Oligosaccharyltransferase Research Articles

Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

SummaryThe Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide1. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates2. Also hepatitis C virus (HCV) remains a major medical problem with 160 million chronically infected patients and only expensive treatment on the market3. Despite distinct differences in pathogenesis and mode of transmission, the two viruses share common replication strategies4. A detailed understanding of the host functions that determine viral infection is lacking. Here, we utilized a pooled CRISPR genetic screening strategy5,6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified ER-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER associated degradation. Dengue virus replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as critical step requiring the OST complex. Moreover, we showed that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects7. In contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA binding proteins and enzymes involved in metabolism. We discovered an unexpected link between intracellular FAD levels and HCV replication. This study shows remarkable divergence in host dependency factors between DENV and HCV and illuminates novel host targets for antiviral therapy.

Origins of Catalytic Specificity in Bacterial Oligosaccharyltransferase

Modification of polypeptides through asparagine (N)-linked glycosylation is involved in a broad range of biological functions. N-linked glycosylation is catalyzed by oligosaccharyltransferase (OST), an endoplasmic reticulum membrane protein, which promotes the formation of an N-glycosidic linkage between the acceptor asparagine and an oligosaccharide donor. Broad polypeptide substrate specificity is characteristic of N-linked glycosylation, resulting from a short consensus sequence: N-X-S/T (where X is any amino acid except P). However, experimental evidence has shown that the N-glycosylation efficiency is affected by the sequence within this consensus motif.We investigated the catalytic specificity of Campylobacter lari OST using molecular dynamics simulations. In OST, a large external loop (EL5) pins the substrate in the binding pocket located between the transmembrane and periplasmic domains while two acidic catalytic residues, E319 and D56 in C. lari, are in position to form hydrogen bonds with the acceptor asparagine and prime it for nucleophilic attack on the oligosaccharide donor. To explore how different substrates affect the efficiency of N-linked glycosylation, four substrates of differing N-glycosylation efficiencies were examined: the optimal consensus sequence (NAT) and three sub-optimal variants (NAS, NFS, and NWS). Our simulations of OST in complex with the optimal substrate (NAT) show a conformational change of EL5 and of the periplasmic domain is necessary to promote optimal hydrogen bond formation between the acceptor asparagine and E319/D56 by restricting side chain motion in the catalytic pocket. In addition, we found that binding of the other three substrates (NAS, NFS, and NWS) affects the structure and dynamics of OST. These changes lower the probability of forming hydrogen bonds with the acceptor asparagine, essential for catalysis, and accelerate substrate release for NFS, providing two mechanisms for modulating the glycosylation efficiency of OST for various consensus sequence substrates.

Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria.

Bacterial N-linking oligosaccharyl transferases (OTase enzymes) transfer lipid-linked glycans to selected proteins in the periplasm and were first described in the intestinal pathogen Campylobacter jejuni, a member of the ε-proteobacteria-subdivision of bacteria. More recently, orthologues from other ε-proteobacterial Campylobacter and Helicobacter species and a δ-proteobacterium, Desulfovibrio desulfuricans, have been described, suggesting that these two subdivisions of bacteria may be a source of further N-linked protein glycosylation systems. Whole-genome sequencing of both ε- and δ-proteobacteria from deep-sea vent habitats, a rich source of species from these subdivisions, revealed putative ORFs encoding OTase enzymes and associated adjacent glycosyltransferases similar to the C. jejuni N-linked glycosylation locus. We expressed putative OTase ORFs from the deep-sea vent species Nitratiruptor tergarcus, Sulfurovum lithotrophicum and Deferribacter desulfuricans in Escherichia coli and showed that they were able to functionally complement the C. jejuni OTase, CjPglB. The enzymes were shown to possess relaxed glycan specificity, transferring diverse glycan structures and demonstrated different glycosylation sequon specificities. Additionally, a permissive D. desulfuricans acceptor protein was identified, and we provide evidence that the N-linked glycan synthesized by N. tergarcus and S. lithotrophicum contains an acetylated sugar at the reducing end. This work demonstrates that deep-sea vent bacteria encode functional N-glycosylation machineries and are a potential source of biotechnologically important OTase enzymes.

Two conserved oligosaccharyltransferase catalytic subunits required for N-glycosylation exist in Spartina alterniflora.

BackgroundAsparagine (N)-linked glycosylation is one of the most crucial post-translational modifications, which is catalyzed in the lumen of the endoplasmic reticulum (ER) by the oligosaccharyltransferase (OST) in eukaryotic cells. Biochemical and genetic assay leads to the identification of the nine subunits (Ost 1–6, Stt3, Swp1 and Wbp1) of the yeast OST and in which Stt3p is proposed playing a central and conserved role in N-glycosylation. Two STT3 isoform genes, STT3A and STT3B, exist in the plant and mammal genomes. OST with different catalytic STT3 isoforms has different enzymatic properties in mammals. The mutation of STT3A in Arabidopsis thaliana causes a salt hypersensitive phenotype the inhibited root growth and swollen root tips suggesting protein N-glycosylation is indispensable for plant growth and development. Spartina alterniflora is widely used for shoreline protection and tidal marsh restoration due to the strong salt tolerance although the exact molecular mechanism is little known. To explore the possible biological roles of N-glycosylation in plant adaptive resistance to salinity stress, we cloned the STT3 genes from S. alterniflora and heterogenously expressed them in Arabidopsis mutant to observe the functional conservation.Results SaSTT3A and SaSTT3B genes were cloned from Spartina alterniflora. SaSTT3A genomic sequences spanned over 23 exons and 22 introns, while SaSTT3B had 6 exons and 5 introns. The gene structures of both genes were conserved among the analyzed plant species. Subcellular localization and transmembrane structure prediction revealed that these two genes had 13 and 11 transmembrane helices respectively. The functional complementation in which the cDNA of SaSTT3A and SaSTT3B driven by CaMV 35S promoter completely or partially rescued Arabidopsis stt3a-2 mutant salt-sensitive phenotype, indicating STT3A functions conservatively between glycophyte and halophyte and N-glycosylation might be involved in plant resistance to salinity.ConclusionsTwo STT3 isoform genes, SaSTT3A and SaSTT3B, were cloned from S. alterniflora and they were evolutionally conserved at gene structure and coding sequences compared with their counterparts. Moreover, SaSTT3 genes could successfully rescue Arabidopsis stt3a-2 salt-sensitive phenotype, suggesting there exists a similar N-glycosylation process in S. alterniflora. Here we provided a first piece of evidence that the N-glycosylation might be involved in salt tolerance of halophyte.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-015-0111-9) contains supplementary material, which is available to authorized users.

Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.

The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the −2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the −2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native ‘QYNST’ sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.

Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation andautoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.

Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides in mammalian cells.

During asparagine (N)-linked protein glycosylation, eukaryotic cells generate considerable amounts of free oligosaccharides (fOSs) in the cytosol. It is generally assumed that such fOSs are produced by the deglycosylation of misfolded N-glycoproteins that are destined for proteasomal degradation or as the result of the degradation of dolichol-linked oligosaccharides (DLOs), which serve as glycan donor substrates in N-glycosylation reactions. The findings reported herein show that the majority of cytosolic fOSs are generated by a peptide:N-glycanase (PNGase) and an endo-β-N-acetylglucosaminidase (ENGase)-independent pathway in mammalian cells. The ablation of the cytosolic deglycosylating enzymes, PNGase and ENGase, in mouse embryonic fibroblasts had little effect on the amount of cytosolic fOSs generated. Quantitative analyses of fOSs using digitonin-permeabilized cells revealed that they are generated by the degradation of fully assembled Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) in the lumen of the endoplasmic reticulum. Because the degradation of Glc3Man9GlcNAc2-PP-Dol is greatly inhibited in the presence of an N-glycosylation acceptor peptide that is recognized by the oligosaccharyltransferase (OST), the OST-mediated hydrolysis of DLO is the most likely mechanism responsible for the production of a large fraction of the cytosolic fOSs.

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Archaeoglobus fulgidus

The genome of the hyperthermophilic archaeon, Archaeoglobus fulgidus, contains three paralogous AglB genes that encode oligosaccharyltransferase (OST) proteins. The OST enzymes catalyze the transfer of an oligosaccharide chain from lipid-linked oligosaccharides (LLO) to asparagine residues in proteins. The detergent-solubilized membrane fractions prepared from cultured A. fulgidus cells contain both OST and LLO. The addition of a peptide containing the glycosylation sequon produced oligosaccharide chains attached to a structurally defined peptide. To facilitate the NMR analysis, the cells were grown in rich medium supplemented with 13C-glucose, to label the LLOs metabolically. The MS analysis of the glycopeptide revealed that the glucose and galactose residues were nearly fully 13C-labeled, but the mannose residues were fractionally labeled with about 20% efficiency. An immunodetection experiment revealed that the longest AglB paralog (AfAglB-L) was expressed in the membrane fractions under our cell culture conditions, while the other two shorter AglB paralogs (AfAglB-S1 and AfAglB-S2) were not. Thus, the oligosaccharide chain analyzed in this study was the product of AfAglB-L. The N-glycan consists of eight hexose residues, as follows:▪The α1,3-linked glucose is an optional residue branching from the distal mannose residue. The MS analysis of the minor HPLC peak of the in vitro oligosaccharyl transfer products also revealed an optional sulfate modification on the glucose residue directly linked to the Asn residue. The present data will be useful for structural and functional studies of the N-glycosylation system of A. fulgidus.

Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.

Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation.

Structure‐to‐Function Characterization of Saccharomyces cerevisiae STT3

The yeast STT3 protein is the catalytic subunit of the octameric protein complex of the oligosaccharyl transferase (OST). This enzyme is responsible for N-linked glycosylation, a highly conserved and essential posttranslational protein modification in eukaryotes. The function of this essential STT3 protein is still not fully understood. We used mutagenesis to elucidate its function based on structure by looking at the effects of mutations on OST complex formation and N-glycosylation of proteins in vivo. Point mutations were generated at the chromosomal level in yeast cells harboring the complementing Leishmania major STT3 (LmSTT3D) protein using site-directed mutagenesis. The effect of the mutations on enzyme activity was evaluated in strains without LmSTT3D. The role of stt3 mutants in complex formation were investigated by immunoblotting and monitoring of OST subunit turnover rates. Glycosylation efficiency of mutants was determined by analyzing glycosylation site occupancy for different substrate proteins by immunoblotting and mass spectrometry. We showed defined residues that form the catalytic center. Mutations of these residues altered N-glycosylation but not complex formation. In contrast, other mutations induce rapid degradation of some OST subunits, indicating complex destabilization, which led to reduced enzyme activity and hypoglycosylation of proteins. These data have led to the identification of the catalytic center of the yeast OST. Our study showed that phenotypic analysis of mutations in subunits of protein complexes has to include assessment of complex stability.

Reduced expression of the oligosaccharyltransferase exacerbates protein hypoglycosylation in cells lacking the fully assembled oligosaccharide donor.

A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc(2)Man(9) as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc(2)Man(9)Glc(3) donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.

Prognostic and therapeutic impact of RPN2-mediated tumor malignancy in non-small-cell lung cancer.

RNA interference (RNAi) is a powerful gene-silencing platform for cancer treatment. Previously, we demonstrated that ribophorin II (RPN2), which is part of the N-oligosaccharyl transferase complex, regulates docetaxel sensitivity and tumor lethal phenotypes in breast cancer. However, the molecular functions and clinical relevance of RPN2 in non-small-cell lung cancer (NSCLC) remain unknown. Here, we examined RPN2 expression in tumor specimens from recurrent NSCLC patients after resection (n = 32 and = 177) and assessed the correlation between RPN2 expression and various clinical features. We also investigated whether RPN2 affects cancer malignancy in vitro and tumor growth and drug resistance in vivo. Our data show that RPN2 expression confers early and distant recurrence as well as poor survival in NSCLC patients. Furthermore, RPN2 silencing suppressed cell proliferation and invasiveness, and increased the sensitivity to chemotherapeutic drugs in vitro. Remarkably, we found that intrinsic apoptosis signaling is the mechanism of cell death involved with RPN2 knockdown. Strikingly, RPN2 silencing repressed tumorigenicity and sensitized the tumors to cisplatin treatment, which led to the longer survival of NSCLC-bearing mice. In conclusion, these data suggest that RPN2 is involved in the regulation of lethal cancer phenotypes and represents a promising new target for RNAi-based medicine against NSCLC.

The Endoplasmic Reticulum-based Acetyltransferases, ATase1 and ATase2, Associate with the Oligosaccharyltransferase to Acetylate Correctly Folded Polypeptides

The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N(ϵ)-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and "select" correctly folded from unfolded/misfolded transiting polypeptides.

Lipid-Linked Oligosaccharides in Membranes Sample Conformations That Facilitate Binding to Oligosaccharyltransferase

Lipid-linked oligosaccharides (LLOs) are the substrates of oligosaccharyltransferase (OST), the enzyme that catalyzes the en bloc transfer of the oligosaccharide onto the acceptor asparagine of nascent proteins during the process of N-glycosylation. To explore LLOs’ preferred location, orientation, structure, and dynamics in membrane bilayers of three different lipid types (dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine), we have modeled and simulated both eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol) and bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol) LLOs, which are composed of an isoprenoid moiety and an oligosaccharide, linked by pyrophosphate. The simulations show no strong impact of different bilayer hydrophobic thicknesses on the overall orientation, structure, and dynamics of the isoprenoid moiety and the oligosaccharide. The pyrophosphate group stays in the bilayer head group region. The isoprenoid moiety shows high flexibility inside the bilayer hydrophobic core, suggesting its potential role as a tentacle to search for OST. The oligosaccharide conformation and dynamics are similar to those in solution, but there are preferred interactions between the oligosaccharide and the bilayer interface, which leads to LLO sugar orientations parallel to the bilayer surface. Molecular docking of the bacterial LLO to a bacterial OST suggests that such orientations can enhance binding of LLOs to OST.

MagT1 helps a glycosylase gain acceptance

![Figure][1] MagT1 (red) localizes to the ER (green) in HeLa cells. Cherepanova et al. describe how an oxidoreductase enzyme promotes the glycosylation of newly synthesized proteins in the ER. Two different oligosaccharyltransferase (OST) complexes glycosylate asparagine-containing

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.

The middle X residue influences cotranslational N-glycosylation consensus site skipping.

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X ≠ P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse–chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B’s role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.

AglB, catalyzing the oligosaccharyl transferase step of the archaeal N‐glycosylation process, is essential in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Sulfolobus acidocaldarius, a thermo-acidophilic crenarchaeon which grows optimally at 76°C and pH 3, exhibits an astonishing high number of N-glycans linked to the surface (S-) layer proteins. The S-layer proteins as well as other surface-exposed proteins are modified via N-glycosylation, in which the oligosaccharyl transferase AglB catalyzes the final step of the transfer of the glycan tree to the nascent protein. In this study, we demonstrated that AglB is essential for the viability of S. acidocaldarius. Different deletion approaches, that is, markerless in-frame deletion as well as a marker insertion were unsuccessful to create an aglB deletion mutant. Only the integration of a second aglB gene copy allowed the successful deletion of the original aglB.