Mechanism of Bacterial Oligosaccharyltransferase

Gerber, Sabina; Lizak, Christian; Michaud, Gaëlle; Bucher, Monika; Darbre, Tamis; Aebi, Markus; Reymond, Jean‐Louis; Locher, Kaspar P.

doi:10.1074/jbc.m112.445940

Cited by 80 publications

(65 citation statements)

References 57 publications

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“…A Cl PglB variant in which the native Q330/R331 residues were rationally replaced with DL glycosylated an AQNAT sequon as efficiently as the Cj PglB DL variant and retained highly efficient glycosylation of a DQNAT sequon. This is in stark contrast to studies where a Cl PglB R331A mutant generates only a very low level of AQNAT glycosylation and significantly reduces glycosylation of a DQNAT sequon 31 . Our results revealed that the adjacent Q330 (R327 in Cj PglB) residue plays an important role in regulating site selection along with R331.…”

Section: Discussioncontrasting

confidence: 86%

“…In stark contrast, the relaxation of our mutants was much more general and potentially more useful for glycoengineering as demonstrated by glycosylation of RNaseA at its native N-X-S/T acceptor site. Recent efforts to further relax the specificity of Cl PglB by swapping the charged residues between the bacterial OST and acceptor peptide resulted in no apparent glycosylation of an RQNAT sequon by Cl PglB R331D/E mutants in vivo 31 . Here, the application of glycoSNAP resulted in a unique instance of charge inversion involving R327 of Cj PglB, where 3 of the 5 sequons most efficiently glycosylated by the DL variant contained Arg in the −2 position.…”

Section: Discussionmentioning

confidence: 99%

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Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

et al. 2014

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The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-glycosylation. This machinery has been functionally transferred into Escherichia coli, thereby enabling convenient mechanistic dissection of the N-glycosylation process in this genetically tractable host. Here, we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the −2 position relative to asparagine. This involved creation of a genetic assay named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that were capable of glycosylating an array of noncanonical acceptor sequences including one in a eukaryotic N-glycoprotein. Collectively, these results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-glycosylation and for engineering designer N-glycosylation biocatalysts.

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Section: Discussioncontrasting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

et al. 2014

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“…This could result from NXT providing a more optimal conformation of the acceptor sequence, with increased nucleophilicity of the asparagine amide group [91]. Furthermore, kinetics studies have demonstrated that the eukaryotic oligosaccharyltransferase enzyme has a higher affinity for NXT sites compared to NXS [95,96]. NXT motifs are also positively selected for in adjacent sequons, and are more likely than NXS to be glycosylated in this setting [97].…”

Section: Discussionmentioning

confidence: 99%

Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth

Smith

Burton

Kilembe³

et al. 2016

PLoS Pathog

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A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

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“…In addition to the structural analysis of PglB from C. lari (PglB Cl ) with a bound substrate peptide, extensive bioinformatics and biochemical studies have provided insight into optimized peptide substrate determinants, 25 the corresponding peptide binding site and potential mechanistic models for asparagine glycosylation. 26 Additionally, computational approaches have been applied to predict the structure of the undecaprenol-diphosphate-heptasaccharide (Und-PP-heptasaccharide) in complex with PglB. 27 …”

Section: Introductionmentioning

confidence: 99%

Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer

2016

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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 C. 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 tags (LBTs), and LRET between the luminescent (LBT)-Tb3+ 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, OTase.

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Mechanism of Bacterial Oligosaccharyltransferase

Cited by 80 publications

References 57 publications

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth

Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer

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