The effects of 2'-fucosyllactose and lacto-N-neotetraose, galacto-oligosaccharides, and maternal human milk oligosaccharide profile on iron absorption in Kenyan infants.

BACKGROUND: Whether prebiotic human milk oligosaccharides (HMO), such as 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT), enhance iron absorption in infants is unknown. Moreover, whether maternal HMO profile affects absorption of iron fortificants or the effects of prebiotic galacto-oligosaccharides (GOS) and/or HMO on iron absorption is uncertain. OBJECTIVES: The aim of this study was to test whether consumption of 3.0 g GOS or HMO enhances iron absorption from iron-fortified maize porridge in partially breastfed Kenyan infants and whether maternal HMO profile modulates these effects. METHODS: In a randomized, prospective crossover study, 55 infants (aged 8-12 mo) were fed test meals fortified with 1 of the following: 1) 5.0 mg iron as 54Fe-labeled ferrous fumarate (FeFum); 2) 5.0 mg iron as 58FeFum and 3.0 g GOS (FeFum+GOS); and 3) 5.0 mg iron as 57FeFum and 2.0 g 2'-FL and 1.0 g LNnT (FeFum+HMO). Fractional iron absorption (FIA) was assessed by erythrocyte incorporation of iron isotopes. HMO profiles were determined by capillary gel electrophoresis with laser-induced florescence detection. Data were analyzed with mixed-effect models, and iron dialyzability was measured in vitro. RESULTS: Of the 55 infants included, 49 were fed as instructed. FIA from the FeFum+GOS group [median (IQR) 22.2% (16.5%-25.9%)] was higher than that from the FeFum group [12.5% (9.5%-20.9%)] (P = 0.005). FIA from the FeFum+HMO group was 13.3% (7.1%-24.4%) and did not differ from the FeFum group (P = 0.923). Maternal HMO profile did not predict FIA or modulate the effects of GOS or HMO on FIA. Iron dialyzability ratios at pH 2 of FeFum+GOS to FeFum and FeFum+HMO to FeFum were 2.1 and 0.9 (P = 0.001 and P = 0.322), respectively. CONCLUSIONS: In Kenyan infants consuming FeFum-fortified maize porridge, co-provision of 3.0 g GOS increased FIA by 78%, whereas co-provision of 3.0 g HMO did not affect FIA. Variations in maternal HMO profile, including secretor and Lewis phenotype, did not predict FIA. These data argue against a physiologic role for 2'-FL and LNnT in facilitating iron absorption in infancy. The study was registered at clinicaltrials.gov as NCT04163406 (https://clinicaltrials.gov/ct2/show/NCT04163406).

Increase of intestinal bacterial sialidase activity exacerbates acute colitis in mice.

The availability of endogenous and dietary carbohydrates in the gastrointestinal tract influences the composition of the gut microbiota. Carbohydrate foraging requires the action of bacterially-encoded glycoside hydrolases, which release mono- and oligosaccharides taken up as carbon sources by multiple microbial taxa. In addition to providing nutrients to the microbiota, the cleavage of host glycans by bacterial glycoside hydrolases may alter the properties of surface glycoproteins involved in cell adhesion and activation processes in the gut lumen. To investigate the impact of bacterial glycoside hydrolase activities on the gut microbial composition and on host glycans during colon inflammation, we increased local glycoside hydrolase activity by supplementing mice with recombinant E. coli expressing specific sialidase, fucosidase and rhamnosidase enzymes during acute colitis induced by dextran sulfate sodium ingestion. Whereas increased fucosidase and rhamnosidase activity did not alter the course of colitis, increased sialidase activity exacerbated disease severity. The effect of increased sialidase activity on inflammation was not caused by changes in the microbial composition given that a similar shift in gut bacteria occurred in all groups of mice supplemented with recombinant E. coli. Increased sialidase activity in the colon of treated mice however significantly altered the distribution of sialic acid on mucosal glycans. Treatment of lamina propria dendritic cells with bacterial sialidase also strongly decreased the density of sialylated ligands to anti-inflammatory siglec lectins, indicating that the remodeling of surface sialylation caused by increased sialidase activity likely accounts for the observed exacerbation of acute colitis in mice.

Glycosylation-Dependent Induction of Programmed Cell Death in Murine Adenocarcinoma Cells.

Altered surface glycosylation is a major hallmark of tumor cells associated with aggressive phenotype and poor prognosis. By recognizing specific carbohydrate motifs, lectins can be applied to distinguish tumor from healthy cells based on the expression of glycosylation-dependent markers. Through their ability to bind to specific carbohydrates, lectins induce cell agglutination and cross-link surface glycoproteins, thereby mediating mitogenic and death-inducing effects in various cell types. The carbohydrate-selective cytotoxic effect of lectins also enables their possible application in therapies targeting cancer cells. To clarify the intracellular pathways mediating cell death induced by a group of plant and fungal lectins, we investigated mouse adenocarcinoma MC-38 cells harboring inactive genes involved in apoptosis, necroptosis and pyroptosis. Treatment of MC-38 cells with wheat germ agglutinin, Maackia amurensis lectin I, and Aleuria aurantia lectin induced multiple cell death pathways through reactions that relied on the autophagy machinery without depending on caspase activation. Furthermore, inhibition of de novo protein synthesis by cycloheximide strongly decreased the cytotoxic response, indicating that the lectins investigated induced cell death via effector molecules that are not expressed under normal circumstances and supporting the non-apoptotic nature of cell death. The broad cytotoxic response to lectins can be beneficial for the development of combination therapies targeting tumor cells. Given that tumors acquire resistance to various cytotoxic treatments because of mutations in cell death pathways, compounds inducing broad cytotoxic responses, such as lectins, represent potent sensitizers to promote tumor cell killing.

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

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.

Significance of fucose in intestinal health and disease.

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.

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

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.

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

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 Bacteroides 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.

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

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 Aleuria aurantia lectin. Bacteroides stercoris was detected in IgG- and lectin-positive fractions and reactivity of A. aurantia lectin was demonstrated for additional Bacteroides species. IgG reactivity to these Bacteroides 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.

Emergence and significance of carbohydrate-specific antibodies.

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.

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

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.

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

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 (n = 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 Bifidobacterium (p = 0.021), Z-scores for length-for-age (p = 0.022) and weight-for-age (p = 0.018), and soluble transferrin receptor (p = 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%) (p = 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.

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

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 (Il10 -/-) 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 Ruminococcus gnavus, which was accompanied by an enhanced cecal concentration of propionate. Decreased activation of immune cells by R. gnavus was confirmed by reconstitution of antibiotic-treated Il10 -/- mice and by stimulation of dendritic cells in vitro. 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 Il10 -/- mice. The expansion of R. gnavus sets a positive microbial environment at the cost of pro-inflammatory Gram-negative bacteria, thereby lowering intestinal inflammation.

Collagen glycosylation.

Despite the ubiquity of collagens in the animal kingdom, little is known about the biology of the disaccharide Glc(α1-2)Gal(ß1-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(ß1-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.

Biallelic COLGALT1 variants are associated with cerebral small vessel disease.

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 ß (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.

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

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-1ß release. Overall, cyclic-carbamate-mediated ligation is useful to study the biology of carbohydrate epitopes on proteins and on cell membranes.

Mechanisms and consequences of intestinal dysbiosis.

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.

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

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.

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

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.

Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.

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.