Detection of calcium binding by Ro 60 multiple antigenic peptides on nitrocellulose membrane using Quin-2-

Abstract

Systemic lupus erythematosus is associated with the production of antibodies to self-constituents, particularly those that target certain specific ribonucleoprotein (RNP) particles. Among these is the Ro RNP particle, composed of a 60,000 molecular weight protein (Ro 60 or SS-A) that is noncovalently associated with at least one of four short uridine-rich RNAs (the hY RNAs). Our earlier work demonstrated that multiple antigenic peptides (MAPs) constructed 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 (SPR), to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also found that calcium is important in mediating this interaction. We hypothesized, therefore, that the Ro 60 antigen is a calcium binding protein. To investigate this we used Ro 60 MAPs 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 the protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, make us propose that the Ro 60 antigen is a calcium binding protein.