Oligosaccharyltransferases Research Articles

Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

Synthesis and characterisation of fluorescent substrates for eukaryotic protein N-glycosylation

Herein we report the synthesis of two fluorescently labelled analogues of C25 dolichol (Dol25) in which the terminal isoprene unit has been replaced by a dansyl or 7-amino-4-trifluoromethylcoumarin fluorophore, a transformation enabled by the regioselective epoxidation of the terminal olefin via its bromohydrin using the van Tamelen procedure. The lipid alcohols were phosphorylated and glycosylated to obtain lipid-linked chitobiose-α-diphosphates and a lipid-linked mannosyl-β-phosphate. Biochemical assays showed that these labelled substrates are accepted by eukaryotic protein N-glycosylation enzymes with rates comparable to the unlabelled substrates, reconstituting a major part of the pathway up to the lipid-linked dodeca-saccharide Glc1Man9GlcNAc2-PP-Dol25 and its transfer to an acceptor peptide catalysed by eukaryotic oligosaccharyltransferases (OSTs), namely the single-subunit OST STT3A from Trypanosoma brucei and the octameric OST complex from Saccharomyces cerevisiae. The fluorescent labels facilitate handling and purification of the lipid-linked glycosyl donors and acceptors and should facilitate further biochemical studies of protein glycosylation enzymes.

Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV

N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

Common and unique features of glycosylation and glycosyltransferases in African trypanosomes.

Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic β3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed.

2020 FASEB Science Research Conference on Microbial Glycobiology, July 13-14, 2020.

2020 FASEB Science Research Conference on Microbial Glycobiology, July 13-14, 2020.

The Lazy Life of Lipid-Linked Oligosaccharides in All Life Domains.

Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein.

Structure-Based Prediction of Polypeptide Substrate Specificities of Glycosyltransferases

Glycans are some of the most abundant and diverse biopolymers, important in a number of biological processes including cell adhesion, receptor activation, molecular trafficking, endocytosis, and signal transduction. Protein glycosylation is primarily mediated by enzymes known as oligosaccharyltransferases (OSTs) and glycosyltransferases (GTs). Polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) are higher eukaryotic retaining GTs that transfer a GalNAc moiety from uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) onto Ser/Thr residues. This enzyme family initiates the most common type of O-glycosylation and thus affects the structure, stability, and function of many proteins. Structure-based prediction tools have the potential to advance our understanding of glycan-related phenomena as well as enable the design of novel glycoconjugates. Here, we present new computational protocols that allow rapid computational screening and scoring of polypeptide substrates of GTs such as GalNAcT2 and human-OGT. Our Rosetta-based algorithm docks a fully flexible glycosylated polypeptide substrate to the catalytic site of the GT, exhaustively sampling polypeptide and glycan conformational space, generating thousands of decoys for each docked polypeptide followed by ranking of decoys using the Rosetta score function. Using this protocol, we are able to predict relative specificities for polypeptide glycosylation for all possible amino acid permutations at substrate positions X-1 and X+1 for GalNAc-T2 and human-OGT. We compared predictions to experimental data (Kightlinger, W. et al., Nat. Chem. Biol.14, 627-635 (2018)) for all 400 polypeptide substrates per enzyme. Using a receiver operating curve analysis, the predictions were found to have an area-under-curve above 0.80, indicating that the method can often distinguish true and false positives. To the best of our knowledge, our protocol is the first example of a structure-based computational protocol for predicting polypeptide substrate specificities of GTs.

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.

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

Construction of green fluorescence protein mutant to monitor STT3B-dependent N-glycosylation.

Oligosaccharyltransferases (OSTs) mediate the enbloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

Analysis of substrate specificity of Trypanosoma brucei oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeast

N-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single-protein OSTs called TbSTT3A, TbSTT3B, and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST, making it an ideal experimental system to study the fundamental properties of OST activity. We characterized the LLO and polypeptide specificity of all three TbOST isoforms and their chimeric forms in the heterologous expression host S. cerevisiae where we were able to apply yeast genetic tools and newly developed glycoproteomics methods. We demonstrated that TbSTT3A accepted LLO substrates ranging from Man5GlcNAc2 to Man7GlcNAc2 In contrast, TbSTT3B required more complex precursors ranging from Man6GlcNAc2 to Glc3Man9GlcNAc2 structures, and TbSTT3C did not display any LLO preference. Sequence differences between the isoforms cluster in three distinct regions. We have swapped the individual regions between different OST proteins and identified region 2 to influence the specificity toward the LLO and region 1 to influence polypeptide substrate specificity. These results provide a basis to further investigate the molecular mechanisms and contribution of single amino acids in OST interaction with its substrates.

Probing Polytopic Membrane Protein-Substrate Interactions by Luminescence Resonance Energy Transfer.

Integral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally determined and computationally predicted structures are urgently needed. Herein we present the application of luminescence resonance energy transfer (LRET) for investigating the interactions of the polytopic membrane-bound oligosaccharyl transferases (OTases) with partner substrates. Monomeric OTases, such as the PglBs from Campylobacter jejuni and Campylobacter lari, catalyze transfer of glycans from membrane-associated undecaprenol diphosphate-linked substrates to proteins in the bacterial periplasm. LRET-based distance measurements are enabled by the inclusion of an encoded N-terminal lanthanide-binding tag (LBT), and LRET between the luminescent (LBT)-Tb(3+) donor complex and fluorescently labeled peptide and glycan substrates provides discrete distance measurements across the span of the membrane. LRET-based measurements of detergent-solubilized PglB from C.lari allowed direct comparison with the distances based on the previously reported the C.lari PglB crystal structure, thereby validating the approach in a defined system. Distance measurements between peptide and glycan substrates and the C.jejuni PglB offer new experimental information on substrate binding to the related, but structurally uncharacterized, eukaryotic OTase.

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.

GlycoSNAP: A High-Throughput Screening Methodology for Engineering Designer Glycosylation Enzymes.

The Campylobacter jejuni protein glycosylation locus (pgl) encodes enzymes for asparagine-linked (N-linked) glycosylation and serves as the prototype for N-glycosylation in bacteria. This pathway has been functionally transferred into Escherichia coli, thereby enabling efficient N-linked glycosylation of acceptor proteins with the C. jejuni heptasaccharide in this genetically tractable host. However, expansion of this bacterial system to encompass the vast biological diversity of potential glycans and target proteins often requires engineering of the non-native enzymes to isolate variants with optimal or altered functions. Here, we describe a genetic assay named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that facilitates engineering of glycosylation enzymes directly in E. coli. To date, this method has been successfully applied to screen (1) combinatorial libraries of bacterial oligosaccharyltransferases (OSTs) for relaxed substrate specificity and (2) acceptor site libraries to identify sequons recognized by natural OSTs as well as engineered variants.

Effect of glucose feeding on the glycosylation quality of antibody produced by a human cell line, F2N78, in fed-batch culture

The human cell line rF2N78 produces an antibody with a high galactosylation ratio which resembles human IgG. However, it has been observed that the aglycosylated antibody starts to appear when glucose is depleted. To determine whether glucose depletion is a main cause for aglycosylation of the antibody, fed-batch cultures of rF2N78 cells were performed using different feeding cocktails (glucose only, nutrient feeding cocktail without glucose, and nutrient feeding cocktail with glucose). In the fed-batch culture with nutrient feeding cocktail without glucose, aglycosylated antibody was produced in a later phase of culture, when glucose was depleted. Approximately 44% of antibodies produced were aglycosylated at the end of culture. In contrast, aglycosylated antibody was not produced in cultures with glucose feeding. The expression levels of oligosaccharyl transferases determined by Western blot analysis were similar among the cultures, suggesting that aglycosylation of the antibody was not due to altered expression of oligosaccharyl transferases under glucose-deficient conditions. Thus, it is likely that glucose deficiency led to insufficiency of the precursor for glycosylation and induced aglycosylation of the antibody. Taken together, glucose feeding in fed-batch cultures successfully prevented occurrence of aglycosylated antibody during the cultures, confirming that glucose depletion is a main cause for aglycosylation of antibody.