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

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

Origin of the different electronic structure of Rh- and Ru-doped Sr2IrO4

One way to induce insulator to metal transitions in the spin-orbit Mott insulator Sr2IrO4 is to substitute iridium with transition metals (Ru, Rh). However, this creates intriguing inhomogeneous metallic states, which cannot be described by a simple doping effect. We detail the electronic structure of the Ru-doped case with angle-resolved photoemission and show that, contrary to Rh, it cannot be connected to the undoped case by a rigid shift. We further identify bands below $E_F$ coexisting with the metallic ones that we assign to non-bonding Ir sites. We rationalize the differences between Rh and Ru by a different hybridization with oxygen, which mediates the coupling to Ir and sensitively affects the effective doping. We argue that the spin-orbit coupling does not control neither the charge transfer nor the transition threshold

Benzo(a)pyrene inhibits the role of the bioturbator Tubifex tubifex in river sediment biogeochemistry

International audienceThe interactions between invertebrates and micro-organisms living in streambed sediments often play key roles in the regulation of nutrient and organic matter fluxes in aquatic ecosystems. However, benthic sedi- ments also constitute a privileged compartment for the accumulation of persistent organic pollutants such as PAHs or PCBs that may affect the diversity, abundance and activity of benthic organisms. The objective of this study was to quantify the impact of sediment contamination with the PAH benzo(a)pyrene on the in- teraction between micro-organisms and the tubificid worm, Tubifex tubifex, which has been recognized as a major bioturbator in freshwater sediments. Sedimentary microcosms (slow filtration columns) contaminated or not with benzo(a)pyrene (3 tested concentrations: 0, 1 and 5 mg kg−1) at the sediment surface were in- cubated under laboratory conditions in the presence (100 individuals) or absence of T. tubifex. Although the surface sediment contaminations with 1 mg kg−1 and 5 mg kg−1 of benzo(a)pyrene did not affect tubificid worm survival, these contaminations significantly influenced the role played by T. tubifex in biogeochemical processes. Indeed, tubificid worms stimulated aerobic respiration, denitrification, dehydrogenase and hydrolytic activities of micro-organisms in uncontaminated sediments whereas such effects were inhibited in sediments polluted with benzo(a)pyrene. This inhibition was due to contaminant-induced changes in bioturbation (and especially bio-irrigation) activities of worms and their resulting effects on microbial processes. This study reveals the importance of sublethal concentrations of a contaminant on ecological processes in river sediments through affecting bioturbator-microbe interactions. Since they affect microbial processes involved in water purification processes, such impacts of sublethal concentrations of pollutants should be more often considered in ecosystem health assessment

Golgi function and dysfunction in the first COG4-deficient CDG type II patient

The conserved oligomeric Golgi (COG) complex is a hetero-octameric complex essential for normal glycosylation and intra-Golgi transport. An increasing number of congenital disorder of glycosylation type II (CDG-II) mutations are found in COG subunits indicating its importance in glycosylation. We report a new CDG-II patient harbouring a p.R729W missense mutation in COG4 combined with a submicroscopical deletion. The resulting downregulation of COG4 expression additionally affects expression or stability of other lobe A subunits. Despite this, full complex formation was maintained albeit to a lower extent as shown by glycerol gradient centrifugation. Moreover, our data indicate that subunits are present in a cytosolic pool and full complex formation assists tethering preceding membrane fusion. By extending this study to four other known COG-deficient patients, we now present the first comparative analysis on defects in transport, glycosylation and Golgi ultrastructure in these patients. The observed structural and biochemical abnormalities correlate with the severity of the mutation, with the COG4 mutant being the mildest. All together our results indicate that intact COG complexes are required to maintain Golgi dynamics and its associated functions. According to the current CDG nomenclature, this newly identified deficiency is designated CDG-IIj

Biometals and glycosylation in humans: Congenital disorders of glycosylation shed lights into the crucial role of Golgi manganese homeostasis

International audienceAbout half of the eukaryotic proteins bind biometals that participate in their structure and functions in virtually all physiological processes, including glycosylation. After reviewing the biological roles and transport mechanisms of calcium, magnesium, manganese, zinc and cobalt acting as cofactors of the metalloproteins involved in sugar metabolism and/or glycosylation, the paper will outline the pathologies resulting from a dysregulation of these metals homeostasis and more particularly Congenital Disorders of Glycosylation (CDGs) caused by ion transporter defects. Highlighting of CDGs due to defects in SLC39A8 (ZIP8) and TMEM165, two proteins transporting manganese from the extracellular space to cytosol and from cytosol to the Golgi lumen, respectively , has emphasized the importance of manganese homeostasis for glycosylation. Based on our current knowledge of TMEM165 structure and functions, this review will draw a picture of known and putative mechanisms regulating manganese homeostasis in the secretory pathway

Anomalies congénitales de la glycosylation (CDG)

La glycosylation est un processus cellulaire complexe conduisant à des transferts successifs de monosaccharides sur une molécule acceptrice, le plus souvent une protéine ou un lipide. Ce processus est universel chez tous les organismes vivants et est très conservé au cours de l’évolution. Chez l’homme, des perturbations survenant au cours d’une ou plusieurs réactions de glycosylation sont à l’origine de glycopathologies génétiques rares, appelées anomalies congénitales de la glycosylation ou congenital disorders of glycosylation (CDG). Cette revue propose de revisiter ces CDG, de 1980 à aujourd’hui, en présentant leurs découvertes, leurs diagnostics, leurs causes biochimiques et les traitements actuellement disponibles

Impact of short-term treatment with telmisartan on cerebral arterial remodeling in SHR.

Chronic hypertension decreases internal diameter of cerebral arteries and arterioles. We recently showed that short-term treatment with the angiotensin II receptor blocker telmisartan restored baseline internal diameter of small cerebral arterioles in spontaneously hypertensive rats (SHR), via reversal of structural remodeling and inhibition of the angiotensin II vasoconstrictor response. As larger arteries also participate in the regulation of cerebral circulation, we evaluated whether similar short-term treatment affects middle cerebral arteries of SHR.Baseline internal diameters of pressurised middle cerebral arteries from SHR and their respective controls, Wistar Kyoto rats (WKY) and responses to angiotensin II were studied in a small vessel arteriograph. Pressure myogenic curves and passive internal diameters were measured following EDTA deactivation, and elastic modulus from stress-strain relationships.Active baseline internal diameter was 23% lower in SHR compared to WKY, passive internal diameter (EDTA) 28% lower and elastic modulus unchanged. Pressure myogenic curves were shifted to higher pressure values in SHR. Telmisartan lowered blood pressure but had no effect on baseline internal diameter nor on structural remodeling (passive internal diameter and elastic modulus remained unchanged compared to SHR). Telmisartan shifted the pressure myogenic curve to lower pressure values than SHR.In the middle cerebral arteries of SHR, short-term treatment with telmisartan had no effect on structural remodeling and did not restore baseline internal diameter, but allowed myogenic tone to adapt towards lower pressure values

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

Influence des glycannes sur la destinée des N-glycoprotéines

Le processus de N-glycosylation prend place dans la lumière du reticulum endoplasmique(RE) et correspond au transfert en bloc d'un précurseur glycannique de type Glc3Man9GlcNAc2 sur les protéines en cours de synthèse.Une fois transféré, ce glycanne va subir un élagage progressif,conduisant dans la lumière du RE à l'apparition de nombreuses structures glycanniques de type oligomannosidique. Depuis quelques années, il a été montré que ces structures sont impliquées dans la destinée des glycoprotéines aussi bien dans la mise en conformation, la dégradation, la sécrétion et même dans l'accès au protéasome. Alors que la glycosylation est extrêmement conservée au cours de l'évolution, de nombreux paramètres intra ou extracellulaires peuvent perturber ce processus. Dans de nombreux cas,le transfert de glycannes tronqués de type Man5GlcNAc2 est alors observé. Le but de notre travail, a été d'étudier l'influence du transfert de ces populations glycanniques tronquées sur la destinée des N-glycoprotéines. Dans la première partie de notre étude nous avons comparé la destinée des N-glycoprotéines portant des glycannes de type Man5GlcNAc2 avec celles portant des glycannes de type Man9GlcNAc2, lorsque ces deux populations sont synthétisées au sein d'un même RE. Alors que la populationà Man9GlcNAc2 est majoritairement exportée dans l'appareil de Golgi pour être transformée en type complexe, celle à Man5GlcNAc2 est convertie en Man4GlcNAc2 et retenue dans la lumière du RE.En prenant les oligosaccharides solubles accumulés dans le cytosol comme témoins de la dégradation des glycoprotéines, nous avons démontré que la population à Man5GlcNAc2 était préférentiellement dégradée par rapport à celleà Man9GlcNAc2 bien que,comme nous l'avons montré, le contrôle qualité soit aussi efficace sur une population glycoprotéique à Man5GlcNAc2 que sur une populationà Man9GlcNAc2. La deuxième partie de nos recherches a porté sur l'éventuelle relation entre une réponse cellulaire aux protéines mal conformées (l'UPR) et la structure du glycanne transférée sur la protéine. A l'aide d'une lignée cellulaire mutante de glycosylation qui transfère un oligosaccharide tronqué Glc3Man5GlcNAc2 sur les protéines, nous avons révélé que le niveau transcriptionnel et traductionnel de la Protéine chaperonne BiP (pris comme marqueur de la réponse UPR) était plus élevé que dans une lignée de type sauvage.Ceci démontre que l'UPR est activée de façon constitutive dans cette lignée mutante de glycosylation. D'une façon intéressante, nous avons montré que le niveau de cette réponse pouvait être affecté par l'emploi de kifunensine (inhibiteur de mannosidases de classe l). En effet, en présence de kifunensine, le niveau de l'UPR est diminué ce qui est corrélé par l'augmentation de la sécrétion des glycoprotéines. Nos résultats démontrent non seulement une relation étroite entre la qualité du glycanne transféré sur la protéine et le niveau de l'UPR, mais également l'implication d'une étape de démannosylation dans le contrôle qualité des N-glycoprotéines. Cette étape, présente à la fois dans les cellules sauvages et mutantes de glycosylation génère une population de glycoprotéines à Man4GlcNAc2, retenue dans la lumière du RE, qui avant d'être dégradée, permet le déclenchement de la réponse UPR.LILLE1-BU (590092102) / SudocSudocFranceF