Specificity and modularity of flagellin nonulosonic acid glycosyltransferases

Kint, Nicolas; Unay, Jovelyn; Viollier, Patrick H.

doi:10.1016/j.tim.2021.10.005

Cited by 7 publications

(15 citation statements)

References 14 publications

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“…Once flagellin subunits are translated, they are exported through the flagellar protein secretory apparatus along the hollow flagellar filament for polymerization at its growing tip. Glycosylation typically occurs post‐translationally on serine or threonine residues of flagellin by highly specific flagellin glycosyltransferases (fGTs; Szymanski & Wren, 2005; Kint et al , 2022). Unlike the pilus‐specific glycosyltransferases that execute the glycosylation only after the acceptor protein has been translocated across the cytoplasmic membrane (Valguarnera et al , 2016; Harding & Feldman, 2019), the fGTs are soluble enzymes that act on flagellin in the cytoplasm before their secretion through the flagellar apparatus.…”

Section: Introductionmentioning

confidence: 99%

Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

2023

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Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide‐activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O‐glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco‐profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin‐binding domain that identifies the acceptor.

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Section: Introductionmentioning

confidence: 99%

Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

2023

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“…The FlmG class of fGTs are suitable subjects for molecular dissection of the underlying specificity determinants because of their conspicuous two-domain architecture that is recognizable by simple primary structure (sequence) comparisons, even without tertiary structural analysis. In fact, the (predicted) bilobed FlmG structure [15] had previously prompted us to determine that the N-terminal TPR domain of FlmG Cc confers flagellin (acceptor) recognition, whereas the GT-B domain cannot bind flagellin [19]. Hypothesizing that the GT-B domain could act as determinant for the donor, we considered a simple division of labor model between the two parts of FlmG accounting for the bipartite specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Once flagellin subunits are translated, they are exported through the flagellar protein secretory apparatus along the hollow flagellar filament for polymerization at its growing tip. Glycosylation typically occurs post-translationally on serine or threonine residues of flagellin by highly specific and flagellin glycosyltransferases (fGTs)[15, 16]. Unlike the pilus-specific glycosyltransferases that execute the glycosylation only after the acceptor protein has been translocated across the membrane [17, 18], the fGTs are soluble enzymes that act on flagellin in the cytoplasm before their secretion through the flagellar apparatus.…”

Section: Introductionmentioning

confidence: 99%

“…Unlike the pilus-specific glycosyltransferases that execute the glycosylation only after the acceptor protein has been translocated across the membrane [17, 18], the fGTs are soluble enzymes that act on flagellin in the cytoplasm before their secretion through the flagellar apparatus. Two types of fGTs have been described to date, the Maf- and FlmG-type [15]. It is thought that these fGTs accept CMP-activated forms of Pse or Leg as glycosyl donors and then join the Pse or Leg moiety to the flagellin acceptor molecule that they bind directly.…”

Section: Introductionmentioning

confidence: 99%

“…Conversely, expression of FlmG and the FljK flagellin in heterologous hosts producing Pse resulted in FljK modification [19]. Sequence analysis predicts a simple 2-domain organization for FlmG: an N-terminal tetratrico-peptide-repeat (TPR) domain and a C-terminal GT-B type glycosyltransferase domain [15]. Bacterial-two-hybrid (BACTH) assays revealed that the TPR domain can directly bind flagellin, whereas the GT-B domain cannot [19].…”

Section: Introductionmentioning

confidence: 99%

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Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

Kint

Dubois

Viollier

2022

Preprint

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Glycosylation of surface structures diversifies cells chemically and physically. Sialic acids commonly serve as glycosyl donors, particularly pseudaminic (Pse) or legionaminic acid (Leg) that prominently decorate eubacterial and archaeal surface layers or appendages. We investigated a new class of FlmG protein glycosyltransferases that modify flagellin, the structural subunit of the flagellar filament. Functional insulation of orthologous Pse and Leg biosynthesis pathways accounted for the flagellin glycosylation specificity and motility conferred by the cognate FlmG in the α-proteobacteria Caulobacter crescentus and Brevundimonas subvibrioides, respectively. Exploiting these functions, we conducted genetic glyco-profiling to classify Pse or Leg biosynthesis pathways and we used heterologous reconstitution experiments to unearth a signature determinant of Leg biosynthesis in eubacteria and archaea. These findings and our chimeric FlmG analyses reveal two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that accepts either Leg or Pse and that uses specialized flagellin-binding domain to identify the substrate.

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Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae

Hadjineophytou,

Loh,

Koomey

et al. 2024

Proteomics

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Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O‐linked protein glycosylation is conserved yet closely related Neisseria species express O‐oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data‐Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus‐level protein glycosylation.

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Specificity and modularity of flagellin nonulosonic acid glycosyltransferases

Cited by 7 publications

References 14 publications

Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

Stereoisomer‐specific reprogramming of a bacterial flagellin sialyltransferase

Genetic glyco-profiling and rewiring of insulated flagellin glycosylation pathways

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae

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