ProGlyProt ID BC140 Organism Information Organism Name Escherichia coli K12 Domain Bacteria Classification Family: Enterobacteriaceae Order: "Enterobacteriales" Class: Gammaproteobacteria Division or phylum: "Proteobacteria" Taxonomic ID (NCBI) 83333 Genome Sequence (s) GeneBank U00096.2 EMBL U00096 Gene Information Gene Name flu NCBI Gene ID 946540 GenBank Gene Sequence 946540 Protein Information Protein Name Ag43α (Antigen 43 passenger domain) UniProtKB/SwissProt ID P39180 NCBI RefSeq YP_026164.1 EMBL-CDS AAT48141.1 UniProtKB Sequence >sp|P39180|AG43_ECOLI Antigen 43 OS=Escherichia coli (strain K12) GN=flu PE=1 SV=3 MKRHLNTCYRLVWNHMTGAFVVASELARARGKRGGVAVALSLAAVTSLPVLAADIVVHPG ETVNGGTLANHDNQIVFGTTNGMTISTGLEYGPDNEANTGGQWVQDGGTANKTTVTSGGL QRVNPGGSVSDTVISAGGGQSLQGRAVNTTLNGGEQWMHEGAIATGTVINDKGWQVVKPG TVATDTVVNTGAEGGPDAENGDTGQFVRGDAVRTTINKNGRQIVRAEGTANTTVVYAGGD QTVHGHALDTTLNGGYQYVHNGGTASDTVVNSDGWQIVKNGGVAGNTTVNQKGRLQVDAG GTATNVTLKQGGALVTSTAATVTGINRLGAFSVVEGKADNVVLENGGRLDVLTGHTATNT RVDDGGTLDVRNGGTATTVSMGNGGVLLADSGAAVSGTRSDGKAFSIGGGQADALMLEKG SSFTLNAGDTATDTTVNGGLFTARGGTLAGTTTLNNGAILTLSGKTVNNDTLTIREGDAL LQGGSLTGNGSVEKSGSGTLTVSNTTLTQKAVNLNEGTLTLNDSTVTTDVIAQRGTALKL TGSTVLNGAIDPTNVTLASGATWNIPDNATVQSVVDDLSHAGQIHFTSTRTGKFVPATLK VKNLNGQNGTISLRVRPDMAQNNADRLVIDGGRATGKTILNLVNAGNSASGLATSGKGIQ VVEAINGATTEEGAFVQGNRLQAGAFNYSLNRDSDESWYLRSENAYRAEVPLYASMLTQA MDYDRIVAGSRSHQTGVNGENNSVRLSIQGGHLGHDNNGGIARGATPESSGSYGFVRLEG DLMRTEVAGMSVTAGVYGAAGHSSVDVKDDDGSRAGTVRDDAGSLGGYLNLVHTSSGLWA DIVAQGTRHSMKASSDNNDFRARGWGWLGSLETGLPFSITDNLMLEPQLQYTWQGLSLDD GKDNAGYVKFGHGSAQHVRAGFRLGSHNDMTFGEGTSSRAPLRDSAKHSVSELPVNWWVQ PSVIRTFSSRGDMRVGTSTAGSGMTFSPSQNGTSLDLQAGLEARVRENITLGVQAGYAHS VSGSSAEGYNGQATLNVTF Sequence length 1039 AA Subcellular Location Secreted Function Self-associating autotransporters (SAAT). It is the extracellular passenger domain of the E. coli autotransporter which represents a branch of the type V secretion pathway. Mediates bacterial autoaggregation (Ag43 interacts with Ag43 intercellularly) and biofilm formation as well as adhesion to and invasion of mammalian cells. It causes frizzy colony morphology and cell settling. Protein Structure PDB ID Glycosylation Status Glycosylation Type O (Ser) linked Experimentally Validated Glycosite(s) in Full Length Protein (Signal pepide: 1-52; sequence beyond 551 is cleaved during secretion) T87, T113, S117, S135, T149, T150, T229, T232, T287/T288, S317, S332, S396, S406, S422, T487, S491, S503 Experimentally Validated Glycosite(s ) in Mature Protein T35, T61, S65, S83, T97, T98, T177, T180, T235/T236, S265, S280, S344, S354, S370, T435, S439, S451 Glycosite(s) Annotated Protein Sequence >sp|P39180|AG43_ECOLI Antigen 43 OS=Escherichia coli (strain K12) GN=flu PE=1 SV=3 MKRHLNTCYRLVWNHMTGAFVVASELARARGKRGGVAVALSLAAVTSLPVLAADIVVHPG ETVNGGTLANHDNQIVFGTTNGMTIST*(87)GLEYGPDNEANTGGQWVQDGGTANKT*(113)TVTS*(117)GGL QRVNPGGSVSDTVIS*(135)AGGGQSLQGRAVNT*(149)T(150)LNGGEQWMHEGAIATGTVINDKGWQVVKPG TVATDTVVNTGAEGGPDAENGDTGQFVRGDAVRTTINKNGRQIVRAEGT*(229)ANT*(232)TVVYAGGD QTVHGHALDTTLNGGYQYVHNGGTASDTVVNSDGWQIVKNGGVAGNT*(287)T*(288)VNQKGRLQVDAG GTATNVTLKQGGALVTS*(317)TAATVTGINRLGAFS*(332)VVEGKADNVVLENGGRLDVLTGHTATNT RVDDGGTLDVRNGGTATTVSMGNGGVLLADSGAAVS*(396)GTRSDGKAFS*(406)IGGGQADALMLEKG SS*(422)FTLNAGDTATDTTVNGGLFTARGGTLAGTTTLNNGAILTLSGKTVNNDTLTIREGDAL LQGGSLT*(487)GNGS*(491)VEKSGSGTLTVS*(503)NTTLTQKAVNLNEGTLTLNDSTVTTDVIAQRGTALKL TGSTVLNGAIDPTNVTLASGATWNIPDNATVQSVVDDLSHAGQIHFTSTRTGKFVPATLK VKNLNGQNGTISLRVRPDMAQNNADRLVIDGGRATGKTILNLVNAGNSASGLATSGKGIQ VVEAINGATTEEGAFVQGNRLQAGAFNYSLNRDSDESWYLRSENAYRAEVPLYASMLTQA MDYDRIVAGSRSHQTGVNGENNSVRLSIQGGHLGHDNNGGIARGATPESSGSYGFVRLEG DLMRTEVAGMSVTAGVYGAAGHSSVDVKDDDGSRAGTVRDDAGSLGGYLNLVHTSSGLWA DIVAQGTRHSMKASSDNNDFRARGWGWLGSLETGLPFSITDNLMLEPQLQYTWQGLSLDD GKDNAGYVKFGHGSAQHVRAGFRLGSHNDMTFGEGTSSRAPLRDSAKHSVSELPVNWWVQ PSVIRTFSSRGDMRVGTSTAGSGMTFSPSQNGTSLDLQAGLEARVRENITLGVQAGYAHS VSGSSAEGYNGQATLNVTF Sequence Around Glycosites (21 AA) FGTTNGMTISTGLEYGPDNEA WVQDGGTANKTTVTSGGLQRV GGTANKTTVTSGGLQRVNPGG PGGSVSDTVISAGGGQSLQGR GQSLQGRAVNTTLNGGEQWMH QSLQGRAVNTTLNGGEQWMHE NGRQIVRAEGTANTTVVYAGG QIVRAEGTANTTVVYAGGDQT IVKNGGVAGNTTVNQKGRLQV VKNGGVAGNTTVNQKGRLQVD TLKQGGALVTSTAATVTGINR VTGINRLGAFSVVEGKADNVV VLLADSGAAVSGTRSDGKAFS SGTRSDGKAFSIGGGQADALM ADALMLEKGSSFTLNAGDTAT GDALLQGGSLTGNGSVEKSGS LQGGSLTGNGSVEKSGSGTLT EKSGSGTLTVSNTTLTQKAVN Glycosite Sequence Logo seqlogo Glycosite Sequence Logo Technique(s) used for Glycosylation Detection Labelling with digoxigenin (DIG)-conjugated hydrazide. Subsequently, the DIG-labelled structures were detected with peroxidase-conjugated anti-DIG antibodies.. Technique(s) used for Glycosylated Residue(s) Detection Automated LC-ESI–MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) with CID (collision induced dissociation) and ETD (electron transfer dissociation) after β-elimination. Protein Glycosylation- Implication Glycosylation protects Ag43α against native-state proteolysis and stabilizes it against thermal and chemical denaturation, and also increases its refolding rate. It reduces the stabilizing effect of Ca2+ ions, prevents Ca2+ to promote cell adhesion and inhibits the bacterial amyloid-forming ability of Ag43α. Glycan Information Glycan Annotation Multiple heptose residues. Technique(s) used for Glycan Identification Protein Glycosylation linked (PGL) gene(s) OST Gene Name aah (autotransporter-adhesin-heptosyltransferase) charcaterized based on gene deletion studies OST NCBI Gene ID OST GenBank Gene Sequence OST Protein Name Aah is the heptosyltransferase. Tib C (E. coli (ET OST UniProtKB/ SwissProt ID OST NCBI RefSeq OST EMBL-CDS OST UniProtKB Sequence OST EC Number (BRENDA) OST Genome Context Characterized Accessory Gene(s) PGL Additional Links CAZy Literatures Reference(s) 1) Knudsen, S.K., Stensballe, A., Franzmann, M., Westergaard, U.B. and Otzen, D.E. (2008) Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation. Biochem J, 412, 563-577. [PubMed: 18341480] 2) Sherlock, O., Dobrindt, U., Jensen, J.B., Munk Vejborg, R. and Klemm, P. (2006) Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein. J Bacteriol, 188, 1798-1807. [PubMed: 16484190] Additional Comments Ag43 protein is involved in phase variable biofilm formation. The glycosylated peptides were identified in a region of the protein composed of imperfect 19-amino-acid repeats. Glycosylation by heptoses represents a novel protein modification in eubacteria. Year of Identification 2006 Year of Validation 2008