Identification of kinectin as a novel Behcet's disease-related autoantigen

Background

Lines of evidence support that an autoantibody reaction contributes to the immunological abnormalities underlying Behcet's disease (BD), yet no specific antibody has been reported in the disease. In our previous study, common immune reactivities against unknown cellular proteins were uncovered in 23.1% patient sera from that of 39 Chinese BD patients as determined by immunoblotting.

Objectives

This project set out to identify target antigen(s) in BD in view of the fact that it may provide new inroads into the immunopathogenesis of the disease, and may have serodiagnostic usefulness.

Methods

Immnuoscreening of a ZAP cDNA library followed by transcription/translation in vitro of candidate clones in a reticulocyte lysate system were carried out. The antigenicity of clone product was analyzed by immunoprecipitation using sera from all 39 BD patients in parallel with normal controls and control sera from Lupus and Sjögren syndrome patients. Three truncated gene products spanning the full length of the target protein were expressed in Escherichia coli, and their respective antigenicities in BD were preliminarily analyzed using an ELISA approach. A full-length gene cloned in the pEGFP vector was transfected into HEp-2 cells as an overexpression antigen substrate for immune fluorescent study.

Results

Six independent candidate clones were isolated from a gene library, being identified as overlapping human kinectin cDNA clones. The antigenic identity of the partial gene product from the largest clone of the six was preliminarily verified since nine out of 39 (23.1%) BD patients' sera could immunoprecipitate the translation product, whereas sera from controls showed no reactivity. The antigenicity of kinectin was found to mainly reside in the middle and carboxyl portion of the intact protein, and the overexpression GFP-kinectin showed distinct perinuclear cytoplasmic staining and can be recognized by BD patient sera but not normal sera under a confocal fluorescent microscope.

Conclusions

BD patient sera contain autoantibodies to cellular proteins, and one of the related autoantigens was kinectin, as unraveled by gene library screening, the antigenicity of which mainly resides in the middle and carboxyl portion of the intact protein. Further in-depth work is needed to clarify the significance of kinectin in the disease entity.