The Ro/SSA Complex in Systemic Lupus Erythematosus Patients

Resum

In this work the involved mechanisms between Ro/SSA complex, composed also by the tripartite motif 21 (TRIM21alpha) and trove domain 2 (TROVE2) proteins, with respect to systemic lupus erythematosus (SLE) autoantibodies is studied. The work is divided in three chapters: I- In vitro and in silico analysis of the molecular recognition between lupus autoantibodies and TRIM21alpha Fc Receptor ; II- In vitro evidence of bipolar-bridged immune TROVE2 complexes in the pathogenesis of systemic lupus erythematosus and III- Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus. Samples of lupic patients and health subjects were kindly provided by La Fe hospital, accordingly the required protocols. After its extraction and purification, the immunoglobulin samples were obtained to study in vitro protein interactions and the involved mechanism by using a quartz crystal microbalance with dissipation factor attributions, and dual polarization interferometry. Techniques such, polarization modulation infrared interferometry, x-ray photoelectron interferometry and contact angle measurement were used in order to characterize surfaces. Pre-steady-state analysis revealed an antibody bipolar bridging involved in both TRIM21alpha and TROVE2 proteins. Identification of the main immunodominant human linear epitope for TRIM21alpha was finely mapped using a series of overlapping synthetic polypeptides with a size of 21 amino acids. The epitopes recognised by autoantibodies for this protein spanned the linear sequence from the aminoacid 151 to 183, and a conformational epitope for SLE patients and healthy subjects, respectively. Autoantibodies from lupic patients targeted protein epitopes, allowing health subjects to be discriminated. Major Histocompatibility Complex Class-II binding peptide prediction results corroborated the sequence as the immunodominant linear epitope, mostly coded as the HLA DRB1*1304 allele for SLE patients, and HLA DRB1*0806 for controls. The subdominant epitope corresponded to the PRY-SPRY domain, recently known as mammalian Fc receptor. Finally, the TRIM21alpha protein structure was modeled by a new homology modeling, never before presented. From the TROVE2 protein, the major linear epitope recognized by autoantibodies correspond to the sequence from the aminoacid 160 to 210 for healthy subjects. However, the major epitope in SLE serum is undiscovered. We suggest that the difference between epitopes could correspond to a majority necrosis-induced specificity in SLE patients, and an apoptotic via in healthy subjects. TROVE2 showed the ability to bind to Fcs, depending on alkaline earth cations in solution. The results suggest that the TROVE2-TRIM21alpha binding is a calcium-dependent protein interaction linked through the MIDAS-like motif in the vWFA-like domain. Finally, a pratical consequence of all study was the development of label-free biosensing method, based in microbalance technology, for in vitro diagnostics of systemic lupus erythematosus patients, allowing the premature sensing of autoantibodies against TRIM21alpha and TROVE2 protein, in advance of the clinical illness symptoms appear.