Lack of NKG2D in MAGT1-deficient patients is caused by hypoglycosylation.

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.

COG6-CDG: Novel variants and novel malformation.

BACKGROUND: Deficiency of Conserved Oligomeric Golgi (COG) subunits (COG1-8) is characterized by both N- and O-protein glycosylation defects associated with destabilization and mislocalization of Golgi glycosylation machinery components (COG-CDG). Patients with COG defects present with neurological and multisystem involvement and possible malformation occurrence. Eighteen patients with COG6-CDG (COG6 mutations) were reported to date. We describe a patient with COG6-CDG with novel variants and a novel clinical feature namely a congenital recto-vaginal fistula. METHODS: In-depth serum N- and O-glycosylation structural analyses were conducted by MALDI-TOF mass spectrometry. COG6 variants were identified by a gene panel and confirmed by Sanger sequencing. RESULTS: This female newborn presented with facial dysmorphism, distal arthrogryposis and recurrent stool discharges per vaginam. A double-contrast barium-enema X-ray study revealed a dehiscence (approximately 5 mm) at the anterior wall of the rectal ampoule communicating with the vagina consistent with a recto-vaginal fistula. She had developmental delay, corpus callosum dysgenesis, liver and gastrointestinal involvement, hyperthermia episodes and early demise. Serum N- and O-glycosylation analyses pointed to a profound Golgi disarrangement. We identified two novel variants in COG6: a deletion of 1 bp mutation c.823delA creating a shift in the reading frame and a premature stop codon and a 3 bp deletion (c.1141_1143delCTC) producing an in-frame deletion of 1 amino acid. CONCLUSION: The congenital recto-vaginal fistula is a rare type of anorectal malformation that, to our knowledge, has not been reported in patients with a COG6 defect nor in patients with other COG defects. This study broadens COG6-CDG genetic landscape and spectrum of malformations.

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 a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present 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, the present 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.

Galactose Supplementation in Patients With TMEM165-CDG Rescues the Glycosylation Defects.

CONTEXT: 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 in which the glycosylation process is altered. Patients have decreased galactosylation by serum glycan analysis. There are >100 CDGs, but only specific types are treatable. OBJECTIVE: Galactose has been shown to be beneficial in other CDG types with abnormal galactosylation. The aim of this study was to characterize the effects of galactose supplementation on Golgi glycosylation in TMEM165-depleted HEK293 cells, as well as in 2 patients with TMEM165-CDG and in their cultured skin fibroblast cells. DESIGN AND SETTING: Glycosylation was assessed by mass spectrometry, western blot analysis, and transferrin isoelectrofocusing. PATIENTS AND INTERVENTIONS: Both unrelated patients with TMEM165-CDG with the same deep intronic homozygous mutation (c.792+182G>A) were allocated to receive d-galactose in a daily dose of 1 g/kg. RESULTS: We analyzed N-linked glycans and glycolipids in knockout TMEM165 HEK293 cells, revealing severe hypogalactosylation and GalNAc transfer defects. Although these defects were completely corrected by the addition of Mn2+, we demonstrated that the observed N-glycosylation defect could also be overcome by galactose supplementation. We then demonstrated that oral galactose supplementation in patients with TMEM165-deficient CDG improved biochemical and clinical parameters, including a substantial increase in the negatively charged transferrin isoforms, and a decrease in hypogalactosylated total N-glycan structures, endocrine function, and coagulation parameters. CONCLUSION: To our knowledge, this is the first description of abnormal glycosylation of lipids in the TMEM165 defect and the first report of successful dietary treatment in TMEM165 deficiency. We recommend the use of oral d-galactose therapy in TMEM165-CDG.

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. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted 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 in TMEM165 depleted 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.

Assessing ER and Golgi N-glycosylation process using metabolic labeling in mammalian cultured cells.

Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.

Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway.

We present the first two identified cases of phosphoserine aminotransferase deficiency. This disorder of serine biosynthesis has been identified in two siblings who showed low concentrations of serine and glycine in plasma and cerebrospinal fluid. Clinically, the index patient presented with intractable seizures, acquired microcephaly, hypertonia, and psychomotor retardation and died at age 7 mo despite supplementation with serine (500 mg/kg/d) and glycine (200 mg/kg/d) from age 11 wk. The younger sibling received treatment from birth, which led to a normal outcome at age 3 years. Measurement of phosphoserine aminotransferase activity in cultured fibroblasts in the index patient was inconclusive, but mutational analysis revealed compound heterozygosity for two mutations in the PSAT1 gene--one frameshift mutation (c.delG107) and one missense mutation (c.299A-->C [p.Asp100Ala])--in both siblings. Expression studies of the p.Asp100Ala mutant protein revealed a V(max) of only 15% of that of the wild-type protein.