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ProGlyProt ID
BC167
Organism Information
Organism Name
Pseudomonas aeruginosa Pa5196
Domain
Bacteria
Classification
Family: Pseudomonadaceae
Order: Pseudomonadales
Class: Gammaproteobacteria
Division or phylum: "Proteobacteria"
Taxonomic ID (NCBI)
 
 
Genome Sequence(s)
EMBL
Organism Additional Information
This Gram-negative opportunistic pathogen is responsible for nosocomial pneumonia. It possesses a multitude of virulence factors including type IV pili that mediate adhesion to host cells. It is also the major cause of mortality among cystic fibrosis (CF) patients.
 
 
Gene Information
Gene Name
pilA
 
 
Protein Information
Protein Name
PilA (Type IV pilin)
UniProtKB/SwissProt ID
EMBL-CDS
UniProtKB Sequence
Sequence length
155 AA
Subcellular Location
Surface
Function
Structural unit of the pili that have a role in adherence (colonization of both living and non-living surfaces) and twitching motility. These colonization factors (type IV pili) are also involved in biofilm formation.
 
 
Protein Structure
Homology Model
Homology Model File
 
 
Glycosylation Status
Glycosylation Type
O (Ser/Thr) linked
Experimentally Validated Glycosite(s) in Full Length Protein
(Propeptide: 1-6) T70, T72, S87, S88, S91, S95
Experimentally Validated Glycosite(s ) in Mature Protein
T64, T66, S81, S82, S85, S89
Glycosite(s) Annotated Protein Sequence
>tr|Q8KQ32|Q8KQ32_PSEAE Type IV pilin structural subunit OS=Pseudomonas aerugino
sa GN=pilA PE=3 SV=1 MKAQKGFTLIELMIVVAIIGILAAVAIPAYQDYITRGQVTEAVSLGGGLKSPLA
EYGADK NAWPTLVAPT*(70)AT*(72)PGAGQLNATLVGKYS*(87)S*(88)VDS*(91)TIA
S*(95)
GYPNGQITVTMTQGKASGKKLTFST QDGGSSWACGNASIDGFAGTGTTIDAKYLPNACKP
Sequence Around Glycosites (21 AA)
KNAWPTLVAPTATPGAGQLNA
AWPTLVAPTATPGAGQLNATL
QLNATLVGKYSSVDSTIASGY
LNATLVGKYSSVDSTIASGYP
TLVGKYSSVDSTIASGYPNGQ
KYSSVDSTIASGYPNGQITVT
Glycosite Sequence Logo
Glycosite Sequence Logo
Technique(s) used for Glycosylation Detection
Slower migration on SDS-PAGE than its predicted mass and staining with GlycoProfile III fluorescent-glycoprotein detection kit
Technique(s) used for Glycosylated Residue(s) Detection
Validation of T70 and T72 glycosylated sites using site-directed mutagenesis and ETD-MS (electron transfer dissociation mass spectrometry). Rest of the sites were determined by a combination of nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) and nESI-MS/MS with front-end-collision induced dissociation (nESI-feCID-MS/MS).
Protein Glycosylation- Implication
Modifications of αβ-loop residues Thr64 and Thr66 are important for normal pilus assembly or extension/retraction dynamics. The Thr64Ala and Thr66Ala single and double mutants, as well as the nonglycosylated tfpW mutant, had fewer surface pili and reduced motility compared with the wild type.
 
 
Glycan Information
Glycan Annotation
Linkages: FucNAc-Ser.
Unusual homooligomers of α-1,5-linked D-arabinofuranose (α-1,5-D-Araf). Oligomer length varies from 3 to 8 units with an average length of 6 units. Trisaccharides of α-1,5-D-Araf are the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues.
The pilin glycan is antigenically and chemically identical to that of Mycobacterium.
Microheterogeneity in glycosylation is observed.
BCSDB ID
Technique(s) used for Glycan Identification
NMR spectroscopy including 1H-13C HSQC (heteronuclear single-quantum coherence) and 1H-13C HMBC (heteronuclear multiple bond coherence) and, chirality of glycans identified by GC-MS (gas chromatography-mass spectrometry).
 
 
Protein Glycosylation linked (PGL) gene(s)
OST Gene Name
tfpW
OST Protein Name
TfpW (arabinosyltransferase)
OST UniProtKB/ SwissProt ID
OST EMBL-CDS
OST UniProtKB Sequence
 
 
Literature
Reference(s)
1) Kus, J.V., Kelly, J., Tessier, L., Harvey, H., Cvitkovitch, D.G. and Burrows, L.L. (2008) Modification of Pseudomonas aeruginosa Pa5196 type IV Pilins at multiple sites with D-Araf by a novel GT-C family Arabinosyltransferase, TfpW. J Bacteriol, 190, 7464-7478. [PubMed: 18805982]
2) Voisin, S., Kus, J.V., Houliston, S., St-Michael, F., Watson, D., Cvitkovitch, D.G., Kelly, J., Brisson, J.R. and Burrows, L.L. (2007) Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like alpha-1,5-linked d-Araf oligosaccharides. J Bacteriol, 189, 151-159. [PubMed: 17085575]
3) Kus, J.V., Tullis, E., Cvitkovitch, D.G. and Burrows, L.L. (2004) Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology, 150, 1315-1326. [PubMed: 15133094]
Additional Comments
It is the first report where ETD-MS has been used for bacetrial glycoprotein characterization.
It is also the first report of glycosylation of a bacterial protein with a homooligosaccharide.
Group I P. aeruginosa pilins are glycosylated on a C-terminal Ser residue unlike Neisseria pilin which is glycosylated at a flexible-loop region within the globular domain of the protein.
TfpW (arabinosyltransferase) is the member of the GT-C glycosyltransferase family. Its role was determined by knockout studies. It has got a restricted pilin substrate specificity.
D-Araf is uncommon in prokaryotes. It is mainly present in the arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacterial cell wall including those of M. tuberculosis and M. leprae.
Year of Identification
2004
Year of Validation
2007
 
 
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