Flagellin from<i>Listeria monocytogenes</i>Is Glycosylated with β-O-Linked<i>N</i>-Acetylglucosamine

Schirm, Michael; Kalmokoff, Martin; Aubry, Annie; Thibault, Pierre; Sandoz, Mildred; Logan, Susan M.

doi:10.1128/jb.186.20.6721-6727.2004

Cited by 102 publications

(110 citation statements)

References 49 publications

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confidence: 99%

“…This post-translational modification is also found on purified S-layer protein from this organism. This is in marked contrast to recent studies of bacterial flagellin glycosylation in Campylobacter, Helicobacter, Pseudomonas, and Listeria where each unique glycan was shown to be O-linked to the respective flagellin proteins (12)(13)(14)(15).Although the M. voltae flagellins did not react with glycoprotein-specific stains, these proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDSpolyacrylamide gel suggesting significant post-translational modification. In this study we provide a detailed structural characterization of the unique glycan found on all of the M. voltae flagellin proteins and demonstrate that the S-layer protein is also post-translationally modified with the same novel trisaccharide.…”

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Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

Voisin

Houliston

Kelly

et al. 2005

Journal of Biological Chemistry

131

144

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The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant posttranslational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel Nlinked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of ␤-ManpNAcA6Thr-(1-4)-␤-GlcpNAc3NAcA-(1-3)-␤-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway.Glycosylation of prokaryotic proteins is now well accepted, and examples of N-and O-glycosylation and of attachment of glycosylphosphatidylinositol anchors can now be found in the literature (1, 2). However, in contrast to eukaryotic glycosylation systems, prokaryotic systems display considerable diversity in the structure of the respective glycans and the proximal monosaccharide linkage. As a consequence there is considerable interest in determining the structural and genetic basis of glycan production among these diverse prokaryotic systems.The archaeal flagellum is a unique motility structure that is distinct from the well characterized bacterial flagellum (3). In contrast to bacterial flagellar assembly where newly synthesized flagellin is incorporated at the distal tip of the filament, it is believed that mature archaeal flagellin is incorporated at the base of the filament. In recent studies, the assembly of archaeal flagellum has been shown to more closely resemble a second bacterial motility system, the type IV pilus, where the structural protein pilin is synthesized with an unusual signal peptide and a hydrophobic N terminus. In Archaea, signal peptidases have been shown to cleave a signal peptide of the preflagellin proteins to produce mature flagellin, which is then incorporated into the filament (4). Flagellated archaeal species have one to five flagellin genes organized into a fla locus (3). The marine archaeon Methanococcus voltae has four flagellin structural genes organized in two transcriptional units: one unit contains flaA, whereas the second unit contains flaB1, flaB...

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confidence: 97%

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

Voisin

Houliston

Kelly

et al. 2005

Journal of Biological Chemistry

131

144

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“…The studies have revealed significant diversity in the composition of the attached glycans. The flagellins of C. jejuni and H. pylori are glycosylated with pseudaminic acid (5,7-diacetamideo-3,5,7,9,-tetradeoxy-L-glycero-L-mannononulosonic acid) and related derivatives, whereas Listeria monocytogenes flagellin is glycosylated with GlcNAc, and P. aeruginosa type-a flagellin is glycosylated with a rhamnose-linked complex glycan (37,(42)(43)(44). More recently, flagellin of the plant pathogenic bacteria P. syringae pv.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation Regulates Specific Induction of Rice Immune Responses by Acidovorax avenae Flagellin

Hikita

Takai

Iwano

et al. 2011

Journal of Biological Chemistry

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Plants have a sensitive system that detects various pathogenderived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H 2 O 2 generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. During development, plants are continuously confronted with diverse pathogens. However, plants are resistant to most microbes and rely entirely on plant immune responses for their defense. Plants have evolved a multilayered defense system that can be activated upon pathogen invasion. The first layer recognizes conserved microbial molecules, referred to as microbeassociated molecular patterns, via pattern recognition receptors (1, 2). Microbe-associated molecular pattern-triggered immunity is key to plant innate immunity (3). Successful pathogens can deliver effectors that suppress these immune responses and contribute to pathogen virulence (4). Another layer recognizes pathogen effector molecules through host resistance genes, triggering a rapid defense response that often includes a localized programmed cell death reaction known as the hypersensitive response (5-7).Microbe-associated molecular patterns include structures characteristic of pathogens, such as ␤-glucan, polysaccharide chitin, ergosterol, lipopolysaccharides (LPS), flagellin, and elongation factor Tu (8 -13). Among these microbe-associated molecular patterns, flagellin, a main component of the bacterial flagellum, has been the most extensively studied in regard to the recognition mechanism and signal transduction. Arabidopsis recognizes the most conserved N-terminal domain of flagellin that consists of a 22-amino acid peptide (flg22) 2 (12). Recognition of this elicitor-active domain depends on flagellin sensing 2 (FLS2) (14). FLS2 encodes a receptor-like kinase composed of an extracellular leucine-rich repeat, a single membrane-spanning domain, and a cytoplasmic serine/threonine kinase domain. FLS2 and flg22 were shown to physically interact by chemical cross-linking and immunoprecipitation studies, suggesting that FLS2 determines the specificity in recognizing flagellin (15).Acidovorax avenae is a Gram-negative bacterium that causes a seedling disease that is characterized by the formation of brown stripes on the sheaths of infected plants. A. avenae has a wide host range among monocotyledonous plants; however, individual strains of this pathogen infect only one or a few host species (16). For example, strains isolated from rice, such as K1 and H8301, can infect only rice plants (virulent), whereas the N1141 strain isolated from finger millet cannot infect rice even after it is inoculated into rice tissues (avirulent). ...

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“…Flagellin subunits are covalently modified by monomeric -O-linked N-acetylglucosamine (GlcNAc) residues at three to six sites per subunit (Schirm et al, 2004b). The functional consequence of flagellin glycosylation in L. monocytogenes was investigated by modification of the O-GlcNAc transferase (Lmo0688 renamed to GmaR), which is located just upstream of the flagellin gene (Shen et al, 2006).…”

Section: Listeria Monocytogenesmentioning

confidence: 99%

Flagellar Glycosylation: Current Advances

Hayakawa¹,

Ishizuka²

2012

Glycosylation

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Flagellin fromListeria monocytogenesIs Glycosylated with β-O-LinkedN-Acetylglucosamine

Cited by 102 publications

References 49 publications

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

Glycosylation Regulates Specific Induction of Rice Immune Responses by Acidovorax avenae Flagellin

Flagellar Glycosylation: Current Advances

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