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

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

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

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

Congenital disorders of glycosylation (CDG) are heterogeneous group of genetic protein and lipid glycosylation abnormalities. With some 33 reported patients, MAN1B1-CDG belongs to the more frequent causes of CDG-II. MAN1B1 encodes an α1,2-mannosidase that removes the terminal mannose residue from the middle branch. Several methods have been proposed to characterize the glycosylation changes. In MAN1B1-CDG, the abnormal accumulating N-glycan structures are mostly absent or found in trace amounts in total human serum. To overcome this issue, in this study, we present a straightforward procedure based on the use of Endo-β-N-acetylglucosaminidase H to easily diagnose MAN1B1-CDG patients and mannosidase defects.status: publishe

Identification of phosphorylated oligosaccharides in cells of patients with a Congenital Disorders of Glycosylation (CDG-I)

Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man(5)GlcNAc(2)-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man(5)GlcNAc(2)-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labelling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients' cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn(2+) for optimal activity. In CDG patients' cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.status: publishe

Mutations in MAGT1 lead to a glycosylation disorder with a variable phenotype

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.status: publishe

Manganese-induced turnover of TMEM165

TMEM165 deficiencies lead to one of the Congenital Disorders of Glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In this paper we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether this study identified the Golgi protein TMEM165 as a novel Mn2+ sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+ induced degradation of TMEM165

Clinical features of the seven cases.

<p>Clinical features of P1 at the age of 7 years (A), P2 at the age of 10 years (B, C), P3 at the age 3.5 years (D), P4.1 at the age of 18 years (E), P4.2 at the age of 12 years (F), P5 at the age of 13 years (G, H) and P6 at the age of 5 years (I, J). Note the facial dysmorphism, i.e. hypertelorism with downslanting palpebral fissures (A, D, E, F, G), large, low set ears (A, B, C, D, E, F, G, J), thin upper lip with hypoplastic nasolabial fold (A, B, D, E, F, G, I), tubular nose in P1, P4.1 and P4.2 (A, E, F) and a depressed nasal bridge in patients P2, P3, P5 and P6 (B, D, G, I). Note the truncular obesity (C, E, F, H) and the widely spaced, inverted nipples (B, F, H). Note the pectus excavatum in P5 (H).</p

Clinical features of patients with MAN1B1 deficiency.

<p>+) or absence (−) of each feature is reported for each individual. The following abbreviations are used: NA, not available; yrs: years; F: female; M: male. The presence (</p