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) 287 Genome Sequence (s) EMBL AY112720 Gene Information Gene Name pilA NCBI Gene ID Protein Information Protein Name PilA (Type IV pilin) UniProtKB/SwissProt ID Q8KQ32 NCBI RefSeq EMBL-CDS AAM52059.1 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 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 PDB ID 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 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 seqlogo 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. 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 NCBI Gene ID OST GenBank Gene Sequence OST Protein Name TfpW (arabinosyltransferase) OST UniProtKB/ SwissProt ID Q8KQ31 OST NCBI RefSeq OST EMBL-CDS AAM52060.1 OST UniProtKB Sequence >tr|Q8KQ31|Q8KQ31_PSEAE Putative arabinosyltransferase TfpW OS=Pseudomonas aeruginosa PE=4 SV=1 MKSLWVYLQGGGKKNVLLWVGVLCAVVWGLYGRALFFGYVWDDSILFLDKTDLLNSSISW KVLSEPVLPGTSYLRPLVFLSFFVEFQIFGQSPFVSHAVNIAIFNLNVLLVFWLGSTLAR HTERKYGLLLAFLAALFYAMHPALIESTVWASGRFDLLTTFFILAGLVVYLADIKSTVLR AFLLCLCMAGALLSKELGIVFPVLLLCLWLAKECDCSTQRGGALLVQALFRNGWMLVALI ITAFGYLVVRKHSAGGIYHSPVGLSYIADVVLGQRLPLETLKFYLLQSFLPFQSINVSHP LNEVFERSVYVLLAGQMVVLILVLTVIYQALYRRSAAAWMFFAYLVCLLPVLHIIPLTIG ENLGHERFLTAPLAFLALALVFVRYDRVLSKVGLSGAPLLRVGLVAGLLWLGMAGFTIYS ILPMWTSDASLWAWAYKTHPESKYARYDYLYGALRGGYVELLDKEVSRLQKKYGGLEVPE QILYAHRLISAGDSEGLNYLEGVLQVLPRFHDAENGRVALNDFKHLSSMQIGGAYAVYAE GLSMLRGDAERALKYNKIAEWYLATGEVIPLMYQRSAILYALGDYGQAARIYQDQEGLHY YMSQAVKKDMKRLVEGFCKEKGFPSDPCGELRERGVISKESEARQG OST EC Number (BRENDA) OST Genome Context Characterized Accessory Gene(s) PGL Additional Links CAZy Literatures 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 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 determi Year of Identification 2004 Year of Validation 2007