Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.

New insights into the pathogenicity of TMEM165 variants using structural modeling based on AlphaFold 2 predictions.

TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165's function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.

Metalloglycobiology: The power of metals in regulating glycosylation.

The remarkable structural diversity of glycans that is exposed at the cell surface and generated along the secretory pathway is tightly regulated by several factors. The recent identification of human glycosylation diseases related to metal transporter defects opened a completely new field of investigation, referred to herein as "metalloglycobiology", on how metal changes can affect the glycosylation and hence the glycan structures that are produced. Although this field is in its infancy, this review aims to go through the different glycosylation steps/pathways that are metal dependent and that could be impacted by metal homeostasis dysregulations.

Insights into the regulation of cellular Mn2+ homeostasis via TMEM165.

Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification.

Differential Effects of D-Galactose Supplementation on Golgi Glycosylation Defects in TMEM165 Deficiency.

Glycosylation is a ubiquitous and universal cellular process in all domains of life. In eukaryotes, many glycosylation pathways occur simultaneously onto proteins and lipids for generating a complex diversity of glycan structures. In humans, severe genetic diseases called Congenital Disorders of Glycosylation (CDG), resulting from glycosylation defects, demonstrate the functional relevance of these processes. No real cure exists so far, but oral administration of specific monosaccharides to bypass the metabolic defects has been used in few CDG, then constituting the simplest and safest treatments. Oral D-Galactose (Gal) therapy was seen as a promising tailored treatment for specific CDG and peculiarly for TMEM165-CDG patients. TMEM165 deficiency not only affects the N-glycosylation process but all the other Golgi-related glycosylation types, then contributing to the singularity of this defect. Our previous results established a link between TMEM165 deficiency and altered Golgi manganese (Mn2+) homeostasis. Besides the fascinating power of MnCl2 supplementation to rescue N-glycosylation in TMEM165-deficient cells, D-Gal supplementation has also been shown to be promising in suppressing the observed N-glycosylation defects. Its effect on the other Golgi glycosylation types, most especially O-glycosylation and glycosaminoglycan (GAG) synthesis, was however unknown. In the present study, we demonstrate the differential impact of D-Gal or MnCl2 supplementation effects on the Golgi glycosylation defects caused by TMEM165 deficiency. Whereas MnCl2 supplementation unambiguously fully rescues the N- and O-linked as well as GAG glycosylations in TMEM165-deficient cells, D-Gal supplementation only rescues the N-linked glycosylation, without any effects on the other Golgi-related glycosylation types. According to these results, we would recommend the use of MnCl2 for TMEM165-CDG therapy.

CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking.

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.

SLC10A7, an orphan member of the SLC10 family involved in congenital disorders of glycosylation.

SLC10A7, encoded by the so-called SLC10A7 gene, is the seventh member of a human sodium/bile acid cotransporter family, known as the SLC10 family. Despite similarities with the other members of the SLC10 family, SLC10A7 does not exhibit any transport activity for the typical SLC10 substrates and is then considered yet as an orphan carrier. Recently, SLC10A7 mutations have been identified as responsible for a new Congenital Disorder of Glycosylation (CDG). CDG are a family of rare and inherited metabolic disorders, where glycosylation abnormalities lead to multisystemic defects. SLC10A7-CDG patients presented skeletal dysplasia with multiple large joint dislocations, short stature and amelogenesis imperfecta likely mediated by glycosaminoglycan (GAG) defects. Although it has been demonstrated that the transporter and substrate specificities of SLC10A7, if any, differ from those of the main members of the protein family, SLC10A7 seems to play a role in Ca2+ regulation and is involved in proper glycosaminoglycan biosynthesis, especially heparan-sulfate, and N-glycosylation. This paper will review our current knowledge on the known and predicted structural and functional properties of this fascinating protein, and its link with the glycosylation process.