We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino gcids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with β-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.
Abbreviations used: COPD, chronic obstructive pulmonary disease; DIG, digoxigenin; EK, enterokinase; EST, expressed sequence tag; HA, haemagglutinin; HRP, horseradish peroxidase; hTMT, human transmembrane tryptase; IF, immunofluorescence; IHC, immunohistochemistry; pNA, p-nitroanilide; PI, protease inhibitor; PRSS, protease serine S1 family member; RACE, rapid amplification of cDNA ends; SSC, standard saline citrate; Suc, succinoyl.