New cysteine protease inhibitors in human tears and milk were found and their medical
significance was studied. As the protective components against bacterial infection in eyes, we detected four
kinds of biologically active proteins in normal human tears including three kinds of cysteine protease
inhibitors. Using our reverse zymography of normal tears, the three kinds of cysteine protease inhibitors
were found to be 78, 20 and 15 kDa and were determined to be lactoferrin, VEG protein and cystatin S,
respectively. The C-terminus area 17 mer peptide, Y679-K695 of lactoferrin molecule showed strong
homology with a common active domain of cystatin family and the synthesized peptide itself showed
considerable inhibition of cysteine proteases. Not only disease-specific changes of these inhibitor contents,
but disease-specific new inhibitors were also found in patient tears in special autoimmune diseases. The
characteristic 35 kDa inhibitor band which was detected specifically in the cases of Behcet's disease tears, an
autoimmune disease, was determined to be a lachrymal acidic proline-rich protein family based on the Nterminus
sequence analysis. The 65 kDa inhibitor of tears in Harada's autoimmune-disease was determined
to be a human Ig heavy chain V-III region. Also lactoferrin content in Harada's disease was very low
compared with that of normal tears. Also we found two cathepsin inhibitors, lactoferrin and ?-casein, in milk
of human and bovine using reverse zymography. They may also play a role in bacterio-cidal and viro-cidal
functions in milk. The L133-Q151 in human ?-casein molecule is the active inhibitory domain. It is most
important to know from biological aspects that the concentration of these inhibitors in natural milk can
inhibit cysteine proteases of bacteria. Surprisingly, the 50 times diluted milk inhibited papain completely,
because lactoferrin and casein contents in milk are very high. We want to emphasize that these inhibitors in
milk play a sufficient role in the protection of bacteria.
