Glycosylation Sequon Research Articles

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.

Glycan shield of the ebolavirus envelope glycoprotein GP

The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions.

Reduced sialidase activity of influenza A(H3N2) neuraminidase associated with positively charged amino acid substitutions.

Neuraminidase (NA) inhibitors (NAI), oseltamivir and zanamivir, are the main antiviral medications for influenza and monitoring of susceptibility to these antivirals is routinely done by determining 50 % inhibitory concentrations (IC50) with MUNANA substrate. During 2010-2019, levels of A(H3N2) viruses presenting reduced NAI inhibition (RI) were low (~0.75 %) but varied year-on-year. The highest proportions of viruses showing RI were observed during the 2013-2014, 2016-2017 and 2017-2018 Northern Hemisphere seasons. The majority of RI viruses were found to contain positively charged NA amino acid substitutions of N329K, K/S329R, S331R or S334R, being notably higher during the 2016-2017 season. Sialidase activity kinetics were determined for viruses of RI phenotype and contemporary wild-type (WT) viruses showing close genetic relatedness and displaying normal inhibition (NI). RI phenotypes resulted from reduced sialidase activity compared to relevant WT viruses. Those containing S329R or N329K or S331R showed markedly higher Km for the substrate and Ki values for NAIs, while those with S334R showed smaller effects. Substitutions at N329 and S331 disrupt a glycosylation sequon (NDS), confirmed to be utilised by mass spectrometry. However, gain of positive charge at all three positions was the major factor influencing the kinetic effects, not loss of glycosylation. Because of the altered enzyme characteristics NAs carrying these substitutions cannot be assessed reliably for susceptibility to NAIs using standard MUNANA-based assays due to reductions in the affinity of the enzyme for its substrate and the concentration of the substrate usually used.

Development of an O-polysaccharide based recombinant glycoconjugate vaccine in engineered E. coli against ExPEC O1

Extraintestinal pathogenic Escherichia coli O1 is a frequently identified serotype that causes serious infections and is often refractory to antimicrobial therapy. Glycoconjugate vaccine represents a promising measure to reduce ExPEC infections. Herein, we designed an O1-specific glyco-optimized chassis strain for manufacture of O-polysaccharide (OPS) antigen and OPS-based bioconjugate. Specifically, OPS and OPS-based glycoprotein were synthesized in glyco-optimized chassis strain, when compared to the unmeasurable level of the parent strain. The optimal expression of oligosaccharyltransferase and carrier protein further improved the titer. MS analysis elucidated the correct structure of resulting bioconjugate at routine and unreported glycosylation sequons of carrier protein, with a higher glycosylation efficiency. Finally, purified bioconjugate stimulated mouse to generate specific IgG antibodies and protected them against virulent ExPEC O1 challenge. The plug-and-play glyco-optimized platform is suitable for bioconjugate synthesis, thus providing a potential platform for future medical applications.

Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

A genomic variant of ALPK2 is associated with increased liver fibrosis risk in HIV/HCV coinfected women.

HIV coinfection is associated with more rapid liver fibrosis progression in hepatitis C (HCV) infection. Recently, much work has been done to improve outcomes of liver disease and to identify targets for pharmacological intervention in coinfected patients. In this study, we analyzed clinical data of 1,858 participants from the Women's Interagency HIV Study (WIHS) to characterize risk factors associated with changes in the APRI and FIB-4 surrogate measurements for advanced fibrosis. We assessed 887 non-synonymous single nucleotide variants (nsSNV) in a subset of 661 coinfected participants for genetic associations with changes in liver fibrosis risk. The variants utilized produced amino acid substitutions that either altered an N-linked glycosylation (NxS/T) sequon or mapped to a gene related to glycosylation processes. Seven variants were associated with an increased likelihood of liver fibrosis. The most common variant, ALPK2 rs3809973, was associated with liver fibrosis in HIV/HCV coinfected patients; individuals homozygous for the rare C allele displayed elevated APRI (0.61, 95% CI, 0.334 to 0.875) and FIB-4 (0.74, 95% CI, 0.336 to 1.144) relative to those coinfected women without the variant. Although warranting replication, ALPK2 rs3809973 may show utility to detect individuals at increased risk for liver disease progression.

A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections

ABSTRACT We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.

High efficiency biosynthesis of O-polysaccharide-based vaccines against extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L−1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

Inulin catabolism in Saccharomyces cerevisiae is affected by some key glycosylation sequons of invertase Suc2.

The objective is to study the effect of individual N-glycosylation sequon on Suc2 activity and stability, and improve inulin conversion capacity and ethanol production of yeast by manipulating the key N-glycosylation sequon in Suc2. Based on previous reported data, it could be deduced that both the activity and thermal stability of Suc2 are probably influenced by some key N-glycosylation sequons. Thus, totally 13 N-glycosylation residues of Suc2 were individually deglycosylated by site-directed mutagenesis. Fermentation results indicated that deglycosylation at N4, N78 and N146 sequons improved Suc2 activity and ethanol production of host strains, whereas deglycosylation at N45 showed an opposite effect. Carbohydrate depletion of Suc2 N4 especially endowed the host strain with better ethanol fermentation performance from inulin when the strain was cultured under the higher temperature for 48h, indicating that deglycosylation of N4 might improve the thermal stability of the Suc2. Carbohydrate chains at N4, N45, N78 and N146 played an important role in modulating Suc2 activity and inulin catabolism of the S. cerevisiae strain. These key N-deglycosylation sequons should become potential targets for rational engineering of S. cerevisiae strains to enhance the ethanol production from inulin.

Potential glycoengineered anti-Burkholderia vaccines by exploiting the bacterial O-glycosylation machinery

Previously, we discovered a protein O-glycosylation (ogc) cluster conserved in all Burkholderia species, which glycosylates proteins with a trisaccharide glycan. Sera from Burkholderia-infected patients produce anti-glycan antibodies, suggesting that the Burkholderia protein glycosylation pathway can be exploited for potential vaccine development. Here, we successfully produced two prototypes of anti-Burkholderia vaccines: a recombinant glycoprotein-based vaccine and an E. coli LPS-display vaccine. To generate the former, we constructed a plasmid carrying a chimeric gene encoding three glycosylation sequons fused to the cholera toxin B subunit. The presence of the glycan was observed in recombinant proteins expressed in B. cenocepacia parental strain, but not in proteins expressed by the glycosyltransferase-deficient ΔpglLstrain, as determined by SDS-PAGE and fluorescent lectin blots. For the development of an E. coli LPS-display vaccine, we constructed a plasmid expressing the ogc cluster, which was introduced into an E. coli strain unable to synthesize O-antigen but carrying the O-antigen ligase WaaL. Our results show that the LPS of this strain contained an additional moiety consistent with the B. cenocepacia trisaccharide glycan, as demonstrated by silver-stained LPS gels and lectin blot. This extra moiety was not detected in aΔwaaL mutant. These results suggest that the plasmid was able to provide the necessary functions for the synthesis and membrane translocation of the lipid-linked trisaccharide, which became a substrate for the WaaL ligase and incorporation into the E. coli LPS. Therefore, we demonstrate that the O-glycosylation pathway can be manipulated for the construction of potential anti-Burkholderia vaccines.

An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology Is Protective in an Inhalational Rat Model of Tularemia.

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

Loss and gain of N-linked glycosylation sequons due to single-nucleotide variation in cancer

Despite availability of sequence site-specific information resulting from years of sequencing and sequence feature curation, there have been few efforts to integrate and annotate this information. In this study, we update the number of human N-linked glycosylation sequons (NLGs), and we investigate cancer-relatedness of glycosylation-impacting somatic nonsynonymous single-nucleotide variation (nsSNV) by mapping human NLGs to cancer variation data and reporting the expected loss or gain of glycosylation sequon. We find 75.8% of all human proteins have at least one NLG for a total of 59,341 unique NLGs (includes predicted and experimentally validated). Only 27.4% of all NLGs are experimentally validated sites on 4,412 glycoproteins. With respect to cancer, 8,895 somatic-only nsSNVs abolish NLGs in 5,204 proteins and 12,939 somatic-only nsSNVs create NLGs in 7,356 proteins in cancer samples. nsSNVs causing loss of 24 NLGs on 23 glycoproteins and nsSNVs creating 41 NLGs on 40 glycoproteins are identified in three or more cancers. Of all identified cancer somatic variants causing potential loss or gain of glycosylation, only 36 have previously known disease associations. Although this work is computational, it builds on existing genomics and glycobiology research to promote identification and rank potential cancer nsSNV biomarkers for experimental validation.

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.

Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry

ABSTRACTThe study of influenza virus evolution in humans has revealed a significant role of glycosylation profile alterations in the viral glycoproteins – hemagglutinin (HA) and neuraminidase (NA), in the emergence of both seasonal and pandemic strains. Viral antigenic drift can modify the number and location of glycosylation sites, altering a wide range of biological activities and the antigenic properties of the strain. In view of the key role of glycans in determining antigenicity, elucidating the glycosylation profiles of influenza strains is a requirement towards the development of improved vaccines. Sequence-based analysis of viral RNA has provided great insight into the role of glycosite modifications in altering virulence and pathogenicity. Nonetheless, this sequence-based approach can only predict potential glycosylation sites. Due to experimental challenges, experimental confirmation of the occupation of predicted glycosylation sites has only been carried out for a few strains. Herein, we utilized HCD/CID-MS/MS tandem mass spectrometry to characterize the site-specific profile of HA of an egg-grown H1N1 reference strain (A/New Caledonia/20/1999). We confirmed experimentally the occupancy of glycosylation sites identified by primary sequence analysis and determined the heterogeneity of glycan structures. Four glycosylation sequons on the stalk region (N28, N40, N304 and N498) and four on the globular head (N71, N104, N142 and N177) of the protein are occupied. Our results revealed a broad glycan microheterogeneity, i.e., a great diversity of glycan compositions present on each glycosite. The present methodology can be applied to characterize other viruses, particularly different influenza strains, to better understand the impact of glycosylation on biological activities and aid the improvement of influenza vaccines.

Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction

Escherichia coli cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. coli is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto-induction procedures. To justify the potential for general application of the optimized auto-induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. coli cells.

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X1NX2(S/T), where X1,2 ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linked-glycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria.

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.

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.