Structural determinants for N-linked glycosylation

Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acids that make up 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-X1-N-X2-S/T, where X1,2 ≠ P) 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, while structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glycocompetent 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 (Sec). Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linked glycosylated prior to complete folding and suggest that PglB activity is coupled to Sec-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria. N-linked protein glycosylation is a ubiquitous post-translational modification that influences protein stability, folding and host-cell interactions (1-3). In eukaryotes, this phenomenon occurs at the rough endoplasmic reticulum (RER) membrane. Here, glycans are assembled onto a polyprenol-diphosphate-linked substrate on the cytoplasmic face of the RER membrane and subsequently flipped to the RER luminal face, where the glycans are further extended prior to transfer to an asparagine residue within a glycosylation consensus sequence by the oligosaccharyltransferase (OTase) (4,5). In mammalian cells, N-linked glycosylation is mediated by two hetero-oligomeric OTase complexes that include either the STT3A or STT3B isoform of the catalytic subunit that have distinct functions (6). The mammalian STT3A is found in a complex that associates with the general secretion (Sec) pathway, coordinating cotranslational glycosylation of nascent proteins entering the RER. This macromolecular association is mediated by direct interactions between subunits of the Sec61 translocation http://www.jbc.org/cgi/doi/10.1074/jbc.M116.747121 The latest version is at JBC Papers in Press. Published on August 29, 2016 as Manuscript M116.747121 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 0, 2017 hp://w w w .jb.org/ D ow nladed from Structural determinants for N-linked glycosylation 2 machinery and the OTase subunits (7-9). The STT3B isoform is part of a distinct OTase complex in the RER that is not associated with the Sec translocon, and rather glycosylates sites missed by STT3A in a post-translational and/or post-translocational manner (10,11). Interestingly, in Less is known about the more recently discovered N-linked protein glycosylation pathway of deltaand epsilonproteobacteria (12). To date, the best characterized bacterial N-linked glycosylation system is the Pgl (Protein glycosylation) pathway of Campylobacter jejuni (13,14). C. jejuni is part of the natural gut flora of poultry and cattle; however, it is a human pathogen and one of the leading causes of bacterial gastroenteritis worldwide (15). Studies have shown that protein N-linked glycosylation plays a role in pathogenicity, as this pathway is important for colonization of the gastrointestinal tract of chickens and mice (16,17). The glycosylation process in C. jejuni begins at the cytoplasmic face of the inner membrane, where the glycan, a branched heptasaccharide, is assembled by stepwise addition of monosaccharides onto an undecaprenoldiphosphate (Und-PP) moiety and then flipped to the periplasmic face of the inner membrane. In the periplasm, the heptasaccharide is transferred en bloc to an amide nitrogen of an asparagine located within a consensus sequence (D/E-X1-N-X2-S/T, X1,2 ≠ proline) on an acceptor protein by a single integral membrane oligosaccharyltransferase (OTase), PglB, that is homologous to the catalytic subunit of eukaryotic OTases (STT3) (13,18) (Fig. 1). The enzymes that biosynthesize and transfer this heptasaccharide are chromosomally encoded within a gene locus (pgl locus). Notably, heterologous glycosylation of selected target proteins can be achieved by expression of the pgl locus in Escherichia coli (glycocompetent E. coli) (19). To date, over 60 proteins from C. jejuni have been demonstrated to be N-glycosylated and many of these have predicted Sec signal peptides (20). Previous studies have demonstrated that PglB is capable of glycosylating both folded and unfolded proteins in vitro and in glycocompetent E. coli. For example, AcrA (Cj0367c), a component of the AcrABC (Cj0365-7c) multidrug efflux system of C. jejuni, is a native glycoprotein that can be Nglycosylated in vitro or in glycocompetent E. coli when it is exported to the periplasm through the Sec pathway, which transports proteins in an unfolded conformation, or though the Twin arginine translocation pathway (TAT), which transports fully folded proteins (21,22). Furthermore, Fisher and colleagues demonstrated that in glycocompetent E. coli, proteins carrying engineered glycosylation tags are glycosylated through diverse export pathways (Sec, TAT or SRP (signal recognition particle)), although variations in glycosylation efficiencies were observed (23). These studies show that N-linked glycosylation is compatible with diverse secretory pathways; however, the native spatial and temporal aspects of N-linked glycosylation in the context of bacterial cells remain unclear. Specifically, it is not known whether Nglycosylation and protein translocation are coupled, as they are in eukaryotes. In this work, we used complementary approaches to determine how the structural context of N-linked glycosylation substrates alters glycosylation efficiency. In silico evaluation of Nlinked glycosylation sequons within C. jejuni glycoproteins was performed using structural homology modeling. This analysis showed that N-linked glycosylation sites are predicted to adopt diverse secondary structures. Next, a detailed investigation was performed on a C. jejuni glycoprotein, PEB3 (Cj0289c), whose X-ray crystallographic structure was previously determined (24,25). Using in vitro site-directed mutagenesis, we introduced N-linked glycosylation sequons into selected positions in the sequence of PEB3, and examined the relative glycosylation efficiency at each of these sites. We found that these new glycosylation sites were susceptible to increased levels of modification compared to the wild type protein, which was likely due to higher accessibility of the glycosylation site and partial destabilization of the protein. We also investigated whether the mode of protein substrate translocation of glycosylation substrates into the periplasm had an effect on glycosylation efficiencies. Using a glycocompetent E. coli strain, we found that Sectranslocation of AcrA and PEB3 is important for efficient glycosylation. Moreover, we show for the first time that in the native system, C. jejuni, efficient glycosylation requires the delivery of by gest on N ovem er 0, 2017 hp://w w w .jb.org/ D ow nladed from Structural determinants for N-linked glycosylation 3 protein substrates by the Sec pathway. In light of these findings and the observation that a subset of native glycoproteins contains sequons in structured regions, we postulate that bacterial protein N-linked glycosylation of selected protein substrates is likely coupled to protein translocation, as it is in eukaryotes. RESULTS In silico analysis of glycosylation sites–– Previously, a survey of eukaryotic N-linked glycoproteins structures (X-ray crystallographic data) revealed that N-glycosylation sequons adopt diverse secondary structure conformations (26). Nglycosylation at these sites is possible because glycosylation occurs prior to folding in eukaryotes (27,28). While over 60 glycoproteins have been identified from C. jejuni and Campylobacter lari, only four X-ray crystallographic structures (CjaA, PEB3, JlpA and PglB), and one NMR structure of truncated AcrA have been determined (24,25,2932). Analysis of the four known X-ray crystal structures shows that the bacterial glycosylation consensus sequence also adopts various conformational states (Fig. 2A). These four structures include five solvent-exposed glycosylation sites: one in an a-helix, one in a structured turn between a b-strand and a-helix, and three non-structured loops. Due to the limited number of X-ray crystallographic structures of known C. jejuni glycoproteins, we generated protein models in order to obtain a broader understanding of the conformations of glycosylation sequons in C. jejuni. Using Phyre2 (Protein homology/analogy recognition engine 2) we generated three-dimensional structural models of experimentally determined C. jejuni glycoproteins (33). A total of 35 high confidence models containing 53 glycosylation sequons were obtained and the secondary structure of each residue within the experimentally determined glycosylation sequon was categorized as a-helix, b-strand, structured turn (b, g) or non-structured loop (Fig. 2B and Table S1). In this study, we define a non-structured loop as a region that has more than five residues that are not appropriately positioned to form backbone hydrogen bonding interactions. All residues within glycosylation sequons were predicted to be surface exposed. Interestingly, >50% were found in predicted ahelices, b-strands or turns, while the remaining were found in larger non-structured loops. These analyses highlight the potential structural diversity of bacterial glycosylation sites. In the context of a folded protein, these conformations could interfere with N-glycosylation activity by PglB. Thus, we hypothesized that native glycosylation sites of C. jejuni proteins located in structurally defined regions are poor substrates of PglB, and that partial unfolding is required for glycosylation to occur at these sites. In vitro, PEB3 is a poor glycosylation substrate––To evaluate whether PglB shows a preference for folded or unfolded protein targets, glycosylation efficiencies were evaluated using a purified target substrate. PEB3, a C. jejuni homodimeric surface-associated protein with immunogenic properties and predicted transporter functions (25,34), was selected for these studies because it is a reported soluble glycoprotein and the X-ray crystallographic structure was previously determined, which shows the native glycosylation site in a structurally defined region of the protein (24,25) (Fig. 2A). Relative levels of N-glycosylated PEB3 appear to be straindependent. In C. jejuni 11168, PEB3 is reported to be approximately 50% N-glycosylated (35). In this study, we used C. jejuni 81-176, wherein 80-100% of PEB3 is N-glycosylated. Based on X-ray crystallographic analysis, the glycosylation site of PEB3 is located in a structured exposed loop between a b-strand and a-helix (24,25). Glycosylation efficiencies of purified PEB3, compared to a peptide representing the glycosylation sequon, were assessed using an in vitro radioactivity-based glycosylation assay with a readily accepted tritiated disaccharide donor (Und-PP-diNAcBac-[H]-GalNAc) (36). Purified PglB and glycan donor were incubated with purified PEB3 wild type, a non-glycosylatable mutant of PEB3 (N90Q) or a peptide containing the glycosylation consensus sequence. Glycosylated products were separated from glycan donor using Ni-NTA chromatography and analyzed by liquid scintillation counting to determine the percent of disaccharide incorporated onto the protein. N-glycosylation of the peptide (~87%) far exceeded that of wild type PEB3 (~1%) (Fig. 3A). Though PEB3 wild type exhibited very low levels of N-glycosylation, the by gest on N ovem er 0, 2017 hp://w w w .jb.org/ D ow nladed from Structural determinants for N-linked glycosylation 4 extent was reproducibly higher than background levels of the assay, as determined using PEB3 N90Q. These data show that while PEB3 is an Nglycosylation substrate in vivo, it is not robustly modified in this in vitro glycosylation assay, suggesting that partial destabilization or unfolding is required for efficient glycosylation in the cell. In vitro, partial destabilization of PEB3 increases glycosylation efficiency––To investigate the structural determinants of protein substrates contributing to N-glycosylation, we determined the glycosylation efficiencies of PEB3 variants with repositioned glycosylation sites. The X-ray structure of PEB3 guided the selection of five sites for integration of the glycosylation consensus sequence (Fig. 3B, Table 1). To avoid intrinsic variation of the local sequence, which has been shown to effect glycosylation efficiencies in eukaryotes (37) and prokaryotes (38), we integrated the same, native PEB3 sequence at each selected site (DFNVS). Two sites are located in predicted flexible loops (A179N and I199N) and the other three are found in a structured helix-turnhelix (Q152N), a partially buried loop between an a-helix and b-strand (D68N) and a buried bstrand (G72N). For simplicity, and to ensure incorporation of a single glycan, consensus sequences were introduced by site-directed mutagenesis in a PEB3 mutant lacking the native glycosylated asparagine residue (N90Q). Purification by Ni-NTA chromatography, followed by size-exclusion chromatography confirmed that all PEB3 variants were soluble and monodisperse (Fig. S1). In vitro end-point glycosylation assays with Und-PP-diNAcBac-[H]-GalNAc were performed with the panel of PEB3 variants. Compared to wild type, an increase in the level of the Nglycoprotein was observed in four of the five variants, with the most efficient incorporation into D68N, Q152N and A179N, while only background levels were observed for the fully buried site at G72N (Fig. 3A). Despite the enhanced levels of N-glycosylation for several PEB3 variants (5-15%), they did not achieve Nglycosylation levels comparable to the peptide (87%). The observed increase in glycosylation at these sites may be attributed to increased flexibility due to destabilization of the native fold. To determine the extent of destabilization, average melting temperature (Tm) values of PEB3 variants were measured by thermofluor analysis using SYPRO Orange (39). The average Tm values of all variants were lower (4-5oC) than the wild type, suggesting that these variants were structurally less stable (Fig. 3C and 3D). Together these data confirm that buried glycosylation sites are not targeted by PglB in vitro. The results also suggest that conformational changes to the native glycosylation sites may be necessary to allow for efficient modification. Efficient N-linked glycosylation is dependent on Sec-translocation in glycocompetent E. coli–– While N-linked glycosylation of PEB3 in C. jejuni has been experimentally validated (35), our data indicate that it is a very poor substrate under in vitro conditions. Several factors may contribute to this observation. First, the native glycosylation consensus sequence is located between two defined elements of secondary structure, affording rigidity that may decrease the accessibility of the glycosylation site to PglB. Second, the folded state (local or global) of PEB3 during glycosylation in vivo, which is currently unknown, may impose constraints that preclude efficient glycosylation. Based on the signal sequence prediction software (PRED-TAT), PEB3 is predicted to be delivered to the periplasm via the Sec export pathway, wherein proteins are translocated to the periplasm in an unfolded conformation and fold during or after translocation (40). We postulated that the poor in vitro N-glycosylation levels of wild type PEB3 is due to its folded state and that N-linked glycosylation occurs prior to, or during, folding, either coor post-translocationally. To better understand the dynamics of in vivo glycosylation, we evaluated the glycosylation efficiency of PEB3 when it was translocated via the Sec pathway or through an alternative translocation pathway, the TAT pathway, which transports fully folded proteins and oligomeric protein complexes across the inner membrane into the periplasm (41,42). This experiment helps to evaluate the importance of environmental factors that differ between the in vitro glycosylation assay and the native periplasmic space. This approach was previously used by Kowarik and colleagues to demonstrate that glycosylation of AcrA, which has two glycosylation sites, can occur on folded or by gest on N ovem er 0, 2017 hp://w w w .jb.org/ D ow nladed from Structural determinants for N-linked glycosylation 5 unfolded proteins in glycocompetent E. coli (21). Currently, an X-ray crystallographic structure of full-length AcrA has not yet been determined; however, homology modeling suggests that one glycosylation site is in an a-helix, and the other is in a flexible loop (18). For the current study, we used densitometry analysis to compare relative glycosylation efficiencies of two proteins, PEB3 and AcrA, when they are transported through the Sec and TAT pathways in glycocompetent E. coli. A soluble periplasmic variant of AcrA, which is a native lipoprotein of C. jejuni, was previously produced in E. coli by substituting the native Sec signal sequence of AcrA, including the lipidation site, with the Sec signal sequence of pelB (21). The same strategy was employed for the current experiments. The Sec signal sequence of pelB and TAT signal sequence of ycbk were substituted for the native signal sequence of AcrA and a Cterminal hexahistidine (His6x) tag was added. Both signal sequences are known to direct translocation through the designated machinery (43,44). PEB3 contains a predicted native Sec signal sequence, thus, it was kept intact for Secmediated translocation and was substituted with the ycbk signal sequence for rerouting through that TAT pathway. Each construct was co-expressed in E. coli W3110 together with the pgl locus. Target proteins purified from the periplasmic fraction were analyzed by SDS-PAGE and immunoblotting with anti-His antibody, for detection of both nonglycosylated and glycosylated protein, and anti-Nglycan antibody (R1), which recognizes the C. jejuni heptasaccharide, for detection of glycosylated protein (45). Both glycosylation sites on AcrA were modified as noted by two higher molecular weight bands that reacted with anti-Nglycan antibodies corresponding to mono and diglycosylated AcrA (Figs. 4A and S2A). Previous studies have confirmed these shifts by mass spectrometry (21). Analysis by densitometry indicated a modest, but significant increase in total di-glycosylated Sec-translocated AcrA (19±3%), compared to TAT-translocated AcrA (11±2%) (Fig. 4B). Of note, the slight difference in molecular weights between non-glycosylated Sec and TAT translocated AcrA are due to a linker that was included when constructing the plasmid containing pelB. More strikingly, glycosylation efficiencies of PEB3 were significantly higher for Sectranslocated PEB3 (23±3%), compared to TAT translocated PEB3 (6±1%) (Figs. 4C, 4D and S2B). These results indicate that glycosylation in glycocompetent E. coli is partially dependent on the Sec translocation pathway. Consistent with previous studies in glycocompetent E. coli (19,46), total glycosylation levels of AcrA (50±2%) and PEB3 (23±3%) are low. Several known factors contribute to inefficient glycosylation in glycocompetent E. coli. For example, WecA, a priming glycosyltransferase, can interfere with Und-PPheptasaccharide production by transferring GlcNAc-P from UDP-GlcNAc onto the Und-P carrier thereby providing a non-native substrate for the Pgl pathway, rather than the native Und-PPdiNAcBac (19,47). In addition, in E. coli PglB can compete with WaaL, an O-antigen ligase, for substrates by incorporating O-antigen subunits onto acceptor proteins (48). Efforts to improve glycosylation efficiencies by targeting these enzymes, as well as controlling carbon flux, in E. coli have resulted in only modest increases in Nglycoprotein yields (49,50). In addition to these limitations, we propose that interactions between PglB and its natively-partnered translocon may be lost in E. coli, thus uncoupling protein translocation from N-glycosylation in the periplasm. To address this, we examined the contribution of Sec translocation to Nglycosylation in the native host, C. jejuni. Efficient N-linked glycosylation is dependent on Sec-translocation in C. jejuni––To direct translocation through the Sec or TAT pathways of C. jejuni, expression vectors encoding acrA and peb3 with the Sec or TAT signal sequences were generated. For Sec-mediated translocation the pelB signal sequence from E. coli was used for acrA, and the native signal sequence was used for peb3. For TAT-mediated translocation, acrA and peb3 were modified with the TAT signal sequence from C. jejuni torA (cj0264c), which was previously determined to be a TAT transported protein (51). Analysis of periplasmic purified AcrA demonstrated overall higher levels of N-linked glycosylation for both Sec and TAT translocated protein (91±7% and 81±5%, respectively) compared to the levels observed in glycocompetent E. coli (Figs. 5A, 5B and S2C). While there was no significant difference in total by gest on N ovem er 0, 2017 hp://w w w .jb.org/ D ow nladed from Structural determinants for N-linked glycosylation 6 glycosylation, we observed a statistically significant increase in di-glycosylated AcrA for Sec-dependent translocation, consistent with the glycocompetent E. coli results (Sec: 74±10%, TAT: 35±2%). N-linked glycosylation in C. jejuni was markedly higher for Sec translocated PEB3 (89±9%) than for TAT translocated PEB3 (6±3%) (Figs. 5C, 5D, S2D). These results in C. jejuni are consistent with those in glycocompetent E. coli, although the relative levels of glycosylation differ between the two system. These data support the hypothesis that PglB specificity depends on the substrate protein and the conformation of the glycosylation sequon that is presented as proteins are delivered to the periplasm. To further validate these findings, and to determine whether the correlation between in vitro and in vivo glycosylation efficiencies are consistent, we assessed glycosylation in C. jejuni of the aforementioned PEB3 variant (A179N) that was shown to be N-linked glycosylated more efficiently compared to wild type PEB3 in vitro (Fig. 3A). When PEB3 A179N was expressed with the native Sec signal sequence or TAT signal sequence in C. jejuni, the protein was fully glycosylated for both conditions, confirming the hypothesis that the structural context influences glycosylation efficiency (Figs. 6 and S2E). Together with the results from native PEB3, these findings suggest that coupling with the Sec translocation pathway will be useful for efficient glycosylation of proteins wherein glycosylation sites in the native target are predicted to be structurally defined.