title: Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane.
abstract: Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein.
authors.affiliation: Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA, biji-kurien@omrf.org.
authors.forename: Biji T
authors.initials: BT
authors.lastname: Kurien
authors.forename: Michael P
authors.initials: MP
authors.lastname: Bachmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000378:metabolism; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011145:Polyvinyls / Q000737:chemistry*; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000032:analysis / Q000378:metabolism*; D012394:Rosaniline Dyes / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000378:metabolism; D000099207:SS-A Antigen; D005465:Fluorocarbon Polymers
chemical_list: D002118:Calcium; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D012261:Ribonucleoproteins; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; D000099207:SS-A Antigen; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C004692:coomassie Brilliant Blue