ProGP231

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ProGP ID ProGP231
Validation Status Characterized
Organism Information
Organism NamePseudomonas aeruginosa Pa5196
Domain Bacteria
Classification Family: Pseudomonadaceae
Order: Pseudomonadales
Class: Gammaproteobacteria
Division or phylum: "Proteobacteria"
Taxonomic ID (NCBI) 287
Genome Sequence(s)
EMBL AY112720
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 NamepilA
Protein Information
Protein NamePilA (Type IV pilin)
UniProtKB/SwissProt ID Q8KQ32
EMBL-CDSAAM52059.1
UniProtKB Sequence >tr|Q8KQ32|Q8KQ32_PSEAE Type IV pilin structural subunit OS=Pseudomonas aeruginosa GN=pilA PE=3 SV=1 MKAQKGFTLIELMIVVAIIGILAAVAIPAYQDYITRGQVTEAVSLGGGLKSPLAEYGADK NAWPTLVAPTATPGAGQLNATLVGKYSSVDSTIASGYPNGQITVTMTQGKASGKKLTFST QDGGSSWACGNASIDGFAGTGTTIDAKYLPNACKP
Sequence length 155 AA
Subcellular LocationSurface
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.
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 ProteinT64, T66, S81, S82, S85, S89
Glycosite(s) Annotated Protein Sequence >tr|Q8KQ32|Q8KQ32_PSEAE Type IV pilin structural subunit OS=Pseudomonas aeruginosa GN=pilA PE=3 SV=1 MKAQKGFTLIELMIVVAIIGILAAVAIPAYQDYITRGQVTEAVSLGGGLKSPLAEYGADK NAWPTLVAPT*(70)AT*(72)PGAGQLNATLVGKYS*(87)S*(88)VDS*(91)TIAS*(95)GYPNGQITVTMTQGKASGKKLTFST QDGGSSWACGNASIDGFAGTGTTIDAKYLPNACKP
Sequence Around Glycosites (21 AA) KNAWPTLVAPTATPGAGQLNA
AWPTLVAPTATPGAGQLNATL
QLNATLVGKYSSVDSTIASGY
LNATLVGKYSSVDSTIASGYP
TLVGKYSSVDSTIASGYPNGQ
KYSSVDSTIASGYPNGQITVT
Glycosite Sequence Logo
Technique(s) used for Glycosylation DetectionSlower 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 ID20019
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 NameTfpW (arabinosyltransferase)
OST ProGT IDProGT23
Accessory Gene(s)Progt IDProGT22.1-ProGT22.3
Literature
Additional CommentIt 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 Identification2004
Year of Identification Month Wise2004.5.1
Year of Validation 2007
Reference 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]
Author Voisin, S., Kus, J.V., Houliston, S., St-Michael, F., Watson, D., Cvitkovitch, D.G., Kelly, J., Brisson, J.R. Burrows, L.L.
Research GroupInstitute for Biological Sciences, National Research Council, Ottawa, Canada.
Corresponding Author Burrows, L.L.
ContactInstitute for Biological Sciences, National Research Council, Ottawa, Canada.
Reference 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]
Author Voisin, S., Kus, J.V., Houliston, S., St-Michael, F., Watson, D., Cvitkovitch, D.G., Kelly, J., Brisson, J.R. Burrows, L.L.
Research GroupInstitute for Biological Sciences, National Research Council, Ottawa, Canada.
Corresponding Author Burrows, L.L.
ContactInstitute for Biological Sciences, National Research Council, Ottawa, Canada.
Reference1) 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]
AuthorKus, J.V., Kelly, J., Tessier, L., Harvey, H., Cvitkovitch, D.G. and Burrows, L.L
Research GroupDepartment of Biological Sciences, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, USA.
Corresponding Author Burrows, L.L.
ContactDepartment of Biological Sciences, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, USA.
Reference 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]
Author Burrows, L.L.
Research GroupDepartment of Surgery, University of Toronto, 7142A Elm Wing, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
Corresponding Author Kus, J.V., Tullis, E., Cvitkovitch, D.G. Burrows, L.L.
ContactDepartment of Surgery, University of Toronto, 7142A Elm Wing, 555 University Avenue, Toronto, ON, Canada M5G 1X8.