Identification of Major Linear Epitopes on the sp100 Nuclear PBC Autoantigen by the Gene-Fragment Phage-Display Technology

Abstract

Approximately 20–30% of sera from patients suffering from primary biliary cirrhosis contain autoantibodies against a nuclear protein termed sp100.^[1] By indirect cytoimmuno-fluorescence it was shown that the sp100 autoantigen is distributed in up to 20 dot-like structures per nucleus co-localizing with the so-called nuclear bodies. In western blots these sera react with a protein with an apparent molecular mass of 100kDa. By screening expression libraries with affinity-purified anti-sp100 antibodies we isolated a full-length sp100 cDNA whose sequence exactly matched the previously published sp100 sequence and encodes a protein of 481 amino acids with a deduced molecular mass of 53 kDa.^[2] In an attempt to determine immunoreactive regions on the sp100 antigen with the recently developed gene-fragment phage-display technology we were able to identify a stretch of sixteen amino acids (IKKEKPFSNSKVECQA) at position 296–311 as a major antigenic region (antigenic region 1) on the sp100-autoantigen. A second antigenic region (antigenic region 2) of twenty amino acids in length could be identified between amino acids 332–351 (EGSTDVDEPLEVFISAPRSE). By using immobilized synthetic peptidcs and various sp100-positive PBC patient sera the corresponding epitopes could be shown to be centered around epitope cores of six amino acids (SNSKVE, antigenic region 1) and nine amino acids (EPLEVFISA, antigenic region 2) respectively.

Key Words:

• Primary biliary cirrhosis (PBC)
• Nuclear protein
• sp100
• Autoantigen
• Epitope mapping
• Phage-display libraries