Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structur

Differential effects of lobe A and lobe B of the conserved oligomeric golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2

AbstractMost lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1–6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease

Biosynthèse et dégradation des N-glycoprotéines (un métabolisme intimement lié à la Man2C1)

La plupart des alpha-mannosidases cellulaires présentent un rôle essentiel dans la maturation des N-glycoprotéines. La mannosidase cytosolique (Man2C1) intervient dans le processus de dégradation des N-glycoprotéines mal conformées nommé Endoplasmic Reticulum Associated Degradation process (ERAD). Au cours de la voie ERAD, les glycoprotéines mal conformées sont acheminées vers le cytosol puis dégradées par le complexe protéosomal. Bien que la séquence des événements de cette voie ERAD soit bien décrite, son mode de régulation reste encore peu connu. Nous avons étudié l'influence de la surexpression de la Man2C1 sur le processus de N-glycosylation des protéines. Notre étude s est portée sur une lignée cellulaire dérivée de cellules HeLa surexprimant de manière stable la Man2C1 (HeLa-NAM). Après avoir validé notre modèle d étude, l analyse structurale des oligosaccharides solubles nous a permis de montrer que la surexpression de la Man2C1 conduisait à 1) une accumulation d oligomannosides de type Man2-4GlcNAc1 dans le cytosol et 2) à une augmentation importante du pool de mannose libre intracellulaire. Nous avons également observé une augmentation du taux de glycoprotéines néosynthétisées envoyées dans le processus ERAD. Enfin, nous avons montré que la surexpression de la Man2C1 était accompagnée de la synthèse et le transfert d un oligosaccharide précurseur non glucosylé (Man9GlcNAc2) conduisant à une altération générale de la N-glycosylation des protéines. Ainsi, l ensemble de nos résultats ont permis de montrer le rôle essentiel de la Man2C1 dans le recyclage du mannose endogène intracellulaire. Par ailleurs, ces travaux ont permis de mettre en évidence l importance de la régulation du pool de mannose endogène dans l efficacité de biosynthèse des N-glycoprotéines. Il s agit donc d un nouveau concept dans lequel, pour la première fois, la variation d expression d une enzyme impliquée dans le catabolisme des N-glycoprotéines affecterait également la biosynthèse des N-glycannes.Alpha-mannosidases are key enzymes in the metabolism of N-glycoproteins and most of them are involved in the processing of N-glycans. The cytosolic mannosidase, Man2C1, plays an essential role in the degradation of misfolded N-glycoproteins also called Endoplasmic Reticulum Associated Degradation process (ERAD). Man2C1 is particularly involved in the catabolism of free oligomannosides released in the cytosol. Although the sequential events of ERAD are well described, its regulation remains poorly understood. We investigated the impact of Man2C1 overexpression on ERAD process and protein glycosylation. We first established a stable cell line, called HeLa-NAM overexpressing the Man2C1. After validating our cellular model, we demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man2-4GlcNAc1 glycans in the cytosol. We correlated this accumulation with the synthesis and transfer of incomplete lipid-linked oligosaccharide precursors, which yields an increase in N-glycoprotein en route to the ERAD. Our results suggest that, besides its essential role in oligosaccharide catabolism, Man2C1 represents an important salvage pathway for recycling free mannose. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Impact du PUGNAc sur le catabolisme des N-glycoprotéines

Les Oligosaccharides soluble (OS) sont essentiellement générés durant le processus de dégradation des N-glycoprotéines nouvellement synthétisées et mal conformées (ERAD) et durant la voie turn-over des glycoprotéines matures. Dans le but de déterminer si la modification post-traductionnelle O-GlcNAc est effectivement impliquée dans le processus de dégradation des N-glycoprotéines, nous avons analysé par spectrométrie de masse les OS provenant des cellules CHO après traitement par l inhibiteur PUGNAc. Le PUGNAc ou O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbanate est un inhibiteur puissant de l O-GlcNAcase (OGA) qui catalyse l hydrolyse du résidu O-GlcNAc des résidus sérine et thréonine des protéines O-GlcNAcylées.L analyse par spectrométrie de masse révèle l apparition d une population d OS de structures anormaux dans les cellules CHO suite au traitement par le PUGNAc. Cette population a été identifiée comme ayant des structures possédant des résidus GlcNAc au niveau de leur extrémité non-réductrice issues d une dégradation lysosomale incomplète des glycoprotéines. Contrairement au PUGNAc, le NButGT, un autre inhibiteur de l OGA, n aboutit pas à l apparition de cette population. Ainsi, Nous avons démontré que ces structures s accumulent exclusivement dans la fraction membranaire conséquence de l inhibition des b-hexosaminidases lysosomaux par le PUGNAc. Notre étude avait permis, d un part, de mettre en évidence la capacité du PUGNAc de mimer une maladie de surcharge lysosomale et, d autre part, de montrer un autre aspect des effets indésirables induits par le PUGNAc et qui nécessite d être pris en considération lors de l utilisation de cet inhibiteur.Free oligosaccharides (fOS) are generated as a result of glycoproteins catabolism that occurs in two principal distinct pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N-glycoproteins and the mature N-glycoproteins turnover pathway. We analyzed fOS by Mass spectrometry in PUGNAc CHO treated cells in order to investigate whether O-GlcNAc modified proteins were involved in N-glycoprotein degradation process. The O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbanate (PUGNAc) is a potent inhibitor of the O-GlcNAcase (OGA) catalyzing the cleavage of b-O-linked 2-acetamido-2-deoxy-beta-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Mass spectrometry (MS) analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of the population. These structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal b-hexosaminidases inhibition by PUGNAc. As Lysosomal Storage Disorders (LSD) are characterized by the accumulation of fOS in various tissues, our study evokes that PUGNAc mimics a LSD and shows another off target effects that needs to be taken into account in the use of this drug.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

The Transmembrane Domains of the prM and E Proteins of Yellow Fever Virus Are Endoplasmic Reticulum Localization Signals

The immature flavivirus particle contains two envelope proteins, prM and E, that are associated as a heterodimer. Virion morphogenesis of the flaviviruses occurs in association with endoplasmic reticulum (ER) membranes, suggesting that there should be accumulation of the virion components in this compartment. This also implies that ER localization signals must be present in the flavivirus envelope proteins. In this work, we looked for potential subcellular localization signals in the yellow fever virus envelope proteins. Confocal immunofluorescence analysis of the subcellular localization of the E protein in yellow fever virus-infected cells indicated that this protein accumulates in the ER. Similar results were obtained with cells expressing only prM and E. Chimeric proteins containing the ectodomain of CD4 or CD8 fused to the transmembrane domains of prM or E were constructed, and their subcellular localization was studied by confocal immunofluorescence and by analyzing the maturation of their associated glycans. Although a small fraction was detected in the ER-to-Golgi intermediate and Golgi compartments, these chimeric proteins were located mainly in the ER. The C termini of prM and E form two antiparallel transmembrane α-helices. Interestingly, the first transmembrane passage contains enough information for ER localization. Taken altogether, these data indicate that, besides their role as membrane anchors, the transmembrane domains of yellow fever virus envelope proteins are ER retention signals. In addition, our data show that the mechanisms of ER retention of the flavivirus and hepacivirus envelope proteins are different

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structur

Differential effects of lobe A and lobe B of the COG complex on the stability of B4GALT1 and ST6GAL

Initially described by Jaeken et al. in 1980, Congenital Disorders of Glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the COG complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of total serum N-glycans of COG deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small interfering RNAs strategy in HeLa cells stably expressing GFP tagged B4GALT1 (GalT1-GFP) and ST6GAL1 (SiaT1-GFP), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, while lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1 and ST6GAL1 induced degradation is likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, while lobe A appears to be essential for maintaining the overall Golgi structure.status: publishe

Use of Endoglycosidase H as a diagnostic tool for MAN1B1‐CDG patients

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