The ST6Gal sialyltransferase controls production of the Sia␣2-6Gal␤1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to Tindependent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.Sialyltransferases are a family of glycosyltransferase enzymes that add sialic acid residues during oligosaccharide diversification (reviewed in ref. 1). Sialic acid addition occurs in the Golgi apparatus and generally terminates further oligosaccharide chain elongation. The outer position of sialic acid linkages places these residues in a location to provide key structural determinants in ligand formation for endogenous and pathogenic lectins. Three sialic acid linkage types commonly exist among vertebrates and the corresponding sialyltransferase genes have been previously isolated. The most abundant sialic acid linkage found among mammalian cell surface oligosaccharides is of the ␣2-3 variety and can be produced independently by four sialyltransferases that each, nonetheless, bear unique substrate preferences among glycolipids, asparagine (N)-linked glycans, and serine͞threonine (O)-linked glycans (2). Sialyltransferases have also been found to be developmentally regulated and differentially expressed among various cell types (1-6). For example, expression of ␣2-8 linked sialic acids is much less common than ␣2-3 linkages and appears restricted to a small subset of glycoproteins (7-10).␣2-6-linked sialic acids are also less abundant than ␣2-3-linked forms and are generated by at least four distinct gene products. However, the ST6Gal sialyltransferase appears solely responsible for producing the Sia␣2-6Gal␤1-4GlcNAc (Sia6LacNAc) terminus on various N glycans, and perhaps on some O glycans (1, 11). High levels of ST6Gal RNA have been found to preferentially accumulate in hematopoietic cells, as well as in the liver (3-5). Moreover, ST6Gal gene transcription is regulated by multiple promoters and altered by glucocorticoids and cytokines (12)(13)(14). Although the physiologic role of the ST6Gal sialyltransferase has not been defined previously by available genetic approaches, it has been shown to be unique in producing the ligand for the CD22 lectin molecule expressed on B lymphocytes.CD22 is a transmembrane glycoprotein lectin found exclusively on B lymphocytes and is known to p...

The composition of the gut microbiota is in constant flow under the influence of factors such as the diet, ingested drugs, the intestinal mucosa, the immune system, and the microbiota itself. Natural variations in the gut microbiota can deteriorate to a state of dysbiosis when stress conditions rapidly decrease microbial diversity and promote the expansion of specific bacterial taxa. The mechanisms underlying intestinal dysbiosis often remain unclear given that combinations of natural variations and stress factors mediate cascades of destabilizing events. Oxidative stress, bacteriophages induction and the secretion of bacterial toxins can trigger rapid shifts among intestinal microbial groups thereby yielding dysbiosis. A multitude of diseases including inflammatory bowel diseases but also metabolic disorders such as obesity and diabetes type II are associated with intestinal dysbiosis. The characterization of the changes leading to intestinal dysbiosis and the identification of the microbial taxa contributing to pathological effects are essential prerequisites to better understand the impact of the microbiota on health and disease.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating 0-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of 0-glycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte 0-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by 0-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate 0-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific 0-glycan formation governed by distinct polypeptide GalNAc-Ts.

Rapid shifts in microbial composition frequently occur during intestinal inflammation, but the mechanisms underlying such changes remain elusive. Here we demonstrate that an increased caecal sialidase activity is critical in conferring a growth advantage for some bacteria including Escherichia coli (E. coli) during intestinal inflammation in mice. This sialidase activity originates among others from Bacteroides vulgatus, whose intestinal levels expand after dextran sulphate sodium administration. Increased sialidase activity mediates the release of sialic acid from intestinal tissue, which promotes the outgrowth of E. coli during inflammation. The outburst of E. coli likely exacerbates the inflammatory response by stimulating the production of pro-inflammatory cytokines by intestinal dendritic cells. Oral administration of a sialidase inhibitor and low levels of intestinal α2,3-linked sialic acid decrease E. coli outgrowth and the severity of colitis in mice. Regulation of sialic acid catabolism opens new perspectives for the treatment of intestinal inflammation as manifested by E. coli dysbiosis.

We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition. DOI: https://doi.org/10. 1038/ng.3578 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-130493 Accepted Version Originally published at: van Karnebeek, Clara D M; Bonafé, Luisa; Wen, Xiao-Yan; Tarailo-Graovac, Maja; Balzano, Sara; RoyerBertrand, Beryl; Ashikov, Angel; Garavelli, Livia; Mammi, Isabella; Turolla, Licia; Breen, Catherine; Donnai, Dian; Cormier, Valerie; Heron, Delphine; Nishimura, Gen; Uchikawa, Shinichi; Campos-Xavier, Belinda; Rossi, Antonio; Hennet, Thierry; Brand-Arzamendi, Koroboshka; Rozmus, Jacob; Harshman, Keith; Stevenson, Brian J; Girardi, Enrico; Superti-Furga, Giulio; Dewan, Tammie; Collingridge, Alissa; Halparin, Jessie; Ross, Colin J; Van Allen, Margot I;et al (2016). NANS-mediated synthesis of sialic acid is required for brain and skeletal development. Nature Genetics, 48 (7) insights into the molecular basis of neurocognitive impairment allows for the development and 89 application of targeted therapeutic strategies 5 . Although less frequent than IDD, genetic disorders 90 affecting skeletal development and growth (commonly called the "skeletal dysplasias") are a 91 group of over 500 distinct disorders 6 . Studying their molecular basis has provided precious 92 insights into the many factors necessary for skeletal development, ranging from minerals and 93 structural molecules to enzymes, to signaling molecules and transcription factors 6,7 . We report 94here a genetic disorder presenting with a combination of severe IDD with skeletal dysplasia and 95 short stature. Our data show that its pathogenic basis is an inborn error of metabolism that 96 affects the endogenous synthesis of N-acetyl neuraminic acid (NeuNAc; sialic acid). Exploration 97 of the biochemical and molecular features of this disorder provides new information on the role 98 of sialic acid in the development of brain and bone. 99 100 RESULTS 101 Clinical and radiographic phenotype of N...

Within a few minutes after addition to L929 cells, tumour necrosis factor-alpha (TNF alpha) induced an increase in lucigenin-enhanced chemiluminescence that could be inhibited by superoxide dismutase. The generation of superoxide anion (O2.-) was sensitive to treatment with rotenone, antimycin A and cyanide, indicating that the signal originated from mitochondria. The mechanism of production of O2.- was shown to be independent of ATP synthesis, as uncoupling of this event from mitochondrial electron transport did not alter the generation of O2.- induced by TNF alpha. Chemiluminescence was further dependent on the presence of extracellular calcium, suggesting a role for this cation as a second messenger. This hypothesis was supported by the finding that inhibition of mitochondrial calcium uptake by Ruthenium Red exerted a protective effect on TNF alpha-treated L929 cells. Increased O2.- generation was followed by a marked decrease in mitochondrial dehydrogenase activity and cellular ATP levels, while cell membrane permeability was moderately increased. A role for mitochondrial O2.- generation in TNF alpha cytotoxicity was further supported by the finding that resistant L929 cells had decreased ability to produce O2.- in response to TNF alpha. In addition, we detected a decreased activity of the mitochondrial enzyme succinate dehydrogenase in these cells, suggesting that this component of the respiratory chain might be an important contributor to the TNF alpha-induced generation of O2.-.

Galactose is transferred via several linkages to acceptor structures by galactosyltransferase enzymes. In prokaryotes, galactose is mainly found on lipopolysaccharides and capsular polysaccharides. In eukaryotes, galactosyltransferases, which are localized in the Golgi apparatus, are involved in the formation of several classes of glycoconjugates and in lactose biosynthesis. Although they sometimes catalyze identical reactions, prokaryotic and eukaryotic galactosyltransferases share only little structural similarities. In mammals, 19 distinct galactosyltransferase enzymes have been characterized to date. These enzymes catalyze the transfer of galactose via beta1-4, beta1-3, alpha1-3 and alpha1-4 linkages. The present review focuses on the description of these mammalian galactosyltransferases.

The gut microbiota has been established as an important player influencing many aspects of human physiology. Breast milk, the first diet for an infant, contains human milk oligosaccharides (HMO) that shape the infant’s gut microbiota by selectively stimulating the growth of specific bacteria, especially bifidobacteria. In addition to their bifidogenic activity, the ability of HMO to modulate immune function and the gut barrier makes them prime candidates to restore a beneficial microbiota in dysbiotic adults and provide health benefits. We conducted a parallel, double-blind, randomised, placebo-controlled, HMO-supplementation study in 100 healthy, adult volunteers, consuming chemically produced 2′-O-fucosyllactose (2′FL) and/or lacto-N-neotetraose (LNnT) at various daily doses and mixes or placebo for 2 weeks. All participants completed the study without premature discontinuation. Supplementation of 2′FL and LNnT at daily doses up to 20 g was shown to be safe and well tolerated, as assessed using the gastrointestinal symptoms rating scale. 16S rRNA sequencing analysis showed that HMO supplementation specifically modified the adult gut microbiota with the primary impact being substantial increases in relative abundance of Actinobacteria and Bifidobacterium in particular and a reduction in relative abundance of Firmicutes and Proteobacteria. This study provides the first set of data on safety, tolerance and impact of HMO on the adult gut microbiota. Collectively, the results from this study show that supplementing the diet with HMO is a valuable strategy to shape the human gut microbiota and specifically promote the growth of beneficial bifidobacteria.