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

O-Linked glycosylation in Acanthamoeba polyphaga mimivirus

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 Mimiviru

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

Milk sialyllactose influences colitis in mice through selective intestinal bacterial colonization

The presence of particular oligosaccharides in mother’s milk influences bacterial colonization of the newborn mouse intestine and susceptibility to dextran sodium sulfate-driven colitis

Exposure to Mimivirus collagen promotes arthritis

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 the ORF 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 intra-dermally 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 human healthy 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

Selective proliferation of intestinal Barnesiella under fucosyllactose supplementation in mice

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 disease

ALG12 mannosyltransferase defect in congenital disorder of glycosylation type lg

In the endoplasmic reticulum (ER) of eukaryotes, N-linked glycans are first assembled on the lipid carrier dolichyl pyrophosphate. The GlcNAc2Man9Glc3 oligosaccharide is transferred to selected asparagine residues of nascent polypeptides. Defects along the biosynthetic pathway of N-glycans are associated with severe multisystemic syndromes called congenital disorders of glycosylation. Here, we describe a deficiency in the ALG12 ER α1,6-mannosyltransferase resulting in a novel type of glycosylation disorder. The severe disease was identified in a child presenting with psychomotor retardation, hypotonia, growth retardation, dysmorphic features and anorexia. In the patient's fibroblasts, the biosynthetic intermediate GlcNAc2Man7 oligosaccharide was detected both on the lipid carrier dolichyl pyrophosphate and on newly synthesized glycoproteins, thus pointing to a defect in the dolichyl pyrophosphate-GlcNAc2Man7-dependent ALG12 α1,6 mannosyltransferase. Analysis of the ALG12 cDNA in the CDG patient revealed compound heterozygosity for two point mutations that resulted in the amino acid substitutions T67M and R146Q, respectively. The impact of these mutations on ALG12 protein function was investigated in the Saccharomyces cerevisiae alg12 glycosylation mutant by showing that the yeast ALG12 gene bearing the homologous mutations T61M and R161Q and the human mutant ALG12 cDNA alleles failed to normalize the growth defect phenotype of the alg12 yeast model, whereas expression of the normal ALG12 cDNA complemented the yeast mutation. The ALG12 mannosyltransferase defect defines a new type of congenital disorder of glycosylation, designated CDG-I

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairment

Beta1,3-N-acetylglucosaminyltransferase 1 glycosylation is required for axon pathfinding by olfactory sensory neurons

During embryonic development, axons from sensory neurons in the olfactory epithelium (OE) extend into the olfactory bulb (OB) where they synapse with projection neurons and form glomerular structures. To determine whether glycans play a role in these processes, we analyzed mice deficient for the glycosyltransferase beta1,3-N-acetylglucosaminyltransferase 1 (beta3GnT1), a key enzyme in lactosamine glycan synthesis. Terminal lactosamine expression, as shown by immunoreactivity with the monoclonal antibody 1B2, is dramatically reduced in the neonatal null OE. Postnatal beta3GnT1-/- mice exhibit severely disorganized OB innervation and defective glomerular formation. Beginning in embryonic development, specific subsets of odorant receptor-expressing neurons are progressively lost from the OE of null mice, which exhibit a postnatal smell perception deficit. Axon guidance errors and increased neuronal cell death result in an absence of P2, I7, and M72 glomeruli, indicating a reduction in the repertoire of odorant receptor-specific glomeruli. By approximately 2 weeks of age, lactosamine is unexpectedly reexpressed in sensory neurons of null mice through a secondary pathway, which is accompanied by the regrowth of axons into the OB glomerular layer and the return of smell perception. Thus, both neonatal OE degeneration and the postnatal regeneration are lactosamine dependent. Lactosamine expression in beta3GnT1-/- mice is also reduced in pheromone-receptive vomeronasal neurons and dorsal root ganglion cells, suggesting that beta3GnT1 may perform a conserved function in multiple sensory systems. These results reveal an essential role for lactosamine in sensory axon pathfinding and in the formation of OB synaptic connections