The Compartmentalisation of Phosphorylated Free Oligosaccharides in Cells from a CDG Ig Patient Reveals a Novel ER-to-Cytosol Translocation Process

BACKGROUND: Biosynthesis of the dolichol linked oligosaccharide (DLO) required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5)GlcNAc(2)-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3)Man(9)GlcNAc(2)-PP-dolichol). After transfer of Glc(3)Man(9)GlcNAc(2) onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P). Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I) in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation. METHODS AND PRINCIPAL FINDINGS: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7)GlcNAc(2)-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7)GlcNAc(2)-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7)GlcNAc(2)-P appears in the cytosol without detectable generation of ER luminal Man(7)GlcNAc(2)-P. CONCLUSIONS AND SIGNIFICANCE: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that pyrophosphatase-mediated cleavage of DLO intermediates yields recyclable dolichol-P. The kinetics of cytosolic fOSGN2-P generation from a luminally-generated DLO intermediate demonstrate the presence of a previously undetected ER-to-cytosol translocation process for either fOSGN2-P or DLO

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

BACKGROUND: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. METHODS/PRINCIPAL FINDINGS: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS. CONCLUSIONS/SIGNIFICANCE: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming

Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster

BACKGROUND: Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. PRINCIPAL FINDINGS: In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. CONCLUSION: Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function

In vitro digestion and lactase treatment influence uptake of quercetin and quercetin glucoside by the Caco-2 cell monolayer

BACKGROUND: Quercetin and quercetin glycosides are widely consumed flavonoids found in many fruits and vegetables. These compounds have a wide range of potential health benefits, and understanding the bioavailability of flavonoids from foods is becoming increasingly important. METHODS: This study combined an in vitro digestion, a lactase treatment and the Caco-2 cell model to examine quercetin and quercetin glucoside uptake from shallot and apple homogenates. RESULTS: The in vitro digestion alone significantly decreased quercetin aglycone recovery from the shallot digestate (p < 0.05), but had no significant effect on quercetin-3-glucoside recovery (p > 0.05). Digestion increased the Caco-2 cell uptake of shallot quercetin-4'-glucoside by 2-fold when compared to the non-digested shallot. Despite the loss of quercetin from the digested shallot, the bioavailability of quercetin aglycone to the Caco-2 cells was the same in both the digested and non-digested shallot. Treatment with lactase increased quercetin recovery from the shallot digestate nearly 10-fold and decreased quercetin-4'-glucoside recovery by more than 100-fold (p < 0.05), but had no effect on quercetin recovery from apple digestates. Lactase treatment also increased shallot quercetin bioavailability to the Caco-2 cells approximately 14-fold, and decreased shallot quercetin-4'-glucoside bioavailability 23-fold (p < 0.05). These Caco-2 cells had lactase activity similar to that expressed by a lactose intolerant human. CONCLUSIONS: The increase in quercetin uptake following treatment with lactase suggests that dietary supplementation with lactase may increase quercetin bioavailability in lactose intolerant humans. Combining the digestion, the lactase treatment and the Caco-2 cell culture model may provide a reliable in vitro model for examining flavonoid glucoside bioavailability from foods

Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro

Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources

<p>Abstract</p> <p>Background</p> <p>Because of its size, allohexaploid nature and high repeat content, the wheat genome has always been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based markers have been developed. This study aims at combining the unique genomic resources developed in wheat and barley to decipher the organisation of gene space on wheat chromosome 3B.</p> <p>Results</p> <p>Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome 3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing gradient of gene density from the centromere to the telomeres positively correlated with the number of genes clustered in islands on wheat chromosome 3B.</p> <p>Conclusion</p> <p>Our study shows that novel structural genomics resources now available in wheat and barley can be combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space.</p

Multigenic phylogeny and analysis of tree incongruences in Triticeae (Poaceae)

Background: Introgressive events (e.g., hybridization, gene flow, horizontal gene transfer) and incomplete lineage sorting of ancestral polymorphisms are a challenge for phylogenetic analyses since different genes may exhibit conflicting genealogical histories. Grasses of the Triticeae tribe provide a particularly striking example of incongruence among gene trees. Previous phylogenies, mostly inferred with one gene, are in conflict for several taxon positions. Therefore, obtaining a resolved picture of relationships among genera and species of this tribe has been a challenging task. Here, we obtain the most comprehensive molecular dataset to date in Triticeae, including one chloroplastic and 26 nuclear genes. We aim to test whether it is possible to infer phylogenetic relationships in the face of (potentially) large-scale introgressive events and/or incomplete lineage sorting; to identify parts of the evolutionary history that have not evolved in a tree-like manner; and to decipher the biological causes of genetree conflicts in this tribe. Results: We obtain resolved phylogenetic hypotheses using the supermatrix and Bayesian Concordance Factors (BCF) approaches despite numerous incongruences among gene trees. These phylogenies suggest the existence of 4-5 major clades within Triticeae, with Psathyrostachys and Hordeum being the deepest genera. In addition, we construct a multigenic network that highlights parts of the Triticeae history that have not evolved in a tree-lik