A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis.

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.

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

Genetics of glycosylation in mammalian development and disease.

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.

Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.

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.

Evolutionary rate covariation is pervasive between glycosylation pathways and points to potential disease modifiers.

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.

O-GlcNAc transferase congenital disorder of glycosylation (OGT-CDG): Potential mechanistic targets revealed by evaluating the OGT interactome.

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.

Insights into molecular and cellular functions of the Golgi calcium/manganese-proton antiporter TMEM165.

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 Mn2+ homeostasis regulation. TMEM165 was shown to act as a Ca2+/Mn2+: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 Mn2+ within the Golgi. Nevertheless, Mn2+ insufficiency in the Golgi is compensated by the activity of the ATPase SERCA2. TMEM165 turnover has also been found to be regulated by Mn2+ 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.

Molecular characterization of Rft1, an ER membrane protein associated with congenital disorder of glycosylation RFT1-CDG.

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.

Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

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.

Dysregulated proteome and N-glycoproteome in ALG1-deficient fibroblasts.

Asparagine-linked glycosylation 1 protein is a ß-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 (HexNAc2) trisaccharides (Hex-HexNAc2) and novel tetrasaccharides (NeuAc-Hex-HexNAc2) 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.

Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant.

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.

N-glycoproteomic and proteomic alterations in SRD5A3-deficient fibroblasts.

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.

A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

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.

Glycosphingolipids in congenital disorders of glycosylation (CDG).

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.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology.

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.

Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability.

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.

In vitro treatment with liposome-encapsulated Mannose-1-phosphate restores N-glycosylation in PMM2-CDG patient-derived fibroblasts.

PMM2-CDG is the most common congenital disorder of glycosylation (CDG). Patients with this disease often carry compound heterozygous mutations of the gene encoding the phosphomannomutase 2 (PMM2) enzyme. PMM2 converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P), which is a critical upstream metabolite for proper protein N-glycosylation. Therapeutic options for PMM2-CDG patients are limited to management of the disease symptoms, as no drug is currently approved to treat this disease. GLM101 is a M1P-loaded liposomal formulation being developed as a candidate drug to treat PMM2-CDG. This report describes the effect of GLM101 treatment on protein N-glycosylation of PMM2-CDG patient-derived fibroblasts. This treatment normalized intracellular GDP-mannose, increased the relative glycoprotein mannosylation content and TNFα-induced ICAM-1 expression. Moreover, glycomics profiling revealed that GLM101 treatment of PMM2-CDG fibroblasts resulted in normalization of most high mannose glycans and partial correction of multiple complex and hybrid glycans. In vivo characterization of GLM101 revealed its favorable pharmacokinetics, liver-targeted biodistribution, and tolerability profile with achieved systemic concentrations significantly greater than its effective in vitro potency. Taken as a whole, the results described in this report support further exploration of GLM101's safety, tolerability, and efficacy in PMM2-CDG patients.

Liposome-encapsulated mannose-1-phosphate therapy improves global N-glycosylation in different congenital disorders of glycosylation.

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 Man6 and higher glycans and a decrease in Man5 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.

D-mannose as a new therapy for fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG).

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.

Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.

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-Man5GlcNAc2 (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, NeuAc1Gal1GlcNAc1Man3GlcNAc2, 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.