Proteomics Analysis of Secretory Cysteine Protease Inhibitors and their Practical Application on Oral Health

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This study focused on the proteomics analysis of secretory cysteine protease inhibitors and their practical application on oral health. The results obtained are as follows ;1.A tow dimensional gel electrophoresis system that can separate and detect cysteine protease inhibitors in biological fluids was developed.2.A novel rice cysteine protease inhibitor, oryzacystatin III was cloned and sequenced. It was found that oryzacystain III from the calli of rice shares 56% and 89% amino acid sequence identity, respectively, with oryzacystatin I and oryzacystatin II. The sequence differs from that of oryzacystatin II in the N-terminal region (Gln^7 - Ala^<19>; in the oryzacystatin II numbering), and this region contained glycine residue (Gly^<14>), which is the evolutionarily conserved in the cystatin superfamily. Biochemical properties of oryzacystatins were characterized using recombinant protein.3.Two cysteine protease inhibitors (Eel-CPI-1 and Eel-CPI-2) from skin mucus of the Japanese eel … More (Anguilla japonica) were isolated and characterized. Eel-CPI-1 inhibited papain (K_i=18 nM) and ficin (K_i=120 nM) competitively. Combined with the data on amino acid and sequence analysis, Eel-CPI-1 is identical to the eel lectin, AJL-2.4.A Bacillus subtilis system that can be used to produce large quantities of recombinant human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily was developed. The DNA sequences for promoter and signal of the alkaline endoglucanase gene in Bacillus sp.KSM-S237 were employed to accomplish higher expression levels of recombinant human salivary cystatins by the first use of ΔaprE type mutant strain of B.subtilis. Our system yielded approximately 1.1 gram/liter of r-cystatin S and 0.8 g/liter of r-cystatin SA.5.We have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.6.It was found that human cystatin SA induce gamma-interferon production by CD4-positive T cells. Less