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BACKGROUND: Congenital disorders of glycosylation (CDG) includes ALG8 deficiency, a protein N-glycosylation defect with a broad clinical spectrum. If most of the 15 previously reported patients present an early-onset multisystem severe disease and early death, three patients including the cas princeps, present long-term survival and less severe symptoms. METHODS: In order to further characterize ALG8-CDG, two new ALG8 patients are described and mRNA analyses of the ALG8-CDG cas princeps were effected. RESULTS: One new patient exhibited a hepato-intestinal and neurological phenotype with two novel variants (c.91A > C p.Thr31Pro; c.139dup p.Thr47Asnfs*12). The other new patient, homozygous for a known variant (c.845C > T p.Ala282Val), presented a neurological phenotype with epilepsy, intellectual disability and retinis pigmentosa. The cas princeps ALG8-CDG patient was reported to have two heterozygous frameshift variants predicted to be without activity. We now described a novel ALG8 transcript variant in this patient and the 3D model of the putative encoded protein reveals no major difference with that of the normal ALG8 protein. CONCLUSION: The description of the two new ALG8 patients affirms that ALG8-CDG is a severe disease. In the cas princeps, as the originally described frameshift variants are degraded, the novel variant is promoted and could explain a milder phenotype.

We reported an oligosaccharide diphosphodolichol (DLO) diphosphatase (DLODP) that generates dolichyl-phosphate and oligosaccharyl phosphates (OSPs) from DLO in vitro. This enzyme could underlie cytoplasmic OSP generation and promote dolichyl-phosphate recycling from truncated endoplasmic reticulum (ER)-generated DLO intermediates. However, during subcellular fractionation, DLODP distribution is closer to that of a Golgi apparatus (GA) marker than those of ER markers. Here, we examined the effect of brefeldin A (BFA), which fuses the GA with the ER on OSP metabolism. In order to increase the steady state level of truncated DLO while allowing formation of mature DLO (Glc3Man9GlcNAc2-PP-dolichol), dolichyl-P-mannose Man7GlcNAc2-PP-dolichol mannosyltransferase was partially downregulated in HepG2 cells. We show that BFA provokes GA endomannosidase trimming of Glc3Man9GlcNAc2-PP-dolichol to yield a Man8GlcNAc2-PP-dolichol structure that does not give rise to cytoplasmic Man8GlcNAc2-P. BFA also strikingly increased OSP derived from mature DLO within the endomembrane system without affecting levels of Man7GlcNAc2-PP-dolichol or cytoplasmic Man7GlcNAc2-P. The BFA-provoked increase in endomembrane-situated OSP is sensitive to nocodazole, and BFA causes partial redistribution of DLODP activity from GA- to ER-containing regions of density gradients. These findings are consistent with BFA-provoked microtubule-dependent GA-to-ER transport of a previously reported DLODP that acts to generate a novel endomembrane-situated OSP population.

Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.

International audience; Orexins (orexin-A and orexin-B) are hypothalamic peptides that are produced by the same precursor and are involved in sleep/wake control, which is mediated by two G protein-coupled receptor subtypes, OX1R and OX2R. Ulcerative colitis (UC) is an inflammatory bowel disease, (IBD) which is characterized by long-lasting inflammation and ulcers that affect the colon and rectum mucosa and is known to be a significant risk factor for colon cancer development. Based on our recent studies showing that OX1R is aberrantly expressed in colon cancer, we wondered whether orexin-A could play a role in UC. Immunohistochemistry studies revealed that OX1R is highly expressed in the affected colonic epithelium of most UC patients, but not in the non-affected colonic mucosa. Injection of exogenous orexin-A specifically improved the inflammatory symptoms in the two colitis murine models. Conversely, injection of inactive orexin-A analog, OxB7-28 or OX1R specific antagonist SB-408124 did not have anti-inflammatory effect. Moreover, treatment with orexin-A in DSS-colitis induced OX1R −/− knockout mice did not have any protective effect. The orexin-A anti-inflammatory effect was due to the decreased expression of pro-inflammatory cytokines in immune cells and specifically in T-cells isolated from colonic mucosa. Moreover, orexin-A inhibited canonical NFκB activation in an immune cell line and in intestinal epithelial cell line. These results suggest that orexin-A might represent a promising alternative to current UC therapies.

International audience; Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [(3)H]OSP from [(3)H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.

The primary function of peptide N-glycanase (PNGase) is thought to be the deglycosylation of endoplasmic reticulum associated degradation (ERAD) substrates. However, inhibition of PNGase appears to have little effect upon the destruction rate of many ERAD substrates, and recent data demonstrate deglycosylation-independent functions for PNGase. Whatever the roles of PNGase turn out to be, the identification of a patient presenting with PNGase deficiency will advance our understanding of the importance of this multifunctional protein in human physiology.

Background Type I congenital disorders of glycosylation (CDG-I) are mostly complex multisystemic diseases associated with hypoglycosylated serum glycoproteins. A subgroup harbour mutations in genes necessary for the biosynthesis of the dolichol-linked oligosaccharide (DLO) precursor that is essential for protein N-glycosylation. Here, our objective was to identify the molecular origins of disease in such a CDG-Ix patient presenting with axial hypotonia, peripheral hypertonia, enlarged liver, micropenis, cryptorchidism and sensorineural deafness associated with hypo glycosylated serum glycoproteins. Results Targeted sequencing of DNA revealed a splice site mutation in intron 5 and a non-sense mutation in exon 4 of the dehydrodolichol diphosphate synthase gene (DHDDS). Skin biopsy fibroblasts derived from the patient revealed ~20 % residual DHDDS mRNA, ~35 % residual DHDDS activity, reduced dolichol-phosphate, truncated DLO and N-glycans, and an increased ratio of [2-3H]mannose labeled glycoprotein to [2-3H]mannose labeled DLO. Predicted truncated DHDDS transcripts did not complement rer2-deficient yeast. SiRNA-mediated down-regulation of DHDDS in human hepatocellular carcinoma HepG2 cells largely mirrored the biochemical phenotype of cells from the patient. The patient also harboured the homozygous ALG6(F304S) variant, which does not cause CDG but has been reported to be more frequent in PMM2-CDG patients with severe/fatal disease than in those with moderate presentations. WES did not reveal other strong candidate causal genes. Conclusions We describe a patient presenting with severe multisystem disease associated with DHDDS deficiency. As retinitis pigmentosa is the only clinical sign in previously reported cases, this report broadens the spectrum of phenotypes associated with this condition.

The congenital disorders of glycosylation (CDG) are a recently described group of inherited multisystem disorders characterized by defects predominantly of N- and O-glycosylation of proteins. Cardiomyopathy in CDG has previously been described in several subtypes; it is usually associated with high morbidity and mortality and the majority of cases present in the first 2 years of life. This is the first case with presentation in late childhood and the article reviews current literature. An 11-year-old female with a background of learning difficulties presented in cardiac failure secondary to severe dilated cardiomyopathy. Prior to the diagnosis of CDG, her condition deteriorated; she required mechanical support (Excor Berlin Heart) and was listed for cardiac transplant. Investigations included screening for glycosylation disorders, and isoelectric focusing of transferrin revealed an abnormal type 1 pattern. Analysis of phosphomannomutase and phosphomannose isomerase showed normal enzyme activity, excluding PMM2 (CDG Ia) and MPI (CDG Ib). Lipid-linked oligosaccharide and mutational studies have not yet defined the defect. Despite aggressive therapy there were persistent difficulties achieving adequate anticoagulation and she developed multiple life-threatening thrombotic complications. She was removed from the transplant list and died from overwhelming sepsis 5 weeks following admission. This case emphasizes the need to screen all children with an undiagnosed cardiomyopathy for CDG, regardless of age, and where possible to exclude CDG before the use of cardiac bridging devices. It highlights the many practical and ethical challenges that may be encountered where clinical knowledge and experience are still evolving.

Autophagy is an intracellular pathway induced by starvation, inhibited by nutrients, that is responsible for degradation of long-lived proteins and altered cell organelles. This process is involved in cell maintenance could be induced by antilipolytic drugs and may have anti-aging effects [A. Donati, The involvement of macroautophagy in aging and anti-aging interventions, Mol. Aspects Med. 27 (2006) 455-470]. We analyzed the effect of an intraperitoneal injection of an antilipolytic agent (3,5'-dimethylpyrazole, DMP, 12mg/kg b.w.), that mimics nutrient shortage on autophagy and expression of autophagic genes in the liver of male 3-month-old Sprague-Dawley albino rats. Autophagy was evaluated by observing electron micrographs of the liver autophagosomal compartment and by monitoring protein degradation assessed by the release of valine into the bloodstream. LC3 gene expression, whose product is one of the best known markers of autophagy, was also monitored. As expected, DMP decreased the plasma levels of free fatty acids, glucose, and insulin and increased autophagic vacuoles and proteolysis. DMP treatment caused an increase in the expression of the LC3 gene although this occurred later than the induction of authophagic proteolysis caused by DMP. Glucose treatment rescued the effects caused by DMP on glucose and insulin plasma levels and negatively affected the rate of autophagic proteolysis, but did not suppress the positive regulatory effect on LC3 mRNA levels. In conclusion, antilipolytic drugs may induce both autophagic proteolysis and higher expression of an autophagy-related gene and the effect on autophagy gene expression might not be secondary to the stimulation of autophagic proteolysis.

During protein N-glycosylation in mammalian cells, free oligosaccharides (fOS) are generated from lipid-linked oligosaccharides by a pyrophosphatase activity and oligosaccharyltransferase and from misfolded glycoproteins by peptide:N-glycanase in both the ER and cytoplasm. Trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an endo-beta-N-acetyl-glucosaminidase, and the cytosolic M2C1 mannosidase drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. Transport of fOS out of the ER is normally efficient and if inhibited causes fOS to be secreted via the Golgi apparatus. By contrast, fOS clearance from the cytosol into lysosomes is less efficient resulting in low micromolar concentrations of fOS in the cytoplasm. Structural analysis of cytosolic fOS reveals oligosaccharide families whose relative abundance highlights the importance of different ER-associated degradation (ERAD) pathways for misfolded glycoproteins and suggests that in liver cells substantial amounts of glycoproteins destined for ERAD may transit early compartments of the Golgi apparatus. Glycoprotein quality control and ERAD are controlled by N-glycan/lectin interactions and the fOS trafficking pathway would seem to ensure that fOS do not interfere with these processes which occur in both the ER and cytoplasm. Although Saccharomyces cerevisiae strains harbouring mutations in genes of the yeast fOS metabolic pathway do not display obvious phenotypes, mammalian fOS are quantitatively more important and the processes leading to their regulation are more complex, raising the possibility that distinct phenotypes will be seen in mammalian cells or animals in which fOS metabolism is modified.

Two highly conserved eukaryotic gene products of unknown function showing homology to glycosyltransferases involved in the second steps of bacterial peptidoglycan (Murg) and capsular polysaccharide (Cps14f/Cps14g) biosynthesis have been identified in silico. The amino acid sequence of the eukaryotic protein that is homologous to the lipid acceptor- and membrane-associating N-terminal domain of Murg and the Cps14f beta4-galactosyltransferase enhancer protein is predicted to possess a cleavable signal peptide and transmembrane helices. The other eukaryotic protein is predicted to possess neither transmembrane regions nor a signal peptide but is homologous to the UDP-sugar binding C-terminal domain of Murg and the Cps14g beta4-galactosyltransferase. Both the eukaryotic proteins are encoded by essential genes in Saccharomyces cerevisiae, and down-regulation of either causes growth retardation, reduced N-glycosylation of carboxypeptidase Y, and accumulation of dolichyl-PP-GlcNAc. In vitro studies demonstrate that these proteins are required for transfer of [3H]GlcNAc from UDP-[3H]GlcNAc onto dolichyl-PP-GlcNAc. To conclude, two gene products showing homology to bacterial glycosyltransferases are required for the second step in dolichyl-PP-oligosaccharide biosynthesis.

Free oligosaccharides (fOS) are generated during glycoprotein biosynthesis in mammalian cells. Here we report on the origin and fate of these structures in the yeast Saccharomyces cerevisiae. After metabolic radiolabelling with [2-(3)H]mannose ([2-(3)H]Man) for 30 min, Man(8)GlcNAc(2) was identified as the predominant fOS in this organism, and radioactivity associated with this structure was found to correspond to approximately 1% of that associated with the same structure N -linked to glycoprotein. Despite provoking a fourfold increase in radioactivity associated with lipid-linked oligosaccharide, the protein-synthesis inhibitor cycloheximide blocked [2-(3)H]Man incorporation into both endo-beta-D- N -acetylglucosamine H-sensitive N-glycans and fOS. Peptide:N-glycanase, encoded by the PNG1 gene, was found to be required for the generation of a large proportion of yeast fOS during, and soon after, protein glycosylation. Use of an ams1 Delta strain deficient in the vacuolar alpha-mannosidase revealed this enzyme to be responsible for the slow growth-associated catabolism of fOS. The present paper constitutes the first description of fOS formation in intact S. cerevisiae, and, with the demonstration that fOS are degraded by the vacuolar mannosidase, a novel function for this poorly understood enzyme has been identified.

The underlying causes of type I congenital disorders of glycosylation (CDG I) have been shown to be mutations in genes encoding proteins involved in the biosynthesis of the dolichyl-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-dolichyl) that is required for protein glycosylation. Here we describe a CDG I patient displaying gastrointestinal problems but no central nervous system deficits. Fibroblasts from this patient accumulate mainly Man(9)GlcNAc(2)-PP-dolichyl, but in the presence of castanospermine, an endoplasmic reticulum glucosidase inhibitor Glc(1)Man(9)GlcNAc(2)-PP-dolichyl predominates, suggesting inefficient addition of the second glucose residue onto lipid-linked oligosaccharide. Northern blot analysis revealed the cells from the patient to possess only 10-20% normal amounts of mRNA encoding the enzyme, dolichyl-P-glucose:Glc(1)Man(9)GlcNAc(2)-PP-dolichyl alpha3-glucosyltransferase (hALG8p), which catalyzes this reaction. Sequencing of hALG8 genomic DNA revealed exon 4 to contain a base deletion in one allele and a base insertion in the other. Both mutations give rise to premature stop codons predicted to generate severely truncated proteins, but because the translation inhibitor emetine was shown to stabilize the hALG8 mRNA from the patient to normal levels, it is likely that both transcripts undergo nonsense-mediated mRNA decay. As the cells from the patient were successfully complemented with wild type hALG8 cDNA, we conclude that these mutations are the underlying cause of this new CDG I subtype that we propose be called CDG Ih.

On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.

The primary function of peptide N-glycanase (PNGase) is thought to be the deglycosylation of endoplasmic reticulum associated degradation (ERAD) substrates. However, inhibition of PNGase appears to have little effect upon the destruction rate of many ERAD substrates, and recent data demonstrate deglycosylation-independent functions for PNGase. Whatever the roles of PNGase turn out to be, the identification of a patient presenting with PNGase deficiency will advance our understanding of the importance of this multifunctional protein in human physiology.

We have previously shown that the transcription of the human sucrase-isomaltase (SI) gene was negatively regulated by glucose. Using two clonal metabolic variants of the human colon adenocarcinoma cell line Caco-2 we demonstrate here that: 1) although similar growth-related variations of phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-diphosphatase (F1,6-dPase), pyruvate kinase (PK) and SI mRNA levels are observed, only F1,6-dPase, PK and SI mRNA levels vary in the same way in response to modifications of glucose utilization; and 2) regulatory elements responsible for the glucose-dependent transcription of the SI gene are located within the −370/+30 region of the promoter.

We have previously shown that glucose can exert a repressive effect on the transcription of the sucrase–isomaltase (SI) gene in the differentiated enterocyte-like human colon carcinoma cell lines HT-29 and Caco-2. To characterize the region through which glucose exerts this effect, three different-length fragments of the 5´-flanking region of the human SI gene were linked to the reporter gene luciferase in an episomal vector carrying a hygromycin resistance gene. These fragments were used for transfection into a clone of the Caco-2 cell line, PF11, which has high glucose consumption and only expresses SI at high levels when cultured in the presence of a low supply of glucose. By using the stably transformed PF11 cells grown either in standard high glucose (25 mM) or in low glucose (1 mM) it was possible to show that the smallest fragment of the SI promoter, extending from bases -370 to +30, contains all the information required for the glucose repression of the reporter gene luciferase.

The expression of the brush border-associated hydrolase sucrase-isomaltase was shown to increase from early to late passages of Caco-2 cells, concomitant with a decrease in the rates of glucose consumption. Twenty-six clones were isolated from early (P29) and late (P198) passages of the cell line. These clones show considerable and inverse differences in the levels of sucrase activities and rates of glucose consumption, without marked changes in other features of enterocytic differentiation of the cells (presence of an apical brush border, levels of expression of other brush border-associated hydrolases). Clones with low sucrase-isomaltase expression show a mosaic expression of the enzyme and a 38-fold higher rate of glucose consumption than clones with high sucrase-isomaltase expression. The clones with high expression show an homogeneous apical distribution of the enzyme and 70-fold and 35-fold higher levels of sucrase activities and sucrase-isomaltase mRNA, respectively. In contrast no differences were found from one clone to another in the enrichment of sucrase activity in brush border-enriched fractions as compared to cell homogenates. Switch to low glucose-containing medium (1 mM versus 25 mM in standard culture conditions) of cells with low sucrase-isomaltase results in an increased and more homogeneous expression of the enzyme and a tenfold augmentation of the levels of sucrase-isomaltase mRNA and sucrase activity. These results show that glucose interferes with the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level.

In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.

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.

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the alpha-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH2-terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the Galphai3-dependent control of autophagy. Accordingly, expressing its non-Galphai3-interacting NH2-terminal domain or its Galphai3-interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the Galphai3 protein in the control of a major catabolic pathway.

Type I congenital disorders of glycosylation (CDG I) are diseases presenting multisystemic lesions including central and peripheral nervous system deficits. The disease is characterized by under-glycosylated serum glycoproteins and is caused by mutations in genes encoding proteins involved in the stepwise assembly of dolichol-oligosaccharide used for protein N-glycosylation. We report that fibroblasts from a type I CDG patient, born of consanguineous parents, are deficient in their capacity to add the eighth mannose residue onto the lipid-linked oligosaccharide precursor. We have characterized cDNA corresponding to the human ortholog of the yeast gene ALG12 that encodes the dolichyl-P-Man:Man(7)GlcNAc(2)-PP-dolichyl alpha6-mannosyltransferase that is thought to accomplish this reaction, and we show that the patient is homozygous for a point mutation (T571G) that causes an amino acid substitution (F142V) in a conserved region of the protein. As the pathological phenotype of the fibroblasts of the patient was largely normalized upon transduction with the wild type gene, we demonstrate that the F142V substitution is the underlying cause of this new CDG, which we suggest be called CDG Ig. Finally, we show that the fibroblasts of the patient are capable of the direct transfer of Man(7)GlcNAc(2) from dolichol onto protein and that this N-linked structure can be glucosylated by UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum.

Incorporation into plasmids of genes conferring resistance to aminoglycoside antibiotics such as hygromycin B is currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably transfected with an episomal plasmid containing the hygromycin resistance gene, we observed that transformed cells subcultured in the presence of hygromycin B exhibit, compared with the same cells subcultured in antibiotic-free medium, a sixfold increase in the rates of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and hexose transporter GLUT-2, which are downregulated, contrasting with an upregulation of hexose transporter GLUT-1. This occurs without significant modifications of the differentiation status of the cells, as demonstrated by the normal expression of villin, ZO-1, dipeptidyl peptidase IV, or Na+-K+-ATPase. The plasmid copy number is, however, the same, whether or not the cells are cultured in the presence of hygromycin B. These results draw attention to the need to consider antibiotic-dependent alterations of metabolism and gene expression in transfection experiments.

The effect of cyclic AMP on the expression of the fructose transporter, GLUT5, was studied in Caco-2 cells, a human colon cancer cell line that differentiates spontaneously in culture into cells with the properties of small intestine enterocytes. Treatment of differentiated Caco-2 cells with 50 microM forskolin, which stimulates adenylate cyclase and raises intracellular cyclic AMP levels, increased fructose uptake 2-fold and raised GLUT5 protein and mRNA levels 5- and 7-fold respectively. The increased GLUT5 mRNA levels in forskolin-treated cells are a result of stabilization of GLUT5 mRNA in these cells and increased transcription. The effect of cyclic AMP on GLUT5 transcription was assessed by measuring the activity of human GLUT5 promoter-reporter gene constructs in forskolin-treated differentiated Caco-2 cells. The results showed that forskolin stimulated the activity of the GLUT5-reporter gene constructs and this stimulatory effect was mediated by cis-acting regulatory sequences.

Colon cancer is a major health problem because of its high frequency and the poor outcome of invasive forms, due to their overall resistance to chemotherapy. Although it has been assumed that drug resistance could be associated with some particular cell populations, these have not been characterized yet. It is only in recent years that progress in the field of intestinal cell biology, based on the development of cultured cell lines and availability of immunological and molecular probes, has allowed to characterize cells at the Single cell level and to study their Organization and functions. This has led to the concept of colon cancer cell differentiation. Cellular differentiation, which should not be confused with differentiation of colon cancers as defined by pathologists, is the ability of colon cancer cells to express, in vitro and in vivo, the same morphological and functional characteristics as normal epithelial intestinal cells, i.e. enterocytes or goblet cells. Whether these cells, which behave like normal cells as to their differentiation characteristics and functions, possess particular adaptation properties which allow them to escape the cytotoxic effect of a number of stress conditions, including treatment with anti-cancer drugs, is based on recent experimental data obtained with cultured human colon cancer cell lines. The purpose of this article is to (1) summarize our knowledge on colon cancer cell differentiation and show how experience in the field of cell biology can be transferred to clinical situations and (2) focus on experimental data which suggest that drug resistance is associated with cellular differentiation.

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.

The complete sequence of the 6 kb cDNA and the 5′ genomic structure are reported for the gene coding for the human intestinal brush border hydrolase sucrase-isomaltase. The human sucrase-isomaltase cDNA shows a high level of identity (83%) with that of the rabbit enzyme, indicating that the protein shares the same structural domains in both species. In addition to the previously reported homology with lysosomal alpha-glucosidase, the sucrase and isomaltase subunits also appear to be homologous to a yeast glucoamylase. A 14 kb human genomic clone has been isolated which includes the first three exons and the first two introns of the gene, as well as 9.5 kb 5′ to the major start site of transcription. The first exon comprises 62 bp of untranslated sequence and the second starts exactly at the initiation ATG codon. Typical CAAT and TATA boxes are seen upstream of the first exon. A genetic polymorphism is described which involves a PstI site in the second intron. Southern blotting, sequencing and mRNA studies indicate that the structures of the sucrase-isomaltase gene and its mRNA are unaltered in the two human colon cancer cell lines Caco-2 and HT-29 in comparison with normal human small intestine.

International audience; Treatment for 48 h of differentiated, confluent Caco-2 cells with 2.5 10(-5) M forskolin or 10(-6) M monensin, which produces a significant decrease of the de novo biosynthesis of sucrase-isomaltase, does not change quantitatively the de novo biosynthesis of dipeptidylpeptidase IV. Western blot analysis and silver nitrate staining indicate that neither drug induces any modification in the steady state expression of these two brush border hydrolases. Northern blot analysis shows that the level of dipeptidylpeptidase IV mRNA does not change in treated as compared to control Caco-2 cells. In contrast, forskolin and monensin dramatically decrease the level of sucrase-isomaltase mRNA. These observations suggest a separate regulation of biosynthesis for sucrase-isomaltase and dipeptidylpeptidase IV in intestinal cells. The mechanisms responsible for such a difference are discussed. Among them, the role of glucose metabolism, which is perturbed by both drugs, appears to be of crucial importance.

International audience; We report the nucleotide sequence and derived amino-acid sequence of a cDNA clone encoding the 3' end of human intestinal dipeptidylpeptidase IV (DPP-IV). This cDNA probe identifies a 4 kb mRNA in the human colon cancer cell line Caco-2. We demonstrate here an extensive homology between this human DPP-IV cDNA and the recently published rat liver DPP-IV cDNA. Using the human DPP-IV cDNA to probe genomic DNA from a panel of somatic cell hybrids we have assigned the gene encoding human DPP-IV to chromosome 2.

International audience; Using L-[35S]methionine labeling, SDS-PAGE and Northern blot analysis of sucrase-isomaltase mRNA, two different concentrations of monensin were used to delineate in Caco-2 cells the effect of the drug on the conversion of the high mannose to the complex form of sucrase-isomaltase from its dual effect on the biosynthesis of the enzyme and on the rate of glucose consumption. At 0.1 microM the drug has no effect on the rate of glucose consumption and, although it inhibits the conversion of the high mannose to the complex form of the enzyme, it has no effect on the level of sucrase-isomaltase mRNA and on the amount of neosynthesized enzyme. At 1 microM, in addition to its inhibiting effect on the maturation of the enzyme, monensin provokes concomitantly an increase in the rate of glucose consumption and a decrease in the level of sucrase-isomaltase mRNA and in the amount of neosynthesized enzyme. All these effects are reversible within 48 h after removal of the drug.

Treatment of Caco-2 cells with forskolin (25 microM) or monensin (1 microM) has previously been shown to cause a marked decrease in the level of sucrase-isomaltase (SI) mRNA, without any effect on the expression of dipeptidylpeptidase IV (DPP-IV). In the present work, we report that there is no significant difference in the stability of SI mRNA between control and treated cells. On the other hand, we demonstrate a decrease in the transcription rate of SI mRNA which is sufficient to account for the decrease in the steady-state level of SI mRNA both in forskolin- and monensin-treated Caco-2 cells.

International audience; The biosynthesis of sucrase-isomaltase was compared in enterocyte-like differentiated (i.e., grown in the absence of glucose) and undifferentiated (i.e., grown in the presence of glucose) HT-29 cells. Unlike differentiated cells, in which the enzyme is easily detectable and active, undifferentiated cells display almost no enzyme activity and the protein cannot be detected by means of cell surface immunofluorescence or immunodetection in membrane-enriched fractions or cell homogenates. Pulse experiments with L-[35S]-methionine show that the enzyme is, however, synthesized in these undifferentiated cells. As compared with the corresponding molecular forms in differentiated cells, the high-mannose form of the enzyme in undifferentiated cells is similarly synthesized and has the same apparent Mr. However, its complex form is less labeled and has a lower apparent Mr. Pulse-chase experiments with L-[35S]methionine show that, although the enzyme is synthesized to the same extent in both situations, the high-mannose and complex forms are rapidly degraded in undifferentiated cells, with an apparent half-life of 6 h, in contrast to differentiated cells in which the enzyme is stable for at least 48 h. A comparison of the processing of the enzyme in both situations shows that the conversion of the high-mannose to the complex form is markedly decreased in undifferentiated cells. These results indicate that the absence of sucrase-isomaltase expression in undifferentiated cells is not the consequence of an absence of biosynthesis but rather the result of both an impaired glycosylation and a rapid degradation of the enzyme.

The expression of small intestinal hydrolases associated with the enterocyte brush border membrane was studied in human colon cancers and foetal colons, by means of monoclonal antibodies against human small intestinal sucrase-isomaltase (SI), maltase-glucoamylase (MGA), lactase (L), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPP-IV). The enzymes were visualized by indirect immunofluorescence on cryostat sections of tumors developed in nude mice with 6 human colon carcinoma cell lines (HT-29, Caco-2, SW-480, HRT-18, HCT-8R, and Co-115), of 27 primary colorectal carcinomas from patients, and of human foetal (16 to 20 weeks of gestation) and normal adult small intestines and colons. All 5 monoclonals bound to the brush border of the adult small intestine, but not to that of the adult colon mucosa. Antibodies against SI, APN and DPP-IV also bound to the brush border of the foetal colons, to apical borders in HT-29 and Caco-2 tumors in nude mice, and to brush border-like structures in 7/27 tumors from patients. No binding was observed for MGA and L in either tumors or foetal colons. Binding of anti-SI antibodies to the brush border of the juxta-tumoral mucosal epithelium was observed in 9/11 samples tested. These data indicate that some colon tumors exhibit a typical pattern of enterocytic differentiation which is of foetal type and which involves at least 3 brush border membrane hydrolases. Monoclonal antibodies to small intestinal hydrolases may, therefore, be important tools for identification and characterization of some differentiated colonic tumors.

International audience; Hybridization analysis of mRNA with a cDNA probe for human sucrase-isomaltase, pulse-chase experiments with L-[35S]-methionine followed by SDS-PAGE, and immunofluorescence detection of sucrase-isomaltase were used to analyze the level(s) at which forskolin interferes with the expression of the enzyme in Caco-2 cells in culture. Three effects are observed in Caco-2 cells treated with forskolin: 1) a marked decrease in the level of sucrase-isomaltase mRNA, 2) a marked decrease in the biosynthesis of the enzyme without any alteration of its stability, and 3) an almost total inhibition of its transport to the brush border membrane. All three effects are reversible when the drug is removed from the culture medium, though this reversibility is asynchronous: transport to the brush border membrane resumes after 24 h, sucrase-isomaltase mRNA levels are back to the normal after 5 days, whereas the biosynthesis of the enzyme, although increasing progressively, remains lower than in control cells, even 10 days after removal of the drug. The possibility that some effects are directly dependent on cAMP and others a consequence of changes in glucose metabolism is discussed.

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.

International audience; Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.