Pathogenic DPAGT1 variants in limb-girdle congenital myasthenic syndrome (LG-CMS) associated with tubular aggregates and ORAI1 hypoglycosylation.

AIMS: Limb-girdle congenital myasthenic syndrome (LG-CMS) is a genetically heterogeneous disorder characterized by muscle weakness and fatigability. The LG-CMS gene DPAGT1 codes for an essential enzyme of the glycosylation pathway, a posttranslational modification mechanism shaping the structure and function of proteins. In DPAGT1-related LG-CMS, reduced glycosylation of the acetylcholine receptor (AChR) reduces its localization at the neuromuscular junction (NMJ), and results in diminished neuromuscular transmission. LG-CMS patients also show tubular aggregates on muscle biopsy, but the origin and potential contribution of the aggregates to disease development are not understood. Here, we describe two LG-CMS patients with the aim of providing a molecular diagnosis and to shed light on the pathways implicated in tubular aggregate formation. METHODS: Following clinical examination of the patients, we performed next-generation sequencing (NGS) to identify the genetic causes, analysed the biopsies at the histological and ultrastructural levels, investigated the composition of the tubular aggregates, and performed experiments on protein glycosylation. RESULTS: We identified novel pathogenic DPAGT1 variants in both patients, and pyridostigmine treatment quantitatively improved muscle force and function. The tubular aggregates contained proteins of the sarcoplasmic reticulum (SR) and structurally conformed to the aggregates observed in tubular aggregate myopathy (TAM). TAM arises from overactivation of the plasma membrane calcium channel ORAI1, and functional studies on muscle extracts from our LG-CMS patients evidenced abnormal ORAI1 glycosylation. CONCLUSIONS: We expand the genetic variant spectrum of LG-CMS and provide a genotype/phenotype correlation for pathogenic DPAGT1 variants. The discovery of ORAI1 hypoglycosylation in our patients highlights a physiopathological link between LG-CMS and TAM.

DPAGT1-CDG: Report of Two New Pediatric Patients and Brief Review of the Literature.

Introduction: Congenital glycosylation disorders are multisystem diseases with heterogeneous clinical manifestations caused by defects in the synthesis of the glycan moiety of glycoproteins or glycolipids or the binding of glycans to proteins and lipids. DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) is an initiating protein in the biosynthetic pathway of dolichol-linked oligosaccharides required for protein N-glycosylation. Pathogenic variants in DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) gene cause a rare type of congenital glycosylation disorder called DPAGT1-CDG (formerly CDG-Ij) (OMIM #608093). It is a rare autosomal recessive disease or a milder version with congenital myasthenic syndrome known as DPAGT1-CMS. A severe disease course with hypotonia, cataracts, skeletal deformities, resistant epilepsy, intellectual disability, global developmental delay, premature death has been described in most patients with DPAGT1-CDG. Patient Presentation: We describe two patients with variants in the DPAGT1 gene: an 8-month-old boy with a homozygous, missense DPAGT1:c.339T>G (p.Phe113Leu) novel variant and a 13-year-old female patient with compound heterozygous variants, DPAGT1:c.466C>T (p.Arg156Cys, R156C) and DPAGT1:c.161+5G>A. While the 8-month-old patient was diagnosed with congenital cataract at the age of 1 month, had dysmorphic findings, and epilepsy, clinical symptoms in the other patient appeared later but with more prominent muscle weakness, behavioral disorder, dysmorphic findings, and no epilepsy. Discussion: Cholinesterase inhibitor therapy was found to be effective in patients against muscle weakness, supporting DPAGT1 deficiency as the underlying etiology. We started pyridostigmine treatment in our patient with more pronounced muscle weakness, and we saw its benefit. We aimed to present our patients diagnosed with DPAGT1-CDG due to different variants in the same gene and different clinical presentations, treatment and to compare them with other patients in the literature.

DPAGT1-CDG: Recurrent fetal death.

BACKGROUND: Congenital disorders of glycosylation (CDG) are a series of relatively uncommon genetic disorders, and variants in the dolichyl-phosphate N-acetylglucosamine-1-phosphotransferase (DPAGT1) gene can cause DPAGT1-CDG, which is characterized by multisystem abnormalities: failure to thrive, psychomotor retardation, seizures, etc. PATIENTS: Two fetuses in a nonconsanguineous family recurrently presented with irregular skull morphology, micrognathia, adduction and supination by prenatal ultrasound. They were finally found dead in utero. Pedigree whole exome sequencing revealed novel compound heterozygous variants in the DPAGT1 gene. We also reviewed 11 previous reports associated with DPAGT1-CDG. CONCLUSIONS: We report novel variants in the DPAGT1 gene in two fetuses from the same family with intrauterine death.

Structure-Based Insight on the Mechanism of N-Glycosylation Inhibition by Tunicamycin.

N-glycosylation, a common post-translational modification, is widely acknowledged to have a significant effect on protein stability and folding. N-glycosylation is a complex process that occurs in the endoplasmic reticulum (ER) and requires the participation of multiple enzymes. GlcNAc-1-P-transferase (GPT) is essential for initiating N-glycosylation in the ER. Tunicamycin is a natural product that inhibits N-glycosylation and produces ER stress, and thus it is utilized in research. The molecular mechanism by which GPT triggers N-glycosylation is discussed in this review based on the GPT structure. Based on the structure of the GPT-tunicamycin complex, we also discuss how tunicamycin reduces GPT activity, which prevents N-glycosylation. This review will be highly useful for understanding the role of GPT in the N-glycosylation of proteins, as well as presents a potential for considering tunicamycin as an antibiotic treatment.

Novel DPAGT1 Gene Mutation in Two Twins with Congenital Myasthenic Syndrome and a Review of the Literature.

Congenital myasthenic syndromes (CMS) are rare diseases caused by mutation in genes coding for proteins involved in neuromuscular junction structure and function. DPAGT1 gene mutations are a rare cause of CMS whose clinical evolution and pathophysiological mechanisms have not been clarified completely. We present the case of two twins displaying an infancy-onset predominant limb-girdle phenotype and carrying a novel DPAGT1 mutation associated with unusual histological and clinical findings. CMS can mimic paediatric and adult limb-girdle phenotype, hence neurophysiology plays a fundamental role in the differential diagnosis.

Unique clinical presentations and follow-up outcomes from experience with congenital disorders of glycosylation: PMM2-PGM1-DPAGT1-MPI-POMT2-B3GALNT2-DPM1-SRD5A3-CDG.

OBJECTIVES: Congenital Glycosylation Disorders (CDG) are a large group of inherited metabolic diseases with multi-organ involvement. Herein, we aimed to expand the clinical characteristics of patients with CDG based on our experience with diagnoses and follow-up of CDG patients from different subtypes. METHODS: The clinical and laboratory findings from the last 15 years were reviewed retrospectively in Ege University Child Metabolism and Nutrition Department. RESULTS: There were 8 (57.2 %) females and 6 (42.8 %) males. Diagnoses of the patients were PMM2-CDG (n=4), PGM1-CDG (n=2), DPAGT1-CDG (n=2), SRD5A3-CDG (n=2), MPI-CDG (n=1), POMT2-CDG (n=1), B3GALNT2-CDG (n=1), DPM1-CDG (n=1). The clinical findings of the patients were dysmorphia (85.7 %), developmental delay (85.7 %), intellectual disability (85.7 %), ocular abnormalities (64.2 %), skeletal malformations (64.2 %), failure to thrive (57.1 %), microcephaly (57.1 %), hepatomegaly (35.7 %), hearing loss (35.7 %), seizures (28.5 %), gastrointestinal symptoms (21.4 %), endocrine abnormalities (21.4 %), and cardiac abnormalities (7.1 %). Laboratory findings were abnormal TIEF (92.8 %), abnormal liver enzymes (64.2 %), decreased protein C (64.2 %), decreased antithrombin III (64.2 %), decreased protein S (42.8 %), hypogammaglobulinemia (35.7 %), cerebellar hypoplasia (28.5 %), CK elevation (7.1 %), and hypoglycemia (7.1 %). CONCLUSIONS: This study contributes to the literature by sharing our ultra-rare DPM1-CDG case with less than 20 cases in the literature and expanding the clinical and molecular characteristics of other CDG patients. Hyperinsulinemic hypoglycemia, short stature, hypothyroidism, growth hormone deficiency, hypogammaglobulinemia, pericardial effusion, elevated CK, congenital myasthenia, and anorectal malformation were unique findings that were observed. Cerebello-ocular findings accompanying multi-organ involvement were an essential clue for a possible CDG.

A Dpagt1 Missense Variant Causes Degenerative Retinopathy without Myasthenic Syndrome in Mice.

Congenital disorders of glycosylation (CDG) are a heterogenous group of primarily autosomal recessive mendelian diseases caused by disruptions in the synthesis of lipid-linked oligosaccharides and their transfer to proteins. CDGs usually affect multiple organ systems and vary in presentation, even within families. There is currently no cure, and treatment is aimed at ameliorating symptoms and improving quality of life. Here, we describe a chemically induced mouse mutant, tvrm76, with early-onset photoreceptor degeneration. The recessive mutation was mapped to Chromosome 9 and associated with a missense mutation in the Dpagt1 gene encoding UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase (EC 2.7.8.15). The mutation is predicted to cause a substitution of aspartic acid with glycine at residue 166 of DPAGT1. This represents the first viable animal model of a Dpagt1 mutation and a novel phenotype for a CDG. The increased expression of Ddit3, and elevated levels of HSPA5 (BiP) suggest the presence of early-onset endoplasmic reticulum (ER) stress. These changes were associated with the induction of photoreceptor apoptosis in tvrm76 retinas. Mutations in human DPAGT1 cause myasthenic syndrome-13 and severe forms of a congenital disorder of glycosylation Type Ij. In contrast, Dpagt1tvrm76 homozygous mice present with congenital photoreceptor degeneration without overt muscle or muscular junction involvement. Our results suggest the possibility of DPAGT1 mutations in human patients that present primarily with retinitis pigmentosa, with little or no muscle disease. Variants in DPAGT1 should be considered when evaluating cases of non-syndromic retinal degeneration.

Concise Synthesis of Tunicamycin†V and Discovery of a Cytostatic DPAGT1 Inhibitor.

A short total synthesis of tunicamycin V (1), a non-selective phosphotransferase inhibitor, is achieved via a Büchner-Curtius-Schlotterbeck type reaction. Tunicamycin V can be synthesized in 15 chemical steps from D-galactal with 21 % overall yield. The established synthetic scheme is operationally very simple and flexible to introduce building blocks of interest. The inhibitory activity of one of the designed analogues 28 against human dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1) is 12.5 times greater than 1. While tunicamycins are cytotoxic molecules with a low selectivity, the novel analogue 28 displays selective cytostatic activity against breast cancer cell lines including a triple-negative breast cancer.

LncRNA LINC00467 acted as an oncogene in esophageal squamous cell carcinoma by accelerating cell proliferation and preventing cell apoptosis via the miR-485-5p/DPAGT1 axis.

BACKGROUND AND AIM: Esophageal carcinoma has been regarded as one of the top 10 common malignancies globally. Esophageal squamous cell carcinoma (ESCC) is an important subtype of esophageal carcinoma with approximately 20% survival rate. Long noncoding RNAs were documented to regulate the occurrence or progression of several tumors. However, neither the biological role nor the molecular mechanism of LINC00467 has been explored. This research is aimed to investigating the regulatory mechanism of LINC00467 in ESCC. METHODS: In this study, a series of experiments including reverse transcription-quantitative polymerase chain reaction, Cell Counting Kit-8, luciferase reporter, western blot, and RNA immunoprecipitation were designed and conducted to explore the potential function and mechanism of LINC00467 in ESCC. RESULTS: According to experimental results, we found out upregulated LINC00467 improved cell proliferation, but hindered cell apoptosis. In mechanism, miR-485-5p was predicted, screened out, and validated to combine with LINC00467, which displayed lower expression in ESCC. Additionally, miR-485-5p negatively regulated and directly targeted DPAGT1. Rescue assays suggested that DPAGT1 amplification was able to recover the influence of LINC00467 deficiency on cell proliferation and apoptosis. Furthermore, knockdown of LINC00467 suppressed tumor growth in vivo. CONCLUSION: We proved that LINC00467 acted as an oncogene in ESCC by accelerating cell proliferation and preventing cell apoptosis via miR-485-5p/DPAGT1 axis. This may provide a potential diagnostic marker for ESCC treatment.

DPAGT1-Mediated Protein N-Glycosylation Is Indispensable for Oocyte and Follicle Development in Mice.

Post-translational modification of proteins by N-linked glycosylation is crucial for many life processes. However, the exact contribution of N-glycosylation to mammalian female reproduction remains largely undefined. Here, DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation, is identified to be indispensable for oocyte development in mice. Dpagt1 missense mutation (c. 497A>G; p. Asp166Gly) causes female subfertility without grossly affecting other functions. Mutant females ovulate fewer eggs owing to defective development of growing follicles. Mutant oocytes have a thin and fragile zona pellucida (ZP) due to the reduction in glycosylation of ZP proteins, and display poor developmental competence after fertilization in vitro. Moreover, completion of the first meiosis is accelerated in mutant oocytes, which is coincident with the elevation of aneuploidy. Mechanistically, transcriptomic analysis reveals the downregulation of a number of transcripts essential for oocyte meiotic progression and preimplantation development (e.g., Pttgt1, Esco2, Orc6, and Npm2) in mutant oocytes, which could account for the defects observed. Furthermore, conditional knockout of Dpagt1 in oocytes recapitulates the phenotypes observed in Dpagt1 mutant females, and causes complete infertility. Taken together, these data indicate that protein N-glycosylation in oocytes is essential for female fertility in mammals by specific control of oocyte development.

A practical synthesis of a novel DPAGT1 inhibitor, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) for in vivo studies.

Immunotherapy that targets N-linked glycans has not yet been developed due in large part to the lack of specificity of N-linked glycans between normal and malignant cells. N-Glycan chains are synthesized by the sequential action of glycosyl transferases in the Golgi apparatus. It is an overwhelming task to discover drug-like inhibitors of glycosyl transferases that block the synthesis of specific branching processes in cancer cells, killing tumor cells selectively. It has long been known that N-glycan biosynthesis can be inhibited by disruption of the first committed enzyme, dolichyl-phosphate N-acetylglucosaminephosphotransferase 1 (DPAGT1). Selective DPAGT1 inhibitors have the promising therapeutic potential for certain solid cancers that require increased branching of N-linked glycans in their growth progressions. Recently, we discovered that an anti-Clostridium difficile molecule, aminouridyl phenoxypiperidinbenzyl butanamide (APPB) showed DPAGT1 inhibitory activity with the IC50 value of 0.25 µM. It was confirmed that APPB inhibits N-glycosylation of ß-catenin at 2.5 nM concentration. A sharp difference between APPB and tunicamycin was that the hemolytic activity of APPB is significantly attenuated (IC50 > 200 µM RBC). Water solubility of APPB is >350-times greater than that of tunicamycin (78.8 mg/mL for APPB, <0.2 mg/mL for tunicamycin). A novel DPAGT1 inhibitor, APPB selectively inhibits growth of the solid tumors (e.g. KB, LoVo, SK-OV-3, MDA-MB-432S, HCT116, Panc-1, and AsPC-1) at low µM concentrations, but does not inhibit growth of a leukemia cell (L1210) and the healthy cells (Vero and HPNE) at these concentrations. In vitro metabolic stability using rat liver microsomes indicated that a half-life (t 1/2) of APPB is sufficiently long (>60 min) for in vivo studies (PK/PD, safety profiles, and in vivo efficacy) using animal models. We have refined all steps in the previously reported synthesis for APPB for larger-scale. This article summarizes protocols of gram-scale synthesis of APPB and its physicochemical data, and a convenient DPAGT1 assay. •Remember that the abstract is what readers see first in electronic abstracting & indexing services.•This is the advertisement of your article. Make it interesting, and easy to be understood.•Be accurate and specific, keep it as brief as possible.

Dysregulation of the miR-325-3p/DPAGT1 axis supports HBV-positive HCC chemoresistance.

BACKGROUND: Chemotherapeutic resistance in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC) patients is an unfortunate side effect of standard chemotherapy. This situation necessitates a better understanding of the molecular pathways underlying HBV + HCC chemoresistance in order to aid the development of novel chemotherapeutic targets. METHODS: We generated two doxorubicin (DOX)-resistant HBV + HCC sublines HepG2.2.15 and Huh7-1.3. qRT-PCR was used to evaluate dysregulation in hexosamine pathway genes in chemosensitive and chemoresistant HBV + HCC cell lines in vitro. Western blots, luciferase reporter assays, and in vivo xenograft tumor studies were conducted to reveal the role of the miRNA-325-3p/DPAGT1 axis in HBV + HCC chemoresistance. RESULTS: The hexosamine pathway gene dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1) was found to be upregulated in both DOX-resistant cell lines. Enhancing DPAGT1 activity significantly improved the survival of DOX-resistant cells. Silencing or pharmacological inhibition of DPAGT1 inhibited xenograft tumor growth under DOX-treated conditions. DPAGT1 upregulation was associated with higher levels of stemness-related markers and ATP-binding cassette (ABC) drug efflux transporters in DOX-resistant cell lines. miR-325-3p was found to negatively modulate DPAGT1 expression and phenocopied the effects of DPAGT1 silencing in vitro and in vivo. In HBV + HCC patients treated with transarterial chemoembolization (TACE), high and low levels of tumor DPAGT1 and miR-325-3p expression, respectively, were associated with a poor chemotherapeutic response. CONCLUSIONS: Our findings provide novel insights into the role of miR-325-3p/DPAGT1 axis dysregulation in supporting HBV + HCC chemoresistance.

DPAGT1 Deficiency with Encephalopathy (DPAGT1-CDG): Clinical and Genetic Description of 11 New Patients.

Pathogenic mutations in DPAGT1 cause a rare type of a congenital disorder of glycosylation termed DPAGT1-CDG or, alternatively, a milder version with only myasthenia known as DPAGT1-CMS. Fourteen disease-causing mutations in 28 patients from 10 families have previously been reported to cause the systemic form, DPAGT1-CDG. We here report on another 11 patients from 8 families and add 10 new mutations. Most patients have a very severe disease course, where common findings are pronounced muscular hypotonia, intractable epilepsy, global developmental delay/intellectual disability, and early death. We also present data on three affected females that are young adults and have a somewhat milder, stable disease. Our findings expand both the molecular and clinical knowledge of previously published data but also widen the phenotypic spectrum of DPAGT1-CDG.

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.

Congenital myasthenic syndrome caused by mutations in DPAGT.

Congenital myasthenic syndromes with prominent limb girdle involvement are an important differential diagnosis for congenital myopathies because of the therapeutic considerations. We present a case where accurate diagnosis was delayed for many years. Fluctuations of weakness were misinterpreted as effects of alternative treatments. Weakness was generalised, most prominently in the arms. Fatigability was more prominent in less affected muscles revealed by a positive Simpson test. Stimulation single fibre electromyography confirmed the suspected neuromuscular transmission defect. The marked response to pyridostigmine and cognitive impairment pointed to a myasthenic syndrome due to impaired glycosylation. Two mutations in trans were found in DPAGT1, the gene coding for dolichyl-phosphate N-acetylglucosaminephosphotransferase, one novel, the other previously reported in a rare form of congenital disorder of glycosylation. Gene expression studies revealed that both mutations reduce DPAGT1 expression. Phenotypic features not previously described for DPAGT1 CMS included restricted ocular abduction and long finger flexor contractures.

Protein N-glycosylation in oral cancer: dysregulated cellular networks among DPAGT1, E-cadherin adhesion and canonical Wnt signaling.

N-Linked glycosylation (N-glycosylation) of proteins has long been associated with oncogenesis, but not until recently have the molecular mechanisms underlying this relationship begun to be unraveled. Here, we review studies describing how dysregulation of the N-glycosylation-regulating gene, DPAGT1, drives oral cancer. DPAGT1 encodes the first and rate-limiting enzyme in the assembly of the lipid-linked oligosaccharide precursor in the endoplasmic reticulum and thus mediates N-glycosylation of many cancer-related proteins. DPAGT1 controls N-glycosylation of E-cadherin, the major epithelial cell-cell adhesion receptor and a tumor suppressor, thereby affecting intercellular adhesion and cytoskeletal dynamics. DPAGT1 also regulates and is regulated by Wnt/ß-catenin signaling, impacting the balance between proliferation and adhesion in homeostatic tissues. Thus, aberrant induction of DPAGT1 promotes a positive feedback network with Wnt/ß-catenin that represses E-cadherin-based adhesion and drives tumorigenic phenotypes. Further, modification of receptor tyrosine kinases (RTKs) with N-glycans is known to control their surface presentation via the galectin lattice, and thus increased DPAGT1 expression likely contributes to abnormal activation of RTKs in oral cancer. Collectively, these studies suggest that dysregulation of the DPAGT1/Wnt/E-cadherin network underlies the etiology and pathogenesis of oral cancer.

ALG1-CDG: a new case with early fatal outcome.

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases. The deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase, encoded by the human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG patient is the focus of this paper. The patient's main symptoms were feeding problems and diarrhea, profound hypoproteinemia with massive ascites, muscular hypertonia, seizures refractory to treatment, recurrent episodes of apnoea, cardiac and hepatic involvement and coagulation anomalies. Compound heterozygosity for the mutations c.1145T>C (M382T) and c.1312C>T (R438W) was detected in the patient's ALG1-coding sequence. In contrast to a previously reported speculation on R438W we confirmed both mutations as disease-causing in ALG1-CDG.

Mesdc2 plays a key role in cell-surface expression of Lrp4 and postsynaptic specialization in myotubes.

Low-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre- and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation.