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Glycosylation of proteins and lipids in mammals is essential for embryogenesis and the development of all tissues. Analyses of glycosylation mutants in cultured mammalian cells and model organisms have been key to defining glycosylation pathways and the biological functions of glycans. More recently, applications of genome sequencing have revealed the breadth of rare congenital disorders of glycosylation in humans and the influence of genetics on the synthesis of glycans relevant to infectious diseases, cancer progression and diseases of the immune system. This improved understanding of glycan synthesis and functions is paving the way for advances in the diagnosis and treatment of glycosylation-related diseases, including the development of glycoprotein therapeutics through glycosylation engineering., (© 2024. Springer Nature Limited.)

SRD5A3-CDG is a congenital disorder of glycosylation (CDG) resulting from pathogenic variants in SRD5A3 and follows an autosomal recessive inheritance pattern. The enzyme encoded by SRD5A3, polyprenal reductase, plays a crucial role in synthesizing lipid precursors essential for N-linked glycosylation. Despite insights from functional studies into its enzymatic function, there remains a gap in understanding global changes in patient cells. We sought to identify N-glycoproteomic and proteomic signatures specific to SRD5A3-CDG, potentially aiding in biomarker discovery and advancing our understanding of disease mechanisms. Using tandem mass tag (TMT)-based relative quantitation, we analyzed fibroblasts derived from five patients along with control fibroblasts. N-glycoproteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 3,047 glycopeptides with 544 unique N-glycosylation sites from 276 glycoproteins. Of these, 418 glycopeptides showed statistically significant changes with 379 glycopeptides decreased (P < 0.05) in SRD5A3-CDG patient-derived samples. These included high mannose, complex and hybrid glycan-bearing glycopeptides. High mannose glycopeptides from protocadherin Fat 4 and integrin alpha-11 and complex glycopeptides from CD55 were among the most significantly decreased glycopeptides. Proteomics analysis led to the identification of 5,933 proteins, of which 873 proteins showed statistically significant changes. Decreased proteins included cell surface glycoproteins, various mitochondrial protein populations and proteins involved in the N-glycosylation pathway. Lysosomal proteins such as N-acetylglucosamine-6-sulfatase and procathepsin-L also showed reduced levels of phosphorylated mannose-containing glycopeptides. Our findings point to disruptions in glycosylation pathways as well as energy metabolism and lysosomal functions in SRD5A3-CDG, providing clues to improved understanding and management of patients with this disorder., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mutations in glycosylation pathways, such as N-linked glycosylation, O-linked glycosylation, and GPI anchor synthesis, lead to Congenital Disorders of Glycosylation (CDG). CDG typically present with seizures, hypotonia, and developmental delay but display large clinical variability with symptoms affecting every system in the body. This variability suggests modifier genes might influence the phenotypes. Because of the similar physiology and clinical symptoms, there are likely common genetic modifiers between CDG. Here, we use evolution as a tool to identify common modifiers between CDG and glycosylation genes. Protein glycosylation is evolutionarily conserved from yeast to mammals. Evolutionary rate covariation (ERC) identifies proteins with similar evolutionary rates that indicate shared biological functions and pathways. Using ERC, we identified strong evolutionary rate signatures between proteins in the same and different glycosylation pathways. Genome-wide analysis of proteins showing significant ERC with GPI anchor synthesis proteins revealed strong signatures with ncRNA modification proteins and DNA repair proteins. We also identified strong patterns of ERC based on cellular sub-localization of the GPI anchor synthesis enzymes. Functional testing of the highest scoring candidates validated genetic interactions and identified novel genetic modifiers of CDG genes. ERC analysis of disease genes and biological pathways allows for rapid prioritization of potential genetic modifiers, which can provide a better understanding of disease pathophysiology and novel therapeutic targets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Thorpe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

O-GlcNAc transferase (OGT) is the sole enzyme responsible for the post-translational modification of O-GlcNAc on thousands of target nucleocytoplasmic proteins. To date, nine variants of OGT that segregate with OGT Congenital Disorder of Glycosylation (OGT-CDG) have been reported and characterized. Numerous additional variants have been associated with OGT-CDG, some of which are currently undergoing investigation. This disorder primarily presents with global developmental delay and intellectual disability (ID), alongside other variable neurological features and subtle facial dysmorphisms in patients. Several hypotheses aim to explain the etiology of OGT-CDG, with a prominent hypothesis attributing the pathophysiology of OGT-CDG to mutations segregating with this disorder disrupting the OGT interactome. The OGT interactome consists of thousands of proteins, including substrates as well as interactors that require noncatalytic functions of OGT. A key aim in the field is to identify which interactors and substrates contribute to the primarily neural-specific phenotype of OGT-CDG. In this review, we will discuss the heterogenous phenotypic features of OGT-CDG seen clinically, the variable biochemical effects of mutations associated with OGT-CDG, and the use of animal models to understand this disorder. Furthermore, we will discuss how previously identified OGT interactors causal for ID provide mechanistic targets for investigation that could explain the dysregulated gene expression seen in OGT-CDG models. Identifying shared or unique altered pathways impacted in OGT-CDG patients will provide a better understanding of the disorder as well as potential therapeutic targets., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multistep pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 congenital disorders of glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multispanning membrane protein located in the ER, with its N and C termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Asparagine-linked glycosylation 1 protein is a β-1,4-mannosyltransferase, is encoded by the ALG1 gene, which catalyzes the first step of mannosylation in N-glycosylation. Pathogenic variants in ALG1 cause a rare autosomal recessive disorder termed as ALG1-CDG. We performed a quantitative proteomics and N-glycoproteomics study in fibroblasts derived from patients with one homozygous and two compound heterozygous pathogenic variants in ALG1. Several proteins that exhibited significant upregulation included insulin-like growth factor II and pleckstrin, whereas hyaluronan and proteoglycan link protein 1 was downregulated. These proteins are crucial for cell growth, survival and differentiation. Additionally, we observed a decrease in the expression of mitochondrial proteins and an increase in autophagy-related proteins, suggesting mitochondrial and cellular stress. N-glycoproteomics revealed the reduction in high-mannose and complex/hybrid glycopeptides derived from numerous proteins in patients explaining that defect in ALG1 has broad effects on glycosylation. Further, we detected an increase in several short oligosaccharides, including chitobiose (HexNAc 2 ) trisaccharides (Hex-HexNAc 2 ) and novel tetrasaccharides (NeuAc-Hex-HexNAc 2 ) derived from essential proteins including LAMP1, CD44 and integrin. These changes in glycosylation were observed in all patients irrespective of their gene variants. Overall, our findings not only provide novel molecular insights into understanding ALG1-CDG but also offer short oligosaccharide-bearing peptides as potential biomarkers., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

The Golgi compartment performs a number of crucial roles in the cell. However, the exact molecular mechanisms underlying these actions are not fully defined. Pathogenic mutations in genes encoding Golgi proteins may serve as an important source for expanding our knowledge. For instance, mutations in the gene encoding Transmembrane protein 165 (TMEM165) were discovered as a cause of a new type of congenital disorder of glycosylation (CDG). Comprehensive studies of TMEM165 in different model systems, including mammals, yeast, and fish uncovered the new realm of Mn 2+ homeostasis regulation. TMEM165 was shown to act as a Ca 2+ /Mn 2+ :H + antiporter in the medial- and trans-Golgi network, pumping the metal ions into the Golgi lumen and protons outside. Disruption of TMEM165 antiporter activity results in defects in N- and O-glycosylation of proteins and glycosylation of lipids. Impaired glycosylation of TMEM165-CDG arises from a lack of Mn 2+ within the Golgi. Nevertheless, Mn 2+ insufficiency in the Golgi is compensated by the activity of the ATPase SERCA2. TMEM165 turnover has also been found to be regulated by Mn 2+ cytosolic concentration. Besides causing CDG, recent investigations have demonstrated the functional involvement of TMEM165 in several other pathologies including cancer and mental health disorders. This systematic review summarizes the available information on TMEM165 molecular structure, cellular function, and its roles in health and disease., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Santulli is an Editorial Board Member for JBC and was not involved in the editorial review or the decision to publish this article. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Dolichol is a lipid critical for N-glycosylation as a carrier for activated sugars and nascent oligosaccharides. It is commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires a three-step detour involving additional metabolites, where SRD5A3 catalyzes only the second reaction. The first and third steps are performed by DHRSX, whose gene resides on the pseudoautosomal regions of the X and Y chromosomes. Accordingly, we report a pseudoautosomal-recessive disease presenting as a congenital disorder of glycosylation in patients with missense variants in DHRSX (DHRSX-CDG). Of note, DHRSX has a unique dual substrate and cofactor specificity, allowing it to act as a NAD + -dependent dehydrogenase and as a NADPH-dependent reductase in two non-consecutive steps. Thus, our work reveals unexpected complexity in the terminal steps of dolichol biosynthesis. Furthermore, we provide insights into the mechanism by which dolichol metabolism defects contribute to disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yanagi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man 5 GlcNAc 2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc 1 Gal 1 GlcNAc 1 Man 3 GlcNAc 2 , probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG., (© 2024 SSIEM.)

PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches. We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

PGM1 -linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.

Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations., Patient and Methods: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose., Results: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed., Conclusion: D-mannose is a promising new treatment for FCSK-CDG., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man 6 and higher glycans and a decrease in Man 5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation., Competing Interests: Declaration of competing interest E.M. and M.H.A.I. are principal investigators in a clinical trial sponsored by Glycomine. Other authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17-34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8-12%), with hair and dental abnormalities present in only 4-7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG., Competing Interests: Declaration of competing interest None of the authors have any COIs., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency., Methods: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses., Results: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation., Conclusion: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease., (© 2024 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

BACKGROUNDDiagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODSUsing multistep mass spectrometry-based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag-based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTSOf the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSIONComplement C4-derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDINGU54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance).

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 Mn 2+ 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., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a rare inborn error of metabolism caused by deficiency of the PMM2 enzyme, which leads to impaired protein glycosylation. While the disorder presents with primarily neurological symptoms, there is limited knowledge about the specific brain-related changes caused by PMM2 deficiency. Here, we demonstrate aberrant neural activity in 2D neuronal networks from PMM2-CDG individuals. Utilizing multi-omics datasets from 3D human cortical organoids (hCOs) derived from PMM2-CDG individuals, we identify widespread decreases in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG, as well as impaired mitochondrial structure and abnormal glucose metabolism in PMM2-deficient hCOs, indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in hCOs and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism, comprising over 160 described diseases to this day. CDG are characterized by a dysfunctional glycosylation process, with molecular defects localized in the cytosol, the endoplasmic reticulum, or the Golgi apparatus. Depending on the CDG, N-glycosylation, O-glycosylation and/or glycosaminoglycan synthesis can be affected. Various proteins, lipids, and glycosylphosphatidylinositol anchors bear glycan chains, with potential impacts on their folding, targeting, secretion, stability, and thus, functionality. Therefore, glycosylation defects can have diverse and serious clinical consequences. CDG patients often present with a non-specific, multisystemic syndrome including neurological involvement, growth delay, hepatopathy and coagulopathy. As CDG are rare diseases, and typically lack distinctive clinical signs, biochemical and genetic testing bear particularly important and complementary diagnostic roles. Here, after a brief introduction on glycosylation and CDG, we review historical and recent findings on CDG biomarkers and associated analytical techniques, with a particular emphasis on those with relevant use in the specialized clinical chemistry laboratory. We provide the reader with insights and methods which may help them properly assist the clinician in navigating the maze of glycosylation disorders., (Copyright © 2024. Published by Elsevier Inc.)

Congenital disorders of glycosylation (CDG) are a complex and heterogeneous family of rare metabolic diseases. With a clinical history that dates back over 40 years, it was the recent multi-omics advances that mainly contributed to the fast-paced and encouraging developments in the field. However, much remains to be understood, with targeted therapies' discovery and approval being the most urgent unmet need. In this paper, we present the 2022 state of the art of CDG, including glycosylation pathways, phenotypes, genotypes, inheritance patterns, biomarkers, disease models, and treatments. In light of our current knowledge, it is not always clear whether a specific disease should be classified as a CDG. This can create ambiguity among professionals leading to confusion and misguidance, consequently affecting the patients and their families. This review aims to provide the CDG community with a comprehensive overview of the recent progress made in this field., (© 2023. Institut National de la Santé et de la Recherche Médicale (INSERM).)

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., Competing Interests: Declaration of Competing Interest FOULQUIER reports financial support was provided by French National Research Agency, ENIGMncA project, ANR-21-CE14-0049-01., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Enzymes of the Golgi implicated in N-glycan processing are critical for brain development, and defects in many are defined as congenital disorders of glycosylation (CDG). Involvement of the Golgi mannosidase, MAN2A2 has not been identified previously as causing glycosylation defects., Methods: Exome sequencing of affected individuals was performed with Sanger sequencing of the MAN2A2 transcript to confirm the variant. N-glycans were analysed in patient-derived lymphoblasts to determine the functional effects of the variant. A cell-based complementation assay was designed to assess the pathogenicity of identified variants using MAN2A1/MAN2A2 double knock out HEK293 cell lines., Results: We identified a multiplex consanguineous family with a homozygous truncating variant p.Val1101Ter in MAN2A2. Lymphoblasts from two affected brothers carrying the same truncating variant showed decreases in complex N-glycans and accumulation of hybrid N-glycans. On testing of this variant in the developed complementation assay, we see the complete lack of complex N-glycans., Conclusion: Our findings show that pathogenic variants in MAN2A2 cause a novel autosomal recessive CDG with neurological involvement and facial dysmorphism. Here, we also present the development of a cell-based complementation assay to assess the pathogenicity of MAN2A2 variants, which can also be extended to MAN2A1 variants for future diagnosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: The study of the impact of some inherited defects in glycosylation on the biosynthesis of some lysosomal glycoproteins. Results description: Whole-exome sequencing revealed a homozygous variant; 428G > A; p. (R143K) in SRD5A3 in one patient and a heterozygous one c.46G > A p. (Gly16Arg) in SLC35A2 in the other patient. Both variants were predicted to be likely pathogenic. Lysosome-associated membrane glycoprotein 2 (LAMP2) immunodetection in both cases showed a truncated form of the protein. Cystinosin (CTN) protein appeared as normal and truncated forms in both patients in ratios of the mature to truncated forms of CTN were lower than the control. The levels of the truncated forms of both cellular proteins were higher in the SRD5A3-CDG case compared to the SLC35A2-CDG case. The tetrameric form of cathepsin C (CTSC) was expressed at low levels in both cases with congenital disorder of glycosylation (CDG). SLC35A2-CDG patient had one extra-unknown band while SRD5A3-CDG patient had a missing band of CTSC forms. The expression patterns of lysosomal glycoproteins could be different between different types of CDG., (© 2023. The Author(s).)

Congenital disorders of glycosylation (CDG) are severe metabolic disorders caused by an imbalance in the glycosylation pathway. Phosphomannomutase2 (PMM2-CDG), the most prevalent CDG, is mainly due to the disorder of PMM2. Pathogenic variants in cysteine have been found in various diseases, and cysteine residues have a potential as therapeutic targets. PMM2 harbor six cysteines; the variants Cys9Tyr (C9Y) and Cys241Ser (C241S) of PMM2 have been identified to associate with CDG, but the underlying molecular mechanisms remain uncharacterized. Here, we purified PMM2 wild type (WT), C9Y, and C241S to investigate their structural characteristics and biophysical properties by spectroscopic experiments under physiological temperature and environmental stress. Notably, the variants led to drastic changes in the protein properties and were prone to aggregate at physiological temperature. Meanwhile, PMM2 was sensitive to oxidative stress, and the cysteine pathogenic variants led to obvious aggregate formation and a higher cellular apoptosis ratio under oxidative stress. Molecular dynamic simulations indicated that the pathogenic variants changed the core domain of homomeric PMM2 and subunit binding free energy. Moreover, we tested the potential drug targeting PMM2-celastrol in cell level and explained the result by molecular docking simulation. In this study, we delineated the pathological mechanism of the cysteine substitution in PMM2, which addressed the vital role of cysteine in PMM2 and provided novel insights into prevention and treatment strategies for PMM2-CDG., Competing Interests: The authors declare no competing interests., (Copyright © 2023 Fan Yu et al.)

Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-β signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-β pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-β signaling to microscopic liver fibrosis in NGLY1 patients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are ultrarare, genetically and clinically heterogeneous metabolic disorders. Although the number of identified CDG is growing rapidly, there are few therapeutic options. Most treatments involve dietary supplementation with monosaccharides or other precursors. These approaches are relatively safe, but in many cases, the molecular and biochemical underpinnings are incomplete. Recent studies demonstrate that yeast, worm, fly, and zebrafish models of CDG are powerful tools in screening repurposed drugs, ushering a new avenue to search for novel therapeutic options. Here we present a perspective on compounds that are currently in use for CDG treatment or have a potential to be applied as therapeutics in the near future.

Background: Clinical management guidelines (CMGs) are decision support tools for patient care used by professionals, patients, and family caregivers. Since clinical experts develop numerous CMGs, their technical language hinders comprehension and access by nonmedical stakeholders. Additionally, the views of affected individuals and their families are often not incorporated into treatment guidelines. We developed an adequate methodology for addressing the needs and preferences of family and professional stakeholders regarding CMGs, a recently developed protocol for managing congenital disorders of glycosylation (CDG), a family of rare metabolic diseases. We used the CDG community and phosphomannomutase 2 (PMM2)-CDG CMGs as a pilot to test and implement our methodology., Results: We listened to 89 PMM2-CDG families and 35 professional stakeholders and quantified their CMG-related needs and preferences through an electronic questionnaire. Most families and professionals rated CMGs as relevant (86.5% and 94.3%, respectively), and valuable (84.3% and 94.3%, respectively) in CDG management. The most identified challenges were the lack of CMG awareness (50.6% of families) and the lack of plain language CMG (39.3% of professionals). Concordantly, among families, the most suggested solution was involving them in CMG development (55.1%), while professionals proposed adapting CMGs to include plain language (71.4%). Based on these results, a participatory framework built upon health literacy principles was created to improve CMG comprehension and accessibility. The outputs are six complementary CMG-related resources differentially adapted to the CDG community's needs and preferences, with a plain language PMM2-CDG CMG as the primary outcome. Additionally, the participants established a distribution plan to ensure wider access to all resources., Conclusions: This empowering, people-centric methodology accelerates CMG development and accessibility to all stakeholders, ultimately improving the quality of life of individuals living with a specific condition and raising the possibility of application to other clinical guidelines.

The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N -linked glycosylation. The yeast gene SEC53 encodes a homolog of human PMM2 . We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53- V238M and sec53 -F126L, or wild-type SEC53 . We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1 , which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53 -V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53 -V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles., Competing Interests: RV, MA, MM, JN, RS, EP, GA, GL No competing interests declared, (© 2022, Vignogna et al.)

Congenital disorders of glycosylation (CDG) are a group of more than 160 inborn errors of metabolism affecting multiple pathways of protein and lipid glycosylation. Patients present with a wide range of symptoms and therapies are only available for very few subtypes. Specific nutritional treatment options for certain CDG types include oral supplementation of monosaccharide sugars, manganese, uridine, or pyridoxine. Additional management includes specific diets (i.e., complex carbohydrate or ketogenic diet), iron supplementation, and albumin infusions. We review the dietary management in CDG with a focus on two subgroups: N-linked glycosylation defects and GPI-anchor disorders., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Haas, de Jonge, Koenen, Joosten, Janssen, de Boer, Hendriks, Lefeber and Cambi.)

Rare genetic mutations of the mannosyl-oligosaccharide glucosidase (MOGS) gene affecting the function of the mannosyl-oligosaccharide glucosidase (glucosidase I) are the cause of the congenital disorder of glycosylation IIb (CDG-IIb). Glucosidase I specifically removes the distal α1,2-linked glucose from the protein bound precursor N-glycan Glc3Man9GlcNAc2, which is the initial step of N-glycan maturation. Here, we comparatively analyzed N-glycosylation of the whole serum proteome, serum-derived immunoglobulin G (IgG), transferrin (TF), and α-1-antitrypsin (AAT) of a female patient who is compound heterozygous for 2 novel missense mutations in the MOGS gene, her heterozygous parents, and a sibling with wildtype genotype by multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) at unprecedented depth. Thereby, we detected the CDG-IIb-characteristic non-de-glucosylated N-glycans Glc3Man7-9GlcNAc2 as well as the free tetrasaccharide Glc3-Man in whole serum of the patient but not in the other family members. The N-glycan analysis of the serum proteome further revealed that relative intensities of IgG-specific complex type di-antennary N-glycans with core-fucosylation were considerably reduced in the patient's serum whereas TF- and AAT-characteristic sialylated di- and tri-antennary N-glycans were increased. This finding reflected the hypogammaglobulinemia diagnosed in the patient. We further detected aberrant oligo-mannose (Glc3Man7GlcNAc2) and hybrid type N-glycans on patient-derived IgGs and we attributed this defective glycosylation to be the reason for an increased IgG clearance. This mechanism can explain the hypogammaglobulinemia that is associated with CDG-IIb., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Congenital disorders of glycosylation (CDG) include 150 genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences in multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein-Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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&#95;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., (© 2022 The Authors. Birth Defects Research published by Wiley Periodicals LLC.)

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five complete crystal structures of hPMM2, three for wild-type and two for the p.Thr237Met variant frequently found among Spanish PMM2-CDG patients, free and bound to the essential activator glucose-1,6-bisphosphate (Glc-1,6-P 2 ). In the hPMM2 homodimer, each subunit has a different conformation, reflecting movement of the distal core domain relative to the dimerization cap domain, supporting an opening/closing process during catalysis. Two Mg 2+ ions bind to the core domain, one catalytic and one structural. In the cap domain, the site for Glc-1,6-P 2 is well delineated, while a Cl - ion binding at the intersubunit interface is predicted to strengthen dimerization. Patient-found amino acid substitutions are nonhomogeneously distributed throughout hPMM2, reflecting differential functional or structural importance for various parts of the protein. We classify 93 of 101 patient-reported single amino acid variants according to five potential pathogenetic mechanism affecting folding of the core and cap domains, linker 2 flexibility, dimerization, activator binding, and catalysis. We propose that ~80% and ~50% of the respective core and cap domains substitutions are potential candidates for pharmacochaperoning treatment., (© 2022 SSIEM.)

The insulin-like growth factor-1 (IGF-1) signaling pathway is crucial for the regulation of growth and development. The correct processing of the IGF-1Ea prohormone (proIGF-1Ea) and the IGF-1 receptor (IGF-1R) peptide precursor requires proper N-glycosylation. Deficiencies of N-linked glycosylation lead to a clinically heterogeneous group of inherited diseases called Congenital Disorders of Glycosylation (CDG). The impact of N-glycosylation defects on IGF-1/IGF-1R signaling components is largely unknown. In this study, using dermal fibroblasts from patients with different CDG [PMM2-CDG (n = 7); ALG3-CDG (n = 2); ALG8-CDG (n = 1); GMPPB-CDG (n = 1)], we analyzed the glycosylation pattern of the proIGF-1Ea, IGF-1 secretion efficiency and IGF-1R signaling activity. ALG3-CDG, ALG8-CDG, GMPPB-CDG and some PMM2-CDG fibroblasts showed hypoglycosylation of the proIGF-1Ea and lower IGF-1 secretion when compared with control (CTR). Lower IGF-1 serum concentration was observed in ALG3-CDG, ALG8-CDG and in some patients with PMM2-CDG, supporting our in vitro data. Furthermore, reduced IGF-1R expression level was observed in ALG3-CDG, ALG8-CDG and in some PMM2-CDG fibroblasts. IGF-1-induced IGF-1R activation was lower in most PMM2-CDG fibroblasts and was associated with decreased ERK1/2 phosphorylation as compared to CTR. In general, CDG fibroblasts showed a slight upregulation of Endoplasmic Reticulum (ER) stress genes compared with CTR, uncovering mild ER stress in CDG cells. ER-stress-related gene expression negatively correlated with fibroblasts IGF-1 secretion. This study provides new evidence of a direct link between N-glycosylation defects found in CDG and the impairment of IGF-1/IGF-1R signaling components. Further studies are warranted to determine the clinical consequences of reduced systemic IGF-1 availability and local activity in patients with CDG., (© 2022. The Author(s).)

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

The Golgi is the central organelle of the secretory pathway and it houses the majority of the glycosylation machinery, which includes glycosylation enzymes and sugar transporters. Correct compartmentalization of the glycosylation machinery is achieved by retrograde vesicular trafficking as the secretory cargo moves forward by cisternal maturation. The vesicular trafficking machinery which includes vesicular coats, small GTPases, tethers and SNAREs, play a major role in coordinating the Golgi trafficking thereby achieving Golgi homeostasis. Glycosylation is a template-independent process, so its fidelity heavily relies on appropriate localization of the glycosylation machinery and Golgi homeostasis. Mutations in the glycosylation enzymes, sugar transporters, Golgi ion channels and several vesicle tethering factors cause congenital disorders of glycosylation (CDG) which encompass a group of multisystem disorders with varying severities. Here, we focus on the Golgi vesicle tethering and fusion machinery, namely, multisubunit tethering complexes and SNAREs and their role in Golgi trafficking and glycosylation. This review is a comprehensive summary of all the identified CDG causing mutations of the Golgi trafficking machinery in humans.

Introduction: Congenital disorders of glycosylation (CDGs) are a group of inborn errors of glycan metabolism with multi-systemic manifestations. More than 100 different types of CDGs have been reported. The form involving the asparagine-linked glycosylation 13 (ALG13) gene is an uncommon X-linked form of these pathologies., Objective: To describe the clinical features in one patient with ALG13-CDG and to compare them with previously reported cases., Clinical Case: A 11-years-old boy, child of consangui neous parents, with hypotonia, severe developmental delay, intellectual disability, feeding difficulties, congenital heart disease (patent ductus arteriosus and mitral regurgitation), without epilepsy or coa gulation disorders. The metabolic screening showed unclear results, including N-glycosylation stu dies in plasma that were normal. Therefore, whole-exome sequencing (WES) was performed which identified a previously unreported variant in the ALG13 gene: c.428C > T (p.P143L) in hemizygous state; confirmed by Sanger sequencing. His mother was a carrier of the same variant., Conclusion: This is the first report of a Colombian patient with ALG13-CDG without epilepsy. The findings in this patient broaden the phenotypic spectrum of ALG13-CDG known to date and support that N- glycosylation disorders may be present in normal biochemical analysis. WES has become a cost- effective technique that allows the identification of disease-causing mutations in diseases with a broad phenotypic and genotypic spectrum.

SLC37A4-CDG is an emerging congenital disorder of glycosylation which is characterized by a dominant inheritance and a major coagulopathy originating from the liver. Recent studies took interest in the biochemical alterations found in this CDG and showed that they consisted of multiple glycosylation abnormalities, which result from mislocalization of the endoplasmic reticulum glucose-6-phosphate transporter and associated Golgi homeostasis defects. In this work, we highlight in six affected individuals abnormal patterns for various serum N-glycoproteins and bikunin proteoglycan isoforms, together with specific alterations of the mass spectra of endoglycosidase H-released serum N-glycans. Collectively, these data complement previous findings, help to better delineate SLC37A4-CDG and could present interest in diagnosing this disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Galactose is an essential carbohydrate for cellular metabolism, as it contributes to energy production and storage in several human tissues while also being a precursor for glycosylation. Galactosylated glycoconjugates, such as glycoproteins, keratan sulfate-containing proteoglycans and glycolipids, exert a plethora of biological functions, including structural support, cellular adhesion, intracellular signaling and many more. The biological relevance of galactose is further entailed by the number of pathogenic conditions consequent to defects in galactosylation and galactose homeostasis. The growing number of rare congenital disorders involving galactose along with its recent therapeutical applications are drawing increasing attention to galactose metabolism. In this review, we aim to draw a comprehensive overview of the biological functions of galactose in human cells, including its metabolism and its role in glycosylation, and to provide a systematic description of all known congenital metabolic disorders resulting from alterations of its homeostasis., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)