POSTER NO: 438

Preliminary study on identification of the gene encoding a novel autoantigen targeted by Behcet Disease patient sera

¹Yu Lu, ¹Ping Ye, ¹Shunle Chen, ²Eng M. Tan, ²Edward K.L. Chan
¹Department of Rheumatology, Shanghai Ren Ji Hospital, #145, Shan Dong (c) Road, Shanghai, 200001, China, ²Keck Autoimmune Disease Center, the Scripps Research Institute, La Jolla, California, 92037, U.S.A

Background: Behcet's Disease is a chronic systemic vasculitis disease with unknown cause. Lines of evidence support that immunological abnormalities in both cellular and humoral immunity milieus are actively involved in the disease process. Albeit there are a number of reports on antibodies to microbial agents or self antigens in the disease, these antibodies are largely either non-specific or not well determined. Based on our previous uncovering of common immune reactivities against yet uncharacterized cellular proteins in up to 23.1% (9/39) patient sera from that of a cohort of 39 Chinese Behcet's Disease patients as determined by immunoblotting, this project set out on isolation of relevant gene(s) encoding candidate antigen(s) by immunoscreening strategy using immunoreactive patient serum as probe(s). The rationale is that specific autoantibody(ies) might be present in Behcet's Disease, and identification of cognate antigen(s) may likely provide new inroads into the immunopathogenesis of the disease, novel autoantibody(ies)- antigen(s) may potentially also have serodiagnostic usefulness. Methods: To clone the gene for target antigen, one patient serum (BD4) that demonstrated highest antibody titer in the initial immunoblotting was used to screen a T24 ZAP cDNA expression library from commercial source. Subcloning, restriction mapping, nucleotide sequencing and Genbank database searching were then carried out for characterization of the target gene, followed by gene transcription and translation in vitro using a reticulocyte lysate system. The antigenicity of the isotope-labeled gene product was studied by immunoprecipitation assay using all 39 Behcet's Disease patient sera. Paralleled control study was also carried out using sera from healthy controls, Systemic Lupus Erythematosus as well as Sjogren Syndrome subjects. Results: 6 independent candidate clones were isolated from gene library, and they were identified as overlapping human kinectin cDNA clones, of which, the cDNA insert of the largest clone (BD441.1) spanned ~2/3 of the ~4.6kb full-length kinectin mRNA that codes for a protein of ~156kDa, and an alternative splicing variant has been found in the 3'moiety sequence among the 6 candidate clones. The in vitro gene expression product of BD441.1 showed molecular mass in consistent with its theoretical value, and the antigenic identity of the gene product in Behcet's Disease was preliminarily verified since 9 out of 39 (23.1%) sera including BD4 could immunoprecipitate the translation product (for some sera the result of immunoblotting is in consistent with that of immunoprecipitation, however, the other not), while sera from 20 healthy controls, 20 Systemic Lupus Erythematosus as well as 20 Sjogren Syndrome subjects showed no reactivity in the analysis. In reviewing literatures, kinectin is a conservative integral membrane protein anchored in the endoplasmic reticulum acting as a receptor for the organelle motor kinesin, the gene of which has been assigned to chromosome 14 band q22.1. Conclusions: Behcet's Disease patient sera contain autoantibodies to cellular proteins, and one of the candidate autoantigens was kinectin as unraveled by gene library screening. Further in-depth work is needed to clarify the significance of kinectin and antigens yet-to-be-identified in the disease process.