Identification and characterization of novel ERC-55 interacting proteins: evidence for the existence of several ERC-55 splicing variants; including the cytosolic ERC-55-C.
ERC-55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC-55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC-55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC-55 splicing variants including ERC-55-C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub-cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin-6, kininogen and lysozyme with ERC-55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca(2+)] of approximately 10(-7) M or greater, while calcyclin interaction requires [Ca(2+)] of >10(-5) M. Interaction with peroxiredoxin-6 is independent of Ca(2+). Co-localization of lactoferrin, S100P and calcyclin with ERC-55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC-55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.
Ludvigsen
Maja
M
Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark.
Jacobsen
Christian
C
Maunsbach
Arvid B
AB
Honoré
Bent
B
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006801:Humans; D007704:Kininogens / Q000032:analysis / Q000378:metabolism; D007781:Lactoferrin / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D054465:Peroxiredoxin VI / Q000032:analysis / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D011485:Protein Binding; D019154:Protein Splicing; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000032:analysis / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D007704:Kininogens; D010455:Peptides; C088393:RCN2 protein, human; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C055351:S100A6 protein, human; D054465:Peroxiredoxin VI; D009113:Muramidase; D007781:Lactoferrin