Publications by authors named "Thierry Hennet"

62 Publications

Synthesis of photoactivable oligosaccharide derivatives from 1,2-cyclic carbamate building blocks and study of their interaction with carbohydrate-binding proteins.

Carbohydr Res 2021 Oct 14;508:108399. Epub 2021 Jul 14.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Despite the broad occurrence of carbohydrate-protein interactions in biology, the low binding affinities of such interactions hamper the characterization of carbohydrate binding sites in the absence of three-dimensional structural models. To allow the identification of proteins interacting with specific carbohydrate epitopes, we have developed new photoactivable oligosaccharide probes. Oligosaccharides containing the 1,2-cyclic carbamate group were attached to building blocks with a primary amino group to yield the corresponding urea derivatives. Cyclic carbamates of lactose, and 3- and 2'-fucosyl lactose, were used for the conjugation with building blocks containing photoactivable diazirine, benzophenone or aryl azido groups. The resulting oligosaccharide derivatives were tested for binding to Erythrina cristagalli lectin (ECL), Aleuria aurantia lectin (AAL) and Ulex europaeus agglutinin-I (UEA I). We found that ligands containing an aryl azido photoactivable group were successfully attached to lectins. The photoactivation reaction preserved lectin integrity, as no sign of protein degradation was visible. Mass spectrometric analysis confirmed the covalent binding of between one to three oligosaccharide probes, which matched with the expected carbohydrate-binding properties of the lectins tested. The conjugation of cyclic carbamate-derivatized oligosaccharides with photoactivable aryl azido groups thus represents a convenient approach to study protein-carbohydrate interactions.
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http://dx.doi.org/10.1016/j.carres.2021.108399DOI Listing
October 2021

Significance of fucose in intestinal health and disease.

Mol Microbiol 2021 06 3;115(6):1086-1093. Epub 2021 Feb 3.

Complex Carbohydrate Research Center and Department of Microbiology, University of Georgia, Athens, GA, USA.

The deoxyhexose sugar L-fucose is important for many biological processes within the human body and the associated microbiota. This carbohydrate is abundant in host gut mucosal surfaces, numerous microbial cell surface structures, and some dietary carbohydrates. Fucosylated oligosaccharides facilitate the establishment of a healthy microbiota and provide protection from infection. However, there are instances where pathogens can also exploit these fucosylated structures to cause infection. Furthermore, deficiencies in host fucosylation are associated with specific disease outcomes. This review focuses on our current understanding of the impact of fucosylation within the mucosal environment of the gastrointestinal tract with a specific emphasis on the mediatory effects in host-microbe interactions.
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http://dx.doi.org/10.1111/mmi.14681DOI Listing
June 2021

Limited Neonatal Carbohydrate-Specific Antibody Repertoire Consecutive to Partial Prenatal Transfer of Maternal Antibodies.

Front Immunol 2020 14;11:573629. Epub 2020 Oct 14.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Despite the prominence of carbohydrate-specific antibodies in human sera, data on their emergence and antigen specificities are limited. Whereas maternal IgG are transferred prenatally to the fetal circulation, IgM present in cord blood originate from fetal B lymphocytes. Considering the limited exposure of the fetus to foreign antigens, we assessed the repertoire of carbohydrate-specific antibodies in human cord blood and matched maternal blood samples using glycan arrays. Carbohydrate-specific IgM was absent in cord blood, whereas low cord blood IgG reactivity to glycans was detectable. Comparing IgG reactivities of matched pairs, we observed a general lack of correlation in the antigen specificity of IgG from cord blood and maternal blood due to a selective exclusion of most carbohydrate-specific IgG from maternofetal transfer. Given the importance of intestinal bacteria in inducing carbohydrate-specific antibodies, we analyzed global antibody specificities toward commensal bacteria. Similar IgG reactivities to specific species were detected in matched cord and maternal blood samples, thus pointing to an efficient maternal transfer of anti-microbial IgG. Due to the observed selectivity in maternofetal IgG transfer, the lack of fetal antibodies to carbohydrate epitopes is only partially compensated by maternal IgG, thus resulting in a weak response to carbohydrate antigens in neonates.
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http://dx.doi.org/10.3389/fimmu.2020.573629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591393PMC
June 2021

Intestinal inflammation alters mucosal carbohydrate foraging and monosaccharide incorporation into microbial glycans.

Cell Microbiol 2021 01 6;23(1):e13269. Epub 2020 Oct 6.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Endogenous carbohydrates released from the intestinal mucus represent a constant source of nutrients to the intestinal microbiota. Mucus-derived carbohydrates can also be used as building blocks in the biosynthesis of bacterial cell wall components, thereby influencing host mucosal immunity. To assess the uptake of endogenous carbohydrates by gut microbes in healthy mice and during intestinal inflammation, we applied azido-monosaccharides that can be tracked on bacterial cell walls after conjugation with fluorophores. In interleukin-10 deficient mice, changes in the gut microbiota were accompanied by decreased carbohydrate hydrolase activities and increased lumenal concentrations of host glycan-derived monosaccharides. Tracking of the monosaccharide N-azidoacetylglucosamine (GlcNAz) in caecum bacteria revealed a preferential incorporation of this carbohydrate by Xanthomonadaceae in healthy mice and by Bacteroidaceae in interleukin-10 deficient mice. These GlcNAz-positive Bacteroidaceae fractions mainly belonged to the species B. acidifaciens and B. vulgatus. Growth of Bacteroides species in the presence of specific monosaccharides changed their stimulatory activity toward CD11c dendritic cells. Expression of activation markers and cytokine production was highest after stimulation of dendritic cells with B. vulgatus. The variable incorporation of monosaccharides by related Bacteroides species underline the necessity to investigate intestinal bacteria down to the species level when addressing microbiota-host interactions.
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http://dx.doi.org/10.1111/cmi.13269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757161PMC
January 2021

Increased Antibody Response to Fucosylated Oligosaccharides and Fucose-Carrying Species in Crohn's Disease.

Front Microbiol 2020 14;11:1553. Epub 2020 Jul 14.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Inflammatory bowel disease is associated with intestinal dysbiosis and with elevated antibody production toward microbial epitopes. The underlying processes linking the gut microbiota with inflammation are still unclear. Considering the constant induction of antibodies by gut microbial glycans, the aim of this study was to address whether the repertoire of carbohydrate-specific antibodies is altered in Crohn's disease or ulcerative colitis. IgG and IgM reactivities to oligosaccharides representative of mucosal glycans were tested in blood serum from 20 healthy control subjects, 17 ulcerative colitis patients, and 23 Crohn's disease patients using glycan arrays. An increased IgG and IgM reactivity toward fucosylated oligosaccharides was detected in Crohn's disease but not in ulcerative colitis. To address the antibody reactivity to the gut microbiota, IgG binding to members of a complex intestinal microbiota was measured and observed to be increased in sera of patients with Crohn's disease. Based on the elevated reactivity to fucosylated oligosaccharides, gut bacteria were tested for recognition by the fucose-binding lectin. was detected in IgG- and lectin-positive fractions and reactivity of lectin was demonstrated for additional species. IgG reactivity to these species was significantly increased in inflammatory bowel disease patients, indicating that the increased reactivity to fucosylated oligosaccharides detected in Crohn's disease may be induced by fucose-carrying intestinal bacteria. Enhanced antibody response to fucosylated epitopes may have systemic effects by altering the binding of circulating antibodies to endogenous glycoproteins.
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http://dx.doi.org/10.3389/fmicb.2020.01553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381230PMC
July 2020

Emergence and significance of carbohydrate-specific antibodies.

Genes Immun 2020 08 5;21(4):224-239. Epub 2020 Aug 5.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Carbohydrate-specific antibodies are widespread among all classes of immunoglobulins. Despite their broad occurrence, little is known about their formation and biological significance. Carbohydrate-specific antibodies are often classified as natural antibodies under the assumption that they arise without prior exposure to exogenous antigens. On the other hand, various carbohydrate-specific antibodies, including antibodies to ABO blood group antigens, emerge after the contact of immune cells with the intestinal microbiota, which expresses a vast diversity of carbohydrate antigens. Here we explore the development of carbohydrate-specific antibodies in humans, addressing the definition of natural antibodies and the production of carbohydrate-specific antibodies upon antigen stimulation. We focus on the significance of the intestinal microbiota in shaping carbohydrate-specific antibodies not just in the gut, but also in the blood circulation. The structural similarity between bacterial carbohydrate antigens and surface glycoconjugates of protists, fungi and animals leads to the production of carbohydrate-specific antibodies protective against a broad range of pathogens. Mimicry between bacterial and human glycoconjugates, however, can also lead to the generation of carbohydrate-specific antibodies that cross-react with human antigens, thereby contributing to the development of autoimmune disorders.
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http://dx.doi.org/10.1038/s41435-020-0105-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449879PMC
August 2020

Antiviral potential of 3'-sialyllactose- and 6'-sialyllactose-conjugated dendritic polymers against human and avian influenza viruses.

Sci Rep 2020 01 21;10(1):768. Epub 2020 Jan 21.

Institute of Medical Virology, University of Zurich, 8057, Zurich, Switzerland.

Current treatment options for influenza virus infections in humans are limited and therefore the development of novel antivirals is of high priority. Inhibiting influenza virus attachment to host cells would provide an early and efficient block of the infection and thus, receptor analogs have been considered as options for antiviral treatment. Here, we describe the rapid and efficient synthesis of PAMAM dendrimers conjugated with either 3'-sialyllactose (3SL) or 6'-sialyllactose (6SL) and their potential to inhibit a diverse range of human and avian influenza virus strains. We show in a hemagglutination inhibition (HAI) assay that human IAV strains can be inhibited by (6SL)- and to a lesser extent also by (3SL)-conjugated PAMAM dendrimers. In contrast, avian strains could only be inhibited by (3SL)-conjugated dendrimers. Importantly, the differential sensitivities of human and avian IAV to the two types of sialyllactose-conjugated dendrimers could be confirmed in cell-based neutralization assays. Based on our findings, we suggest to further develop both, (3SL)- and (6SL)-conjugated PAMAM dendrimers, as influenza virus inhibitors.
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http://dx.doi.org/10.1038/s41598-020-57608-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972948PMC
January 2020

Maternal Human Milk Oligosaccharide Profile Modulates the Impact of an Intervention with Iron and Galacto-Oligosaccharides in Kenyan Infants.

Nutrients 2019 Oct 29;11(11). Epub 2019 Oct 29.

Laboratory of Human Nutrition, Department of Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland.

There is little data on human milk oligosaccharide (HMO) composition in Sub-Saharan Africa. Iron fortificants adversely affect the infant gut microbiota, while co-provision of prebiotic galacto-oligosaccharides (GOS) mitigates most of the adverse effects. Whether variations in maternal HMO profile can influence the infant response to iron and/or GOS fortificants is unknown. The aim of this study was to determine HMO profiles and the secretor/non-secretor phenotype of lactating Kenyan mothers and investigate their effects on the maternal and infant gut microbiota, and on the infant response to a fortification intervention with 5 mg iron (2.5 mg as sodium iron ethylenediaminetetraacetate and 2.5 mg as ferrous fumarate) and 7.5 g GOS. We studied mother-infant pairs ( = 80) participating in a 4-month intervention trial in which the infants (aged 6.5-9.5 months) received daily a micronutrient powder without iron, with iron or with iron and GOS. We assessed: (1) maternal secretor status and HMO composition; (2) effects of secretor status on the maternal and infant gut microbiota in a cross-sectional analysis at baseline of the intervention trial; and (3) interactions between secretor status and intervention groups during the intervention trial on the infant gut microbiota, gut inflammation, iron status, growth and infectious morbidity. Secretor prevalence was 72% and HMOs differed between secretors and non-secretors and over time of lactation. Secretor status did not predict the baseline composition of the maternal and infant gut microbiota. There was a secretor-status-by-intervention-group interaction on ( = 0.021), Z-scores for length-for-age ( = 0.022) and weight-for-age ( = 0.018), and soluble transferrin receptor ( = 0.041). In the no iron group, longitudinal prevalence of diarrhea was higher among infants of non-secretors (23.8%) than of secretors (10.4%) ( = 0.001). In conclusion, HMO profile may modulate the infant gut microbiota response to fortificant iron; compared to infants of secretor mothers, infants of non-secretor mothers may be more vulnerable to the adverse effect of iron but also benefit more from the co-provision of GOS.
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http://dx.doi.org/10.3390/nu11112596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893608PMC
October 2019

Alleviation of Intestinal Inflammation by Oral Supplementation With 2-Fucosyllactose in Mice.

Front Microbiol 2019 19;10:1385. Epub 2019 Jun 19.

Institute of Physiology, University of Zurich, Zurich, Switzerland.

Milk oligosaccharides exert a prebiotic action that contributes to the development of the infant gut microbiota during lactation. Given that milk oligosaccharides remain intact after passage through stomach and small intestine, they can potentially influence the composition of the gut microbiota when ingested as dietary supplements after weaning. To address the regulatory effects of specific oligosaccharides in colitis linked to the microbiota composition, we have supplemented interleukin-10 null ( ) mice with four fucosylated and sialylated oligosaccharides. We found that oral supplementation with 2-fucosyllactose significantly decreased the severity of colitis as displayed by reduced inflammatory marker expression, histological and diarrhea scores, an increased epithelial integrity and less pronounced colon shortening. Oral supplementation with 2-fucosyllactose led to a marked expansion of the commensal , which was accompanied by an enhanced cecal concentration of propionate. Decreased activation of immune cells by was confirmed by reconstitution of antibiotic-treated mice and by stimulation of dendritic cells . This study demonstrates that post-weaning administration of specific oligosaccharides can shift the composition of the gut microbiota to lessen chronic inflammation as observed in mice. The expansion of sets a positive microbial environment at the cost of pro-inflammatory Gram-negative bacteria, thereby lowering intestinal inflammation.
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http://dx.doi.org/10.3389/fmicb.2019.01385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593069PMC
June 2019

Collagen glycosylation.

Authors:
Thierry Hennet

Curr Opin Struct Biol 2019 06 26;56:131-138. Epub 2019 Feb 26.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Electronic address:

Despite the ubiquity of collagens in the animal kingdom, little is known about the biology of the disaccharide Glc(α1-2)Gal(β1-O) bound to hydroxylysine across collagens from sponges to mammals. The extent of collagen glycosylation varies by the types of collagen, with basement membrane collagen type IV being more glycosylated than fibrillar collagens. Beyond true collagens, proteins including collagen domains such as the complement protein 1Q and the hormone adiponectin also feature glycosylated hydroxylysine. Collagen glycosylation is initiated in the endoplasmic reticulum by the galactosyltransferases COLGALT1 and COLGALT2. Mutations in the COLGALT1 gene cause cerebral small vessel abnormality and porencephaly, which are common in collagen type IV deficiency. Beyond the strongly conserved Glc(α1-2)Gal(β1-O) glycan, additional forms of collagen glycosylation have been described in the deep-sea worm Riftia pachyptila and in the giant virus Mimivirus, thereby suggesting that further forms of collagen glycosylation are likely to be identified in the future.
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http://dx.doi.org/10.1016/j.sbi.2019.01.015DOI Listing
June 2019

Biallelic COLGALT1 variants are associated with cerebral small vessel disease.

Ann Neurol 2018 12 30;84(6):843-853. Epub 2018 Nov 30.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Objective: Approximately 5% of cerebral small vessel diseases are hereditary, which include COL4A1/COL4A2-related disorders. COL4A1/COL4A2 encode type IV collagen α1/2 chains in the basement membranes of cerebral vessels. COL4A1/COL4A2 mutations impair the secretion of collagen to the extracellular matrix, thereby resulting in vessel fragility. The diagnostic yield for COL4A1/COL4A2 variants is around 20 to 30%, suggesting other mutated genes might be associated with this disease. This study aimed to identify novel genes that cause COL4A1/COL4A2-related disorders.

Methods: Whole exome sequencing was performed in 2 families with suspected COL4A1/COL4A2-related disorders. We validated the role of COLGALT1 variants by constructing a 3-dimensional structural model, evaluating collagen β (1-O) galactosyltransferase 1 (ColGalT1) protein expression and ColGalT activity by Western blotting and collagen galactosyltransferase assays, and performing in vitro RNA interference and rescue experiments.

Results: Exome sequencing demonstrated biallelic variants in COLGALT1 encoding ColGalT1, which was involved in the post-translational modification of type IV collagen in 2 unrelated patients: c.452 T > G (p.Leu151Arg) and c.1096delG (p.Glu366Argfs*15) in Patient 1, and c.460G > C (p.Ala154Pro) and c.1129G > C (p.Gly377Arg) in Patient 2. Three-dimensional model analysis suggested that p.Leu151Arg and p.Ala154Pro destabilized protein folding, which impaired enzymatic activity. ColGalT1 protein expression and ColGalT activity in Patient 1 were undetectable. RNA interference studies demonstrated that reduced ColGalT1 altered COL4A1 secretion, and rescue experiments showed that mutant COLGALT1 insufficiently restored COL4A1 production in cells compared with wild type.

Interpretation: Biallelic COLGALT1 variants cause cerebral small vessel abnormalities through a common molecular pathogenesis with COL4A1/COL4A2-related disorders. Ann Neurol 2018;84:843-853.
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http://dx.doi.org/10.1002/ana.25367DOI Listing
December 2018

Custom Glycosylation of Cells and Proteins Using Cyclic Carbamate-Derivatized Oligosaccharides.

Cell Chem Biol 2017 Nov 21;24(11):1336-1346.e3. Epub 2017 Sep 21.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Electronic address:

The structural complexity of glycosylation restrains the functional characterization of glycans. We present a versatile carbohydrate ligation technique based on the reaction of cyclic carbamates with primary amines. Cyclic-carbamate-derivatized carbohydrates can be added to primary amine-containing molecules in aqueous solution to yield glycoconjugates. This method enabled the presentation of carbohydrate epitopes on live animal cells, as shown by the acquisition of E-selectin binding sites on mouse MC-38 cells decorated with 3-fucosyllactose or 3-fucosyl-3-sialyllactose. Ligation of 3- and 6-sialyllactose to Escherichia coli demonstrated the importance of sialic acid linkages in regulating complement factor H binding. Proteins were modified with oligosaccharides to study their role in stimulating cytokine secretion by dendritic cells, thus pointing to interactions between glycoproteins and phosphoinositide 3-kinase signaling in controlling interleukin-12, tumor necrosis factor alpha and interleukin-1β release. Overall, cyclic-carbamate-mediated ligation is useful to study the biology of carbohydrate epitopes on proteins and on cell membranes.
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http://dx.doi.org/10.1016/j.chembiol.2017.08.012DOI Listing
November 2017

Mechanisms and consequences of intestinal dysbiosis.

Cell Mol Life Sci 2017 08 28;74(16):2959-2977. Epub 2017 Mar 28.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

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.
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http://dx.doi.org/10.1007/s00018-017-2509-xDOI Listing
August 2017

Oral supplementation of healthy adults with 2'-O-fucosyllactose and lacto-N-neotetraose is well tolerated and shifts the intestinal microbiota.

Br J Nutr 2016 Oct 10;116(8):1356-1368. Epub 2016 Oct 10.

2Department of Medicine,Zealand University Hospital,Lykkebækvej 1,DK-4600 Køge,Denmark.

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.
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http://dx.doi.org/10.1017/S0007114516003354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082288PMC
October 2016

Glycosylation site occupancy in health, congenital disorder of glycosylation and fatty liver disease.

Sci Rep 2016 10 11;6:33927. Epub 2016 Oct 11.

Institute of Physiology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.

Glycosylation is an integral part in health and disease, as emphasized by the growing number of identified glycosylation defects. In humans, proteins are modified with a diverse range of glycoforms synthesized in complex biosynthetic pathways. Glycosylation disorders have been described in congenital disorders of glycosylation (CDG) as well as in acquired disease conditions such and non-alcoholic fatty liver disease (NAFLD). A hallmark in a subset of CDG cases is the reduced glycosylation site occupancy of asparagine-linked glycans. Using an optimized method protocol, we determined the glycosylation site occupancy from four proteins of hepatic and lymphatic origin from CDG and NAFLD patients. We found variable degrees of site occupancy, depending on the tissue of origin and the disease condition. In CDG glycosylation sites of IgG2 and IgA1 were occupied to normal levels. In NAFLD haptoglobin and transferrin glycosylation sites were hyper-glycosylated, a property qualifying for its use as a potential biomarker. Furthermore, we observed, that glycosylation sites of liver-originating transferrin and haptoglobin are differentially occupied under physiological conditions, a further instance not noticed in serum proteins to date. Our findings suggest the use of serum protein hyperglycosylation as a biomarker for early stages of NAFLD.
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http://dx.doi.org/10.1038/srep33927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057071PMC
October 2016

Giant mimivirus R707 encodes a glycogenin paralogue polymerizing glucose through α- and β-glycosidic linkages.

Biochem J 2016 Oct 18;473(20):3451-3462. Epub 2016 Jul 18.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Acanthamoeba polyphaga mimivirus is a giant virus encoding 1262 genes among which many were previously thought to be exclusive to cellular life. For example, mimivirus genes encode enzymes involved in the biosynthesis of nucleotide sugars and putative glycosyltransferases. We identified in mimivirus a glycogenin-1 homologous gene encoded by the open reading frame R707. The R707 protein was found to be active as a polymerizing glucosyltransferase enzyme. Like glycogenin-1, R707 activity was divalent-metal-ion-dependent and relied on an intact DXD motif. In contrast with glycogenin-1, R707 was, however, not self-glucosylating. Interestingly, the product of R707 catalysis featured α1-6, β1-6 and α1-4 glycosidic linkages. Mimivirus R707 is the first reported glycosyltransferase able to catalyse the formation of both α and β linkages. Mimivirus-encoded glycans play a role in the infection of host amoebae. Co-infection of Acanthamoeba with mimivirus and amylose and chitin hydrolysate reduced the number of infected amoebae, thus supporting the importance of polysaccharide chains in the uptake of mimivirus by amoebae. The identification of a glycosyltransferase capable of forming α and β linkages underlines the peculiarity of mimivirus and enforces the concept of a host-independent glycosylation machinery in mimivirus.
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http://dx.doi.org/10.1042/BCJ20160280DOI Listing
October 2016

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.

J Biol Chem 2016 08 11;291(35):18514-24. Epub 2016 Jul 11.

From the Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland

Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability.
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http://dx.doi.org/10.1074/jbc.M116.723379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000096PMC
August 2016

NANS-mediated synthesis of sialic acid is required for brain and skeletal development.

Nat Genet 2016 07 23;48(7):777-84. Epub 2016 May 23.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

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.
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http://dx.doi.org/10.1038/ng.3578DOI Listing
July 2016

Breastfed at Tiffany's.

Trends Biochem Sci 2016 06 16;41(6):508-518. Epub 2016 Apr 16.

Department of Physiology and Zurich Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, Zürich, Switzerland.

The importance of breast milk for the growing infant is undisputed; breastfeeding decreases infantile mortality by tenfold and decreases the incidence of infectious diseases. Despite its recognized benefits, the structural richness of breast milk has also impeded the characterization of the multiple effects of milk components on infant physiology. However, the important roles of some components of breast milk are beginning to be dissected. For instance, molecules such as immunoglobulin A (IgA) and milk oligosaccharides protect from gastrointestinal infections and influence the development of the gut microbiota. Deciphering the complex composition of breast milk brings to light multifaceted contributions that combine to make breast milk the ultimate personalized medicine.
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http://dx.doi.org/10.1016/j.tibs.2016.02.008DOI Listing
June 2016

Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice.

Nat Commun 2015 Aug 25;6:8141. Epub 2015 Aug 25.

Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich CH-8057, Switzerland.

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.
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http://dx.doi.org/10.1038/ncomms9141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560832PMC
August 2015

Congenital disorders of glycosylation: a concise chart of glycocalyx dysfunction.

Trends Biochem Sci 2015 Jul 31;40(7):377-84. Epub 2015 Mar 31.

Institute of Physiology, University of Zurich, CH-8057 Zurich, Switzerland.

Glycosylation is a ubiquitous modification of lipids and proteins. Despite the essential contribution of glycoconjugates to the viability of all living organisms, diseases of glycosylation in humans have only been identified over the past few decades. The recent development of next-generation DNA sequencing techniques has accelerated the pace of discovery of novel glycosylation defects. The description of multiple mutations across glycosylation pathways not only revealed tremendous diversity in functional impairments, but also pointed to phenotypic similarities, emphasizing the interconnected flow of substrates underlying glycan assembly. The current list of 100 known glycosylation disorders provides an overview of the significance of glycosylation in human development and physiology.
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http://dx.doi.org/10.1016/j.tibs.2015.03.002DOI Listing
July 2015

O-Linked glycosylation in Acanthamoeba polyphaga mimivirus.

Glycobiology 2014 Aug 2;24(8):703-14. Epub 2014 May 2.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, Zurich 8057, Switzerland.

Acanthamoeba polyphaga mimivirus is a member of the giant nucleocytoplasmic large DNA viruses, infecting various Acanthamoeba spp. The genomes of giant viruses encode components previously thought to be exclusive to cellular life, such as proteins involved in nucleic acid and protein synthesis. Recent work on enzymes involved in carbohydrate biosynthesis and metabolism show that instead of utilizing host cell resources, Mimivirus produces its own glycosylation machinery. To obtain a more detailed view of glycosylation in Mimivirus, we developed a periodate oxidation-based method to selectively enrich Mimivirus surface glycoproteins. O-Glycosylation in Mimivirus glycoproteins was identified by permethylation and matrix-assisted laser desorption/ionization-mass spectrometry analyses of beta-eliminated glycans. We sequenced 26 previously undescribed O-glycans, most of which contain glucose as their reducing end saccharide. These data will facilitate future studies on the functional significance of glycosylation in Mimivirus.
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http://dx.doi.org/10.1093/glycob/cwu034DOI Listing
August 2014

Decoding breast milk oligosaccharides.

Swiss Med Wkly 2014 Feb 19;144:w13927. Epub 2014 Feb 19.

Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, SWITZERLAND;

Oligosaccharides represent a significant fraction of breast milk, reaching up to 20 g/l in early milk. Human milk oligosaccharides comprise close to 200 structures, which are not absorbed by the intestinal tissue and have no nutritional value for the breastfed infant. Early studies conducted around 1930 already attributed a prebiotic activity to milk oligosaccharides by showing their stimulatory effects on the growth of specific intestinal microbiota. In addition, milk oligosaccharides contribute to the defence against enteric pathogens by acting as soluble decoys preventing the adhesion of viruses and bacteria to their carbohydrate mucosal receptors. The structural complexity of milk oligosaccharides hampers the assignment of specific functions to single carbohydrates. The application of mouse models allows the investigation of unique milk oligosaccharides in the context of intestinal microbiota and mucosal immunity. In this respect, our recent work has demonstrated that uptake of the milk oligosaccharide 3-sialyllactose increases the inflammatory response observed in different colitis models. The proinflammatory action of 3-sialyllactose was attributed on the one hand to the modulation of intestinal bacterial groups, and on the other hand to a direct stimulatory effect on CD11c+ dendritic cells. The availability of pure oligosaccharides in large amounts will soon enable the study of these compounds in humans in the context of intestinal and metabolic disorders associated to various forms of dysbiosis.
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http://dx.doi.org/10.4414/smw.2014.13927DOI Listing
February 2014

Selective proliferation of intestinal Barnesiella under fucosyllactose supplementation in mice.

Br J Nutr 2014 May 10;111(9):1602-10. Epub 2014 Jan 10.

Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.

The oligosaccharides 2-fucosyllactose and 3-fucosyllactose are major constituents of human breast milk but are not found in mouse milk. Milk oligosaccharides have a prebiotic action, thus affecting the colonisation of the infant intestine by microbiota. To determine the specific effect of fucosyllactose exposure on intestinal microbiota in mice, in the present study, we orally supplemented newborn mice with pure 2-fucosyllactose and 3-fucosyllactose. Exposure to 2-fucosyllactose and 3-fucosyllactose increased the levels of bacteria of the Porphyromonadaceae family in the intestinal gut, more precisely members of the genus Barnesiella as analysed by 16S pyrosequencing. The ability of Barnesiella to utilise fucosyllactose as energy source was confirmed in bacterial cultures. Whereas B. intestinihominis and B. viscericola did not grow on fucose alone, they proliferated in the presence of 2-fucosyllactose and 3-fucosyllactose following the secretion of linkage-specific fucosidase enzymes that liberated lactose. The change in the composition of intestinal microbiota mediated by fucosyllactose supplementation affected the susceptibility of mice to dextran sulphate sodium-induced colitis, as indicated by increased resistance of mice subjected to 2-fucosyllactose supplementation for 6 weeks. The present study underlines the ability of specific milk oligosaccharides to change the composition of intestinal microbiota and thereby to shape an intestinal milieu resilient to inflammatory diseases.
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http://dx.doi.org/10.1017/S0007114513004200DOI Listing
May 2014

Recombinant expression of hydroxylated human collagen in Escherichia coli.

Appl Microbiol Biotechnol 2014 May 21;98(10):4445-55. Epub 2013 Dec 21.

Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.
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http://dx.doi.org/10.1007/s00253-013-5447-zDOI Listing
May 2014

Exposure to mimivirus collagen promotes arthritis.

J Virol 2014 Jan 30;88(2):838-45. Epub 2013 Oct 30.

Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.

Collagens, the most abundant proteins in animals, also occur in some recently described nucleocytoplasmic large DNA viruses such as Mimiviridae, which replicate in amoebae. To clarify the impact of viral collagens on the immune response of animals exposed to Mimiviridae, we have investigated the localization of collagens in Acanthamoeba polyphaga mimivirus particles and the response of mice to immunization with mimivirus particles. Using protein biotinylation, we have first shown that viral collagen encoded by open reading frame L71 is present at the surface of mimivirus particles. Exposure to mimivirus collagens elicited the production of anti-collagen antibodies in DBA/1 mice immunized intradermally with mimivirus protein extracts. This antibody response also targeted mouse collagen type II and was accompanied by T-cell reactivity to collagen and joint inflammation, as observed in collagen-induced arthritis following immunization of mice with bovine collagen type II. The broad distribution of nucleocytoplasmic large DNA viruses in the environment suggests that humans are constantly exposed to such large virus particles. A survey of blood sera from healthy human subjects and from rheumatoid arthritis patients indeed demonstrated that 30% of healthy-subject and 36% of rheumatoid arthritis sera recognized the major mimivirus capsid protein L425. Moreover, whereas 6% of healthy-subject sera recognized the mimivirus collagen protein L71, 22% of rheumatoid arthritis sera were positive for mimivirus L71. Accordingly, our study shows that environmental exposure to mimivirus represents a risk factor in triggering autoimmunity to collagens.
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http://dx.doi.org/10.1128/JVI.03141-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911627PMC
January 2014

Milk oligosaccharide sialyl(α2,3)lactose activates intestinal CD11c+ cells through TLR4.

Proc Natl Acad Sci U S A 2013 Oct 7;110(43):17444-9. Epub 2013 Oct 7.

Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, 8057 Zürich, Switzerland.

Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10(-/-) mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10(-/-) mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10(-/-);St3gal4(-/-) mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10(-/-) mice to lactating St3gal4(-/-) mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c(+) dendritic cells and induced production of cytokines required for expansion of TH1 and TH17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c(+) cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.
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http://dx.doi.org/10.1073/pnas.1306322110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808656PMC
October 2013

The role of milk sialyllactose in intestinal bacterial colonization.

Adv Nutr 2012 May 1;3(3):483S-8S. Epub 2012 May 1.

Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Switzerland.

Milk oligosaccharides influence the composition of intestinal microbiota and thereby mucosal inflammation. Some of the major milk oligosaccharides are α2,3-sialyllactose (3SL) and α2,6-sialyllactose, which are mainly produced by the sialyltransferases ST3GAL4 and ST6GAL1, respectively. Recently, we showed that mice fed milk deficient in 3SL were more resistant to dextran sulfate sodium-induced colitis. By contrast, the exposure to milk containing or deficient in 3SL had no impact on the development of mucosal leukocyte populations. Milk 3SL mainly affected the colonization of the intestine by clostridial cluster IV bacteria.
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http://dx.doi.org/10.3945/an.111.001651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3649486PMC
May 2012

Diseases of glycosylation beyond classical congenital disorders of glycosylation.

Authors:
Thierry Hennet

Biochim Biophys Acta 2012 Sep 9;1820(9):1306-17. Epub 2012 Feb 9.

Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Switzerland.

Background: Diseases of glycosylation are rare inherited disorders, which are often referred to as congenital disorders of glycosylation (CDG). Several types of CDG have been described in the last decades, encompassing defects of nucleotide-sugar biosynthesis, nucleotide-sugar transporters, glycosyltransferases and vesicular transport. Although clinically heterogeneous, most types of CDG are associated with neurological impairments ranging from severe psychomotor retardation to moderate intellectual disabilities. CDG are mainly caused by defects of N-glycosylation, owing to the simple detection of under-glycosylated serum transferrin by isoelectric focusing.

Scope Of Review: In the last years, several disorders of O-glycosylation, glycolipid and glycosaminoglycan biosynthesis have been described, which are known by trivial names not directly associated with the family of CDG. The present review outlines 64 gene defects affecting glycan biosynthesis and modifications, thereby underlining the complexity of glycosylation pathways and pointing to unexpected phenotypes and functional redundancies in the control of glycoconjugate biosynthesis.

Major Conclusions: The increasing application of whole-genome sequencing techniques unravels new defects of glycosylation, which are associated to moderate forms of mental disabilities.

General Significance: The knowledge gathered through the investigation of CDG increases the understanding of the functions associated to protein glycosylation in humans. This article is part of a Special Issue entitled Glycoproteomics.
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http://dx.doi.org/10.1016/j.bbagen.2012.02.001DOI Listing
September 2012
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