IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA. A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA. J. Biol. Chem. (0021-9258) 2006 imex curation Wines BD., Willoughby N., Fraser JD., Hogarth PM. BioCreative - Critical Assessment of Information Extraction systems in Biology The sequence of this IgA1 Fc cDNA fragment differs from reference sequences (accession: NG_001019, six single nucleotide polymorphisms, BC092449, seven single nucleotide polymorphisms) resulting in two amino acid differences, L271M and R392H (standard IgA1 Bur numbering), consistent with the Dakiki IgA1 being a putative allelic variant. Here the sequence corresponding to the second accession number has been used as it is longer. This corresponds to a recent entry in Uniprot (Q569J1). The R392H corresponds to position 393 and the L271M to position 411. b.wines@ari.unimelb.edu.au 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Wines BD., Willoughby N., Fraser JD., Hogarth PM. BioCreative - Critical Assessment of Information Extraction systems in Biology The sequence of this IgA1 Fc cDNA fragment differs from reference sequences (accession: NG_001019, six single nucleotide polymorphisms, BC092449, seven single nucleotide polymorphisms) resulting in two amino acid differences, L271M and R392H (standard IgA1 Bur numbering), consistent with the Dakiki IgA1 being a putative allelic variant. Here the sequence corresponding to the second accession number has been used as it is longer. This corresponds to a recent entry in Uniprot (Q569J1). The R392H corresponds to position 393 and the L271M to position 411. b.wines@ari.unimelb.edu.au Accepted 2006-AUG-29 by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA. A competitive mechanism for staphylococcal toxin SSL7 inhibiting the leukocyte IgA receptor, Fc alphaRI, is revealed by SSL7 binding at the C alpha2/C alpha3 interface of IgA. J. Biol. Chem. (0021-9258) 2006 imex curation Wines BD., Willoughby N., Fraser JD., Hogarth PM. BioCreative - Critical Assessment of Information Extraction systems in Biology The sequence of this IgA1 Fc cDNA fragment differs from reference sequences (accession: NG_001019, six single nucleotide polymorphisms, BC092449, seven single nucleotide polymorphisms) resulting in two amino acid differences, L271M and R392H (standard IgA1 Bur numbering), consistent with the Dakiki IgA1 being a putative allelic variant. Here the sequence corresponding to the second accession number has been used as it is longer. This corresponds to a recent entry in Uniprot (Q569J1). The R392H corresponds to position 393 and the L271M to position 411. b.wines@ari.unimelb.edu.au 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro facs fluorescence-activated cell sorting FACS Flow cytometry facs predetermined predetermined participant predetermined Wines BD., Willoughby N., Fraser JD., Hogarth PM. BioCreative - Critical Assessment of Information Extraction systems in Biology The sequence of this IgA1 Fc cDNA fragment differs from reference sequences (accession: NG_001019, six single nucleotide polymorphisms, BC092449, seven single nucleotide polymorphisms) resulting in two amino acid differences, L271M and R392H (standard IgA1 Bur numbering), consistent with the Dakiki IgA1 being a putative allelic variant. Here the sequence corresponding to the second accession number has been used as it is longer. This corresponds to a recent entry in Uniprot (Q569J1). The R392H corresponds to position 393 and the L271M to position 411. b.wines@ari.unimelb.edu.au Accepted 2006-AUG-29 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation q9zfs5_staau set1 NCTC7878_00468 FPP26_02075 protein protein staau Staphylococcus aureus MKLKTLAKATLALGLLTTGVITSEGQAVQAAEKQERVQHLHDIRDLHRYYSSESFEYSNVSGKVENYNGSNVVRFNPKDQNHQLFLLGKDKEQYKEGLQGQNVFVVQELIDPNGRLSTVGGVTKKNNKTSETNTPLFVNKVNGEDLDASIDSFLIQKEEISLKELDFKIRQQLVNNYGLYKGTSKYGKIIINLKDENKVEIDLGDKLQFERMGDVLNSKDIRGISVTINQI 1710902412510102 q8r3h6_mouse Ighg AU044919 protein protein mouse Mus musculus Mouse MEWPCILLFLLSVTEGVHSQVQLLQSGPELVKPGASVKISCRASGYAFSKSWMNWVKRRPGKGLEWIGRIFPGDGDTHYSGKFQGKAKLTADKSSVTAFLQLTSLTSEDSAVYFCARDSDYGDYFDDWGQGATVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK 8608B57C6CD2874A q569j1_human IGHA1 protein protein human Homo sapiens Human MDTLCSTLLLLTIPSWVLSQITLKESGPALVKPTQTLTLTCTFSGFSLSSSGLSVGWIRQPPGKALEWLALIYWNDDKRHRPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHKYSGSWNAFDIWGQGTMVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY The sequence has been withdrawn from uniprot. 5B037BEE5B5B2ADF set1-igha1 1 5 unspecified role unspecified role bait bait transferrin transferrin receptor-IgA Fc fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 3 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association 1 The binding to this immobilized IgA2 of SSL7 at different concentrations was recorded (Fig. 1B) after 3000 s when equilibrium was being approached. Plotting the equilibrium binding responses fitted best to a single binding site with an apparent affinity of 5.0 1.5 nM(n 5, Fig. 1C, one representative experiment KD 6.8 0.8 nM). KD 6.8 plus or minus 0.8 E-9 M The igha1 protein was fused to the human transferrin receptor (TfR). The TfR-IgA Fc fusion protein was expressed in insect cells as a baculovirus expression vector was used. For the IgA1 Fc sequence the authors state there are two differences in sequence and have mapped L271M against the sequence corresponding to NG_001019 and R392H against the sequence corresponding to BC092449, which is a longer sequence. set1-igha1-1 2 5 unspecified role unspecified role bait bait ala463arg mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain A R leu278met,leu279ile mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L M certain certain sequence position certain certain certain sequence position certain L I pro_leu_ala_phe461-464his_asn_his_tyr mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain PLAF HNHY region transferrin receptor-IgA Fc fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 3 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association 2 B and D, 4 The igha1 protein was fused to the human transferrin receptor (TfR). For the IgA1 Fc sequence the authors state there are two differences in sequence and have mapped L271M against the sequence corresponding to NG_001019 and R392H against the sequence corresponding to BC092449, which is a longer sequence. The TfR-IgA Fc fusion protein was expressed in insect cells as a baculovirus expression vector was used. The Fc region of the human IgA1 derived from Dakiki cells was fused to the N-terminal region of the transferrin receptor which is a type II integral membrane protein. This fusion protein was expressed on the surface of CHOP cells (CHO cells expressing the early genes of the polyoma virus). The binding of biotinylated rSSL7 to WT IgA fusion protein was readily detectable (Fig. 4B, MFI 350 40, n 4). In contrast, the level of binding of SSL7 to the A442R mutant IgA Fc fusion protein (MFI 4.5 0.2) was indistinguishable from the binding to the mock-trans-fected cells (MFI 4.3 0.2). It is notable that an experiment with higher surface expression of IgA Fc fusion protein (Fig. 2), with correspondingly higher SSL7 binding to the WT (Fig. 2B, MFI 980 21), did reveal some low level binding of SSL7 to the A442R mutant protein (MFI 8.4 0.4, 0.15% relative to WT 100%, mock MFI 6.9 0.3). The greatly reduced SSL7 binding paralleled the reduced binding of the Fc RI-Ig fusion protein, which bound surface-expressed WT IgA Fc (MFI 29.2 0.2) but not the A442R mutant (MFI 6.5 0.5, mock MFI 6.5 0.1 (Fig. 4C)). The other mutations predicted to affect Fc RI binding also ablated SSL7 binding. The alternative mutation in the FG loop of domain 3 PLAF440-443HNHY abolished both SSL7 binding (MFI 4.7 0.2, mock 4.3 0.2) and Fc RI-Ig binding to levels equivalent to that of the mock transfections (Fig. 4C). Mutation of residues Leu-257 and Leu-258 in the C 2 AB helix/loop of IgA Fc also affected both SSL7 (MFI 35 3, 9% of WT) and Fc RI-Ig binding (MFI 8.1 0.8, 7% of WT). Mutation of the N-linked glycosylation site (N263T) did not affect SSL7 binding (MFI 330 19, 94 6% of WT) and only modestly reduced Fc RI-Ig binding (MFI 24 2, 80 6% of WT). The surface expression of the WT and mutant IgA fusion proteins was determinedby staining with anti-IgA FITC (Fig. 2A) and the rSSL7 (Fig. 2B), and Fc RI-Ig (Fig. 2C) binding to the same transfectants was also determined. In agreement with our previous data the binding of Fc RI-Ig to WT IgA Fc fusion protein expressing cells (mean fluorescent intensity (MFI) 38.6 2) was inhibited 90% in the presence of 2 M (40 g/ml) rSSL7 (MFI 8 2, background staining MFI 5 1, Fig. 2D). igha1-ighg 2 4 unspecified role unspecified role prey prey region fused to FcalphaRI ectodomains fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region Fc region of mouse IgG2b sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. 5 unspecified role unspecified role bait bait ala463arg mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain A R pro_leu_ala_phe461-464his_asn_his_tyr mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain PLAF HNHY leu278met,leu279ile mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain L M certain certain sequence position certain certain certain sequence position certain L I asn284thr mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain N T region transferrin receptor-IgA Fc fusion protein fusion protein fusion protein undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 C and D, 4 For the IgA1 Fc sequence the authors state there are two differences in sequence and have mapped L271M against the sequence corresponding to NG_001019 and R392H against the sequence corresponding to BC092449, which is a longer sequence. The TfR-IgA Fc fusion protein was expressed in insect cells as a baculovirus expression vector was used. The Fc region of the human IgA1 derived from Dakiki cells was fused to the N-terminal region of the transferrin receptor which is a type II integral membrane protein. This fusion protein was expressed on the surface of CHOP cells (CHO cells expressing the early genes of the polyoma virus). The alternative mutation in the FG loop of domain 3 PLAF440-443HNHY abolished both SSL7 binding (MFI 4.7 0.2, mock 4.3 0.2) and Fc RI-Ig binding to levels equivalent to that of the mock transfections (Fig. 4C). Mutation of residues Leu-257 and Leu-258 in the C 2 AB helix/loop of IgA Fc also affected both SSL7 (MFI 35 3, 9% of WT) and Fc RI-Ig binding (MFI 8.1 0.8, 7% of WT). Mutation of the N-linked glycosylation site (N263T) did not affect SSL7 binding (MFI 330 19, 94 6% of WT) and only modestly reduced Fc RI-Ig binding (MFI 24 2, 80 6% of WT). The alternative mutation in the FG loop of domain 3 PLAF440-443HNHY abolished both SSL7 binding (MFI 4.7 0.2, mock 4.3 0.2) and Fc RI-Ig binding to levels equivalent to that of the mock transfections (Fig. 4C). Mutation of residues Leu-257 and Leu-258 in the C 2 AB helix/loop of IgA Fc also affected both SSL7 (MFI 35 3, 9% of WT) and Fc RI-Ig binding (MFI 8.1 0.8, 7% of WT). Mutation of the N-linked glycosylation site (N263T) did not affect SSL7 binding (MFI 330 19, 94 6% of WT) and only modestly reduced Fc RI-Ig binding (MFI 24 2, 80 6% of WT).by staining with anti-IgA FITC (Fig. 2A) and the rSSL7 (Fig. 2B), and Fc RI-Ig (Fig. 2C) binding to the same transfectants was also determined. In agreement with our previous data the binding of Fc RI-Ig to WT IgA Fc fusion protein expressing cells (mean fluorescent intensity (MFI) 38.6 2) was inhibited 90% in the presence of 2 M (40 g/ml) rSSL7 (MFI 8 2, background staining MFI 5 1, Fig. 2D).