title: Interaction of calcium and Ro60: increase of antigenicity.
abstract: The structural and functional integrity of the cell is largely maintained by protein-protein interactions. Recently, we demonstrated that multiple antigenic peptides (MAPs) constructed from 60 kDa Ro sequence could be used to show intramolecular and intermolecular protein-protein interaction within the 60 kDa Ro ribonucleoprotein particle. We were interested in understanding the mechanism of this binding and hypothesized that this interaction might be mediated through divalent metal ions. The 60 kDa Ro-MAPs failed to interact with purified 60 kDa Ro in the presence of EDTA or EGTA when analyzed by Ouchterlony or surface plasmon resonance (SPR) analysis. When purified 60 kDa Ro was incubated with various metal ions such as Cu2+, Mg2+, Zn2+ and Ca2+, and analyzed by Ouchterlony or SPR for binding to specific 60 kDa Ro-MAPs only Ca2+ ions significantly increased the binding. It was interesting to note that recombinant 60 kDa Ro formed precipitin lines with Ro-MAPs only in the presence of Ca2+ ions. Anti-Ro60 containing SLE sera bound to recombinant Ro60 strongly when incubated in the presence of Ca2+ ions but not in the absence of Ca2+ ions. Using SPR analysis we also found that native Ro60 binds to La only in the presence of Ca2+. These data imply that Ca2+ induces a more native tertiary structure to recombinant 60 kDa Ro and makes it more antigenic. Thus, the observed intramolecular and intermolecular interactions and antigen-antibody interactions could be Ca2+ ion mediated conformational interactions, and we propose that 60 kDa Ro is a calcium binding protein.
authors.affiliation: Arthritis and Immunology Program, Oklahoma Medical Research Foundation (OMRF), 825 NE 13th Street, MS# 38, Oklahoma City, OK 73104, USA.
authors.forename: R Hal
authors.initials: RH
authors.lastname: Scofield
authors.forename: Biji T
authors.initials: BT
authors.lastname: Kurien
authors.forename: Joame K
authors.initials: JK
authors.lastname: Gross
authors.forename: Natalie B
authors.initials: NB
authors.lastname: Reed
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Taylor
authors.forename: Nicolas
authors.initials: N
authors.lastname: Dominguez
authors.forename: Padmaja
authors.initials: P
authors.lastname: Mehta
authors.forename: Joel M
authors.initials: JM
authors.lastname: Guthridge
authors.forename: Michael
authors.initials: M
authors.lastname: Bachmann
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology / Q000378:metabolism*; D001324:Autoantigens / Q000276:immunology / Q000378:metabolism*; D015551:Autoimmunity / Q000276:immunology / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D008180:Lupus Erythematosus, Systemic / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D020733:RNA, Small Cytoplasmic / Q000276:immunology / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors; D000099207:SS-A Antigen
chemical_list: D000941:Antigens; D001324:Autoantigens; D002118:Calcium; D010455:Peptides; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; C116006:RO60 protein, human; D000099207:SS-A Antigen