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

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

Mutations in MAGT1 lead to a glycosylation disorder with a variable phenotype.

Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. We identified two patients with defective serum transferrin glycosylation and mutations in the MAGT1 gene. These patients present with a phenotype that is mainly characterized by intellectual and developmental disability. MAGT1 has been described to be a subunit of the oligosaccharyltransferase (OST) complex and more specifically of the STT3B complex. However, it was also claimed that MAGT1 is a magnesium (Mg2+) transporter. So far, patients with mutations in MAGT1 were linked to a primary immunodeficiency, characterized by chronic EBV infections attributed to a Mg2+ homeostasis defect (XMEN). We compared the clinical and cellular phenotype of our two patients to that of an XMEN patient that we recently identified. All three patients have an N-glycosylation defect, as was shown by the study of different substrates, such as GLUT1 and SHBG, demonstrating that the posttranslational glycosylation carried out by the STT3B complex is dysfunctional in all three patients. Moreover, MAGT1 deficiency is associated with an enhanced expression of TUSC3, the homolog protein of MAGT1, pointing toward a compensatory mechanism. Hence, we delineate MAGT1-CDG as a disorder associated with two different clinical phenotypes caused by defects in glycosylation.

Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis.

Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc3Man9GlcNAc2-LLO but not Man9GlcNAc2-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST-dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST-dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.

Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpesvirus responsible for oral lesions and herpesviral encephalitis. The HSV-1 envelope contains N-glycosylated proteins involved in infection and that are candidate drug targets. NGI-1 is a small-molecule inhibitor of oligosaccharyltransferase (OST) complexes STT3A-OST and STT3B-OST, which catalyze cotranslational and post-translational N-glycosylation, respectively. Because host OSTs attach HSV-1 glycans, NGI-1 might have anti-HSV-1 activity. We evaluated HSV-1 function using NGI-1 and human embryonic kidney 293 knockout lines for OST isoform-specific catalytic and accessory subunits. N-glycosylation of 2 representative envelope proteins (gC and gD) was primarily dependent upon STT3A-OST, but to a large extent replaceable by STT3B-OST. Knockouts impairing STT3A- or STT3B-OST activity, by themselves, did not appreciably affect HSV-1 function (plaque-forming units, normalized to viral particles measured by unglycosylated capsid protein VP5 content). However, with cells lacking STT3B-OST activity (missing the catalytic subunit STT3B or the oxidoreductase subunits magnesium transporter 1/tumor suppressor candidate 3) and thus solely dependent upon STT3A-OST for N-glycosylation, NGI-1 treatment resulted in HSV-1 having cell type-dependent dysfunction (affecting infectivity with Vero cells much more than with the 293 lines). Ablation of post-translational N-glycosylation can therefore make HSV-1 infectivity, and possibly masking of immunogenic peptide epitopes by glycans, highly sensitive to pharmacological inhibition of cotranslational N-glycosylation.-Lu, H., Cherepanova, N. A., Gilmore, R., Contessa, J. N., Lehrman, M. A. Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.

Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum.

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. N-linked oligosaccharides are important for protein folding and stability, biosynthetic quality control, intracellular traffic and the physiological function of many N-glycosylated proteins. In metazoan organisms, the oligosaccharyltransferase is composed of a catalytic subunit (STT3A or STT3B) and a set of accessory subunits. Duplication of the catalytic subunit gene allowed cells to evolve OST complexes that act sequentially to maximize the glycosylation efficiency of the large number of proteins that are glycosylated in metazoan organisms. We will summarize recent progress in understanding the mechanism of (a) cotranslational glycosylation by the translocation channel associated STT3A complex, (b) the role of the STT3B complex in mediating cotranslational or posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex, and (c) the role of the oxidoreductase MagT1 in STT3B-dependent glycosylation of cysteine-proximal acceptor sites.

Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity.

BACKGROUND: Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood. RESULTS: Mutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202. CONCLUSIONS: We conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

Adaptive maternal immune deviations as a ground for autism spectrum disorders development in children.

Autism is a vexed problem today. Overall, there is a high frequency of birth children (1:80 - 1:150) with late diagnosed autism spectrum disorders (ASD) and this trend is getting progressively stronger. The causes for the currently increased frequency of ASD and the pathogenesis of ASD are not fully understood yet. One of the most likely mechanisms inducing ASD may be a maternal immune imprinting. This phenomenon is based on transplacental translocation of maternal antibodies of IgG class and, as a consequence, on the epigenetic "tuning" of immune system of the fetus and child. This mechanism provides development of child's anti-infection resistance before meeting with microorganisms, but it can be also a cause of inborn pathology including the ASD appearance. The quantitative changes in maternal blood serum autoantibodies depend on a specific microbial population, or are induced by environmental chemical pollutants in association with some individual features of the maternal metabolism. These immune changes are adaptive in most cases for the maternal organism, but can be pathogenic for the fetus in some cases. We discuss in the present paper the possibilities to predict the risk from abnormal development of nervous system in fetus and early diagnosis of ASD in high-risk group of children.

Mutational analysis of the CG recognizing DNA methyltransferase SssI: insight into enzyme-DNA interactions.

To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)-methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5'-CG-3'), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved S145 (motif IV), R232 (motif VIII, QxRxR), and T313 (variable region, conserved TL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, ENV) and R230 (motif VIII, QxRxR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and R230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases.

Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae.

Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal-to-C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y-derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.

Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites.

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia.

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon.

In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an STT3A-/- phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2- and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes.

Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex.

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV.IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.

Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation.

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. Metazoan organisms express two oligosaccharyltransferase complexes that are composed of a catalytic subunit (STT3A or STT3B) assembled with a shared set of accessory subunits and one to two complex specific subunits. siRNA mediated knockdowns of STT3A and STT3B in HeLa cells have shown that the two OST complexes have partially non-overlapping roles in N-linked glycosylation. However, incomplete siRNA mediated depletion of STT3A or STT3B reduces the impact of OST complex loss, thereby complicating the interpretation of experimental results. Here, we have used the CRISPR/Cas9 gene editing technology to create viable HEK293 derived cells lines that are deficient for a single catalytic subunit (STT3A or STT3B) or two STT3B-specific accessory subunits (MagT1 and TUSC3). Analysis of protein glycosylation in the STT3A, STT3B and MagT1/TUSC3 null cell lines revealed that these cell lines are superior tools for investigating the in vivo role and substrate preferences of the STT3A and STT3B complexes.

Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins.

Stabilization of protein tertiary structure by disulfides can interfere with glycosylation of acceptor sites (NXT/S) in nascent polypeptides. Here, we show that MagT1, an ER-localized thioredoxin homologue, is a subunit of the STT3B isoform of the oligosaccharyltransferase (OST). The lumenally oriented active site CVVC motif in MagT1 is required for glycosylation of STT3B-dependent acceptor sites including those that are closely bracketed by disulfides or contain cysteine as the internal residue (NCT/S). The MagT1- and STT3B-dependent glycosylation of cysteine-proximal acceptor sites can be reduced by eliminating cysteine residues. The predominant form of MagT1 in vivo is oxidized, which is consistent with transient formation of mixed disulfides between MagT1 and a glycoprotein substrate to facilitate access of STT3B to unmodified acceptor sites. Cotranslational N-glycosylation by the STT3A isoform of the OST, which lacks MagT1, allows efficient modification of acceptor sites in cysteine-rich protein domains before disulfide bond formation. Thus, mammalian cells use two mechanisms to achieve N-glycosylation of cysteine proximal acceptor sites.

Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods.

The impact of bulky carcinogen-DNA adducts positioned at or near recognition sites (CpG) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In the present study, we employed site-specifically modified 30-mer oligodeoxyribonucleotides containing stereoisomeric benzo[a]pyrene diol epoxide (B[a]PDE)-derived guanine (B[a]PDE-N(2)-dG) or adenine (B[a]PDE-N(6)-dA) adducts of different conformations as substrates of the catalytic domain of murine Dnmt3a (Dnmt3a-CD). The fluorescence of these lesions was used to examine interactions between Dnmt3a-CD and DNA. In B[a]PDE-DNA•Dnmt3a-CD complexes, the intensity of fluorescence of the covalently bound B[a]PDE residues is enhanced relative to the protein-free value when the B[a]PDE is positioned in the minor groove [(+)- and (-)-trans-B[a]PDE-N(2)-dG adducts in the CpG site] and when it is intercalated on the 5'-side of the CpG site [(+)-trans-B[a]PDE-N(6)-dA adduct]. The fluorescence of B[a]PDE-modified DNA•Dnmt3a-CD complexes exhibits only small changes when the B[a]PDE is intercalated with base displacement in (+)- and (-)-cis-B[a]PDE-N(2)-dG adducts and without base displacement in the (-)-trans-B[a]PDE-N(6)-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N(2)-dG adducts, regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N(2)-dG adducts within the CpG site. The observed changes in fluorescence and methylation rates are consistent with the flipping of the target cytosine and a catalytic loop motion within the DNA•Dnmt3a-CD complexes. In the presence of the regulatory factor Dnmt3L, an enhancement of both methylation rates and fluorescence was observed, which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.

Mechanism of CpG DNA methyltransferases M.SssI and Dnmt3a studied by DNA containing 2-aminopurine.

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase-DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme-substrate complexes were observed.