TMEM165 : a new regulator of Golgi Mn2+ homeostasis involved in congenital disorders of glycosylation

Les anomalies congénitales de la glycosylation (CDG) sont des maladies génétiques rares caractérisées par une glycosylation aberrante des protéines. Récemment, un sous-groupe de CDG dues à des perturbations de l'homéostasie de l’appareil de Golgi a fait son apparition. En 2012, notre équipe a identifié TMEM165 comme étant une protéine golgienne impliquée dans les CDG, mais dont les fonctions biologiques et cellulaires demeurent inconnues. Au cours de mon doctorat, nous avons montré que l'homéostasie golgienne du Mn2+ était altérée en absence de TMEM165. Alors que de forts défauts de glycosylation, en particulier des défauts de galactosylation, ont été observés dans des cellules déficientes en TMEM165, nous avons découvert que la supplémentation en Mn2+ était suffisante pour rétablir une glycosylation normale. De façon intéressante, nous avons également démontré que ce défaut de glycosylation pouvait également être supprimé par une supplémentation en galactose. Fort de cette observation, la supplémentation orale en galactose a été testée chez des patients déficients en TMEM165 et il a été prouvé que ce traitement améliorait significativement les paramètres biochimiques et cliniques. De plus, nous avons mis en évidence que la stabilité de TMEM165 était altérée en présence d'une concentration élevée de Mn2+. En effet, nous avons montré que l'exposition à des concentrations élevées de Mn2+ entraînait une dégradation lysosomale rapide de TMEM165. Dans l'ensemble, notre étude montre que TMEM165 est (i) un acteur clé de la glycosylation golgienne en régulant finement l'homéostasie du Mn2+ et (ii) une nouvelle protéine de l’appareil de Golgi sensible au manganèse.Congenital Disorders of Glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogeneous group, a significant subgroup due to Golgi homeostasis defects is emerging. Our team previously identified TMEM165 as a Golgi protein involved in CDG. But despite strong efforts, the biological and cellular functions of TMEM165 were not known so far. During my thesis, we highlighted that Golgi Mn2+ homeostasis was impaired due to TMEM165 deficiency. While strong glycosylation defects, especially galactosylation defects, were observed in TMEM165 depleted cells, we discovered that Mn2+ supplementation was sufficient to fully restore a normal glycosylation. Interestingly, we also demonstrated that the observed glycosylation defects in mammalian cells could be overcome by galactose supplementation. Strong of this observation, oral galactose supplementation in TMEM165 deficient patients was assayed and this treatment was proven to significantly improve biochemical and clinical parameters. Moreover, we highlighted TMEM165 as a novel Golgi protein whose stability is altered in the presence of high manganese concentration. Indeed, we showed that exposure to high Mn2+ concentrations led to a rapid lysosomal degradation of TMEM165. Altogether, our study points TMEM165 as (i) a key player in Golgi glycosylation by finely regulating Golgi Mn2+ homeostasis and (ii) a novel Golgi protein sensitive to manganese

The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi.

The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency

Involvement of thapsigargin– and cyclopiazonic acid–sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn 2+

International audienceCongenital disorders of glycosylation are severe inherited diseases in which aberrant protein glycosylation is a hallmark. Transmembrane protein 165 (TMEM165) is a novel Golgi transmembrane protein involved in type II congenital disorders of glycosylation. Although its biologic function is still a controversial issue, we have demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi Mn2+ homeostasis defect. The goal of this study was to delineate the cellular pathway by which extracellular Mn2+ rescues N-glycosylation in TMEM165 knockout (KO) cells. We first demonstrated that after extracellular exposure, Mn2+ uptake by HEK293 cells at the plasma membrane did not rely on endocytosis but was likely done by plasma membrane transporters. Second, we showed that the secretory pathway Ca2+-ATPase 1, also known to mediate the influx of cytosolic Mn2+ into the lumen of the Golgi apparatus, is not crucial for the Mn2+-induced rescue glycosylation of lysosomal-associated membrane protein 2 (LAMP2). In contrast, our results demonstrate the involvement of cyclopiazonic acid- and thapsigargin (Tg)-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+. Interestingly, overexpression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b isoform in TMEM165 KO cells partially rescues the observed LAMP2 glycosylation defect. Overall, this study indicates that the rescue of Golgi N-glycosylation defects in TMEM165 KO cells by extracellular Mn2+ involves the activity of Tg and cyclopiazonic acid-sensitive pumps, probably the SERCA pumps.-Houdou, M., Lebredonchel, E., Garat, A., Duvet, S., Legrand, D., Decool, V., Klein, A., Ouzzine, M., Gasnier, B., Potelle, S., Foulquier, F. Involvement of thapsigargin- and cyclopiazonic acid-sensitive pumps in the rescue of TMEM165-associated glycosylation defects by Mn2+

Dissection of TMEM165 function in Golgi glycosylation and its Mn2+ sensitivity

International audienc

Galactose supplementation in TMEM165-CDG patients rescues the glycosylation defects

TMEM165 deficiency is a severe multisystem disease that manifests with metabolic, endocrine and skeletal involvement. It leads to one type of congenital disorders of glycosylation (CDG), a rapidly growing group of inherited diseases where the glycosylation process is altered. Patients have decreased galactosylation by serum glycan analysis. There are over one hundred CDGs but only specific types are treatable.status: publishe

Glycosylation abnormalities in Gdt1p/TMEM165 deficient cells result from a defect in Golgi manganese homeostasis

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1is the ortholog ofTMEM165in yeast, bothgdt1Δ null mutant yeasts andTMEM165depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+)supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+)sensitivity was altered inTMEM165depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+)homeostasis.status: publishe

Glycosylation abnormalities in Gdt1p/TMEM165 deficient cells result from a defect in Golgi manganese homeostasis.

Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1is the ortholog ofTMEM165in yeast, bothgdt1Δ null mutant yeasts andTMEM165depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+)supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+)sensitivity was altered inTMEM165depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+)homeostasis

Investigating the function of Gdt1p in yeast Golgi glycosylation

International audienc

Impaired glucose-1,6-biphosphate production due to bi-allelic PGM2L1 mutations is associated with a neurodevelopmental disorder.

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism