bhf-ucl mayer-2008-2 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting weight by staining molecular weight estimation by staining weight by staining Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins The COS -1 line in fig. 1B is a control that shows no interaction when PCSK9 is not present in the conditioned medium (pIRES-V5) seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-3 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins AnxA2-His6 was confirmed to be human through correspondence with the authors and by looking at the EMD bioscience catalog where they bought it from. seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-4 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-5 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip ms participant Identification by mass spectrometry ms participant Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-6 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. in vitro In vitro electrophoresis electrophoretic mobility-based method electrophoresis western blot western blot Immuno blot Immuno blot Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-7 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting sequence tag sequence tag identification sequence tag Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins COS-1 cell lysates were separated by a first horizontal dimension using a wide pH range isoelectrofocusing gel strip and then vertically by SDS-PAGE. After the SDS-PAGE, the proteins were electrotransferred onto a nitrocellulose membrane and probed by Far Western with anti-V5 antibodies. seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation mayer-2008-8 Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 confocal microscopy confocal microscopy anti-tag immunocytochemistry anti-tag immunocytochemistry Mayer G., Poirier S., Seidah N.G. Cardiac - Interactions involving cardiac related proteins seidahn@ircm.qc.ca J. Biol. Chem. (0021-9258) 2008 Only protein-protein interactions imex curation pcsk9_human Proprotein convertase subtilisin/kexin type 9 Subtilisin/kexin-like protease PC9 Neural apoptosis-regulated convertase 1 Proprotein convertase 9 PCSK9 NARC1 PSEC0052 protein protein human Homo sapiens Human MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ mint homomint May be implicated in the differentiation of cortical neurons and may play a role in cholesterol homeostasis Defects in PCSK9 are the cause of familial hypercholesterolemia 3 (FH3) [MIM:603776]. FH3 inheritance is autosomal dominant anxa2_human Annexin A2 ANXA2 ANX2 ANX2L4 CAL1H LPC2D Annexin-2 Annexin II Lipocortin II Calpactin I heavy chain Chromobindin-8 p36 Protein I Placental anticoagulant protein IV Calpactin-1 heavy chain protein protein human Homo sapiens Human MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSGDFRKLMVALAKGRRAEDGSVIDYELIDQDARDLYDAGVKRKGTDVPKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYDSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYQKALLYLCGGDD anxa2_chlae_protein Annexin A2 protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey similar to P07355 pcsk9-anxa2 1520988 n/a 1520995 unspecified role unspecified role bait bait conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Q8NBP7:p.Gln554Glu mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520996 unspecified role unspecified role prey prey cell lysate cell lysate crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 physical association physical association false false false Fig. 3A, 4B pcsk9-anxa2-2 1520988 n/a 1520995 unspecified role unspecified role bait bait conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520996 unspecified role unspecified role prey prey purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminus s tag s tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 physical association physical association false false false Fig. 3D Purified AnxA2 was submitted to electrophoresis under reducing and non-reducing conditions. PCSK9 binds in a dose-dependent manner AnxA2 in both situations. Under reducing conditions only binding of the monomeric form of Anxa-His to Pcsk9 was observed. pcsk9-anxa2-7 1520988 n/a 1520995 unspecified role unspecified role bait bait conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520996 unspecified role unspecified role prey prey purified purified n-terminus s tag s tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 physical association physical association false false false Fig. 3D Purified AnxA2 was submitted to electrophoresis under reducing and non-reducing conditions. PCSK9 binds in a dose-dependent manner AnxA2 in both situations. pcsk9-anxa2-6 1520988 n/a 1520995 unspecified role unspecified role bait bait conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520996 unspecified role unspecified role prey prey cell lysate cell lysate c-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false r1 domain required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false P07355:p.[Arg77Ala;Arg78Ala;Lys80Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P07355:p.[Arg77Ala;Arg78Ala;Lys80Ala;Lys81Ala;Lys88Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P07355:p.[Arg77Ala;Arg78Ala;Lys80Ala;Lys81Ala] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. physical association physical association false false false Fig. 9 B, C Individual deletions of R2, R3 and R4 reduce the ability to bind to PCSK9 Exchanging the 49-75aa fragment in AnxA2 with the 58-84aa fragment of AnxA1 significantly reduces the AnxA2 ability to bind PCSK9. (Fig. 9C) anxa2-pcsk9-1 1520989 n/a 1520995 unspecified role unspecified role prey prey conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520996 unspecified role unspecified role bait bait purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminus s tag s tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) physical association physical association false false false Fg. 3B pcsk9-anxa2-1 1520990 n/a 1520995 unspecified role unspecified role bait bait c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1520996 unspecified role unspecified role prey prey c-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false physical association physical association false false false Fig. 3C pcsk9-anxa2chlae-1 1520991 n/a 1520995 unspecified role unspecified role bait bait c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1520997 unspecified role unspecified role prey prey physical association physical association false false false Fig. 2 A,E anxa2-anxa2 1520992 n/a 1520996 unspecified role unspecified role unspecified role unspecified role purified purified n-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false n-terminus s tag s tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 3D reducing conditions pcsk9-anxa2chlae-3 1520993 n/a 1520995 unspecified role unspecified role bait bait conditioned medium conditioned medium c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 n/a 1520997 unspecified role unspecified role prey prey cell lysate cell lysate chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 physical association physical association false false false Fig. 2C, D, E anxa2-pcsk9-2 1520994 n/a 1520995 unspecified role unspecified role unspecified role unspecified role c-terminus v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 1520996 unspecified role unspecified role unspecified role unspecified role c-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false colocalization colocalization false false false Fig. 5