Detection of disease–specific cysteine protease inhibitors in tears.

Abstract

Abstract: : Purpose: Cysteine protease is one of the essential protein for bacteria and viruses to live. Detection of cysteine protease inhibitors in human tears of normal people, Behcet's disease and Harada's disease was done at the molecular level. Methods: Tears of normal people, Behcet's disease and Harada's disease were taken with glass–capillaries. After diluting these tears 2 times with Tris–buffer, reverse zymography for the detection of cysteine protease inhibitors developed by Katunuma was done. The structures of inhibitors were determined by the method of amino acid sequence analysis. The antibody for 31 kDa detected later was taken from immunized rabbits and the confirmation was done by western blatting method. Results: 1) Three kinds of cysteine protease inhibitors were detected in normal tear. They were cystatin S of 15 kDa, von Ebner's gland protein of 20 kDa and lactoferrin of 72 kDa. 2) New cysteine protease inhibitor of 31 kDa was found in Behcet's disease. 3) Another new cysteine protease inhibitor of 65 kDa was found in Harada's disease. 4) The antibody was confirmed as specific to the 31 kDa. Conclusions: 1) The three kinds of cysteine protease inhibitors in tears will be working as anti–infectious components. 2) New cysteine protease inhibitors of 31 kDa and 65 kDa are disease–specific proteins for Behcet's disease and Harada's disease, respectively. 3) These specific proteins will give us accurate and easy diagnostic kits and help us to understand the pathogenesis of these diseases.

Keywords: autoimmune disease • lacrimal gland • protein purification and characterization