis was retarded. Ganglioside development was equally delayed in the cerebellum and the brainstem of Ts19 mice. Since in Ts19 mice, morphogenesis of several brain regions is similarly delayed by 2 days, these results confirm the usefulness of gangliosides as biochemical markers for brain maturation. In contrast to brain gangliosides, the ganglioside composition of the Ts19 livers was clearly distinguished from that of control livers. Total ganglioside-bound sialic acid was increased by 35–50% in Ts19 livers. This elevation in ganglioside content not explicable by a simple delay in development was mainly due to an increase in GD3 and fraction 2, which is likely to contain GD1a and GD1b. In contrast, GM2 which increased considerably with age in control mice persisted on a low level in Ts19 livers. Comparable alterations of the ganglioside pattern were neither observed in the spleen nor in the heart of Ts19 mice. The data presented give additional evidence that ganglioside synthesis in the liver is under a different regulation mechanism than that in the brain, heart, and spleen.

Total and ganglioside-bound sialic acid content of lymphocytes from non-insulin-dependent diabetic patients

The total and ganglioside-bound sialic acid content of unfractionated blood lymphocytes obtained from normal-weight healthy subjects (n = 17) and non-insulin-dependent diabetic patients on oral hypoglycemic drugs arbitrarily divided into normal-weight (n = 11), moderately overweight (n = 14) and severely overweight (n = 17) subgroups were examined. In normal-weight diabetics the level of lipid-bound sialic acid (gangliosides) of lymphocytes was found to be reduced (p less than 0.01). A more pronounced decrease (p less than 0.001) was observed for the lymphocyte total sialic acid of normal-weight and moderately overweight diabetics as compared to healthy subjects. In severely overweight non-insulin-dependent diabetics the decrease of total sialic acid was less pronounced compared to that found in healthy subjects. Whether this finding was due to obesity in these patients remains to be clarified.

Glycosphingolipids in lectin-resistant variants of mouse Lewis lung carcinoma cells.

Neutral glycolipids and gangliosides from murine Lewis lung carcinoma cell line LL2 and its lectin-resistant variants, differing in metastatic properties, were studied by fast-atom-bombardment mass spectrometry (FAB-MS), exoglycosidase treatment and an immunostaining procedure. The neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, asialo GM2, globoside and a glycolipid with a preliminary structure of Hex-Hexl-4HexNAc-Hex-Hex-Cer. The major gangliosides were GM3, GM2, GM1 and GD1a. No qualitative differences in glycosphingolipid expression were found between the metastatic cell lines (LL2 and LL2AAA) and the weakly metastatic variants (LL25, LL28, LL230 and LL2RCA II). Some quantitative differences were observed between the cell lines, e.g., in the level of ganglioside-bound sialic acid, which was not apparently correlated with the metastatic capacities.

Effect of denervation on a cholinergic-specific ganglioside antigen (Chol-1) present in Torpedo electromotor presynaptic plasma membranes.

The presence of Chol-1, an antigen identified in the plasma membrane of cholinergic electromotor nerve terminals of Torpedo marmorata, was investigated in Torpedo electric organ after 3, 6, and 9 weeks' denervation. Denervation was monitored by the cessation of stimulus-evoked discharge potentials, by the reduction in nerve terminals seen morphologically, and by the decrease in ACh and ChAT contents. The content of ganglioside-bound sialic acid did not show any appreciable change with time. Some modification of ganglioside pattern on TLC was observed after 9 weeks' denervation. The presence of Chol-1 after denervation was assayed by its activity in inhibiting the selective complement-induced lysis of the cholinergic subpopulation of guinea pig cortical synaptosome which is mediated by the anti-Chol-1 antiserum. Denervation did not affect Chol-1 immunoreactivity although it did alter the distribution of the immunoreactivity among gangliosides. The possible significance of the results is discussed.

Nervous system ganglioside composition of normothermic and hibernating dormice ( Glis glis)

The ganglioside pattern of seven different regions, olfactory bulb, forebrain cortex, midbrain (corpora quadrigemina), cerebellum, brain stem, pons and spinal cord, of nervous system of normothermic and hibernating dormice ( Glis glis) were investigated by two dimensional thin layer chromatography and densitometric quantification. Up to thirty different ganglioside spots were resolved, fifteen of which belonging to alkali labile species. Alkali labile gangliosides were present in all the regions obtained from normothermic animals, and their content, expressed as percentage of total ganglioside-bound sialic acid, ranged from a minimum of 10.2% in olfactory bulb, to a maximum of 30.1% in spinal cord. The most abundant alkali labile gangliosides were O-Ac-GT1b, O-Ac-GQ1b and an unidentified one, we coded I3. Alkali labile gangliosides were practically undetectable in hibernating dormice. They could be recognized only in brain stem, 3.3% and olfactory bulb, 0.6%.

Changes in the Concentration and Composition of Human Brain Gangliosides with Aging

Ganglioside content and composition were studied in whole brains from 9 neurologically normal male individuals ranging from 25 to 85 years in age. The content of ganglioside-bound sialic acid decreased from 1,070 to 380 micrograms/g fresh tissue at 85 years. Ten individual ganglioside fractions were identified on high-performance thin-layer chromatography, seven of which were quantified. With age, ganglioside composition shifted to a more polar pattern due to an increase in the relative concentration of the more polar fractions GQ1b, GT1b and GD1b and a decrease in GD1a and GM1. Except for GQ1b, the absolute concentration of all gangliosides decreased with age. All changes were more pronounced in younger ages. Results are discussed in relation to structural changes occurring in the aging brain, and the involvement of gangliosides is suggested.

Elevated ganglioside concentration in serum and peripheral blood lymphocytes from multiple sclerosis patients in remission

The ganglioside concentration in pooled serum from 20 patients with clinically definite multiple sclerosis (MS) was determined and compared with that in pooled serum from a similar number of healthy blood donors. There was a significant increase in the concentration of ganglioside-bound sialic acid from 691 ± 57 μg/100 ml in the control sera to 926 ± 83 μg/100 ml in MS patients' sera. The profile of individual gangliosides in the two groups was identical, the four main structures being G M3, G D3 and G D1a and G T1b. The ganglioside pattern and concentration in peripheral blood lymphocytes derived from MS patients and controls was identical with the predominant G M3, and small proportions of G D3. MS lymphocytes also showed a 39% increase in ganglioside content over control lymphocytes. The implication of such pronounced ganglioside increases is discussed with regard to the impaired immunocompetence of lymphocytes reported in MS.