Related Experiment Videos
A novel serine protease predominately expressed in macrophages
Cailin Chen1, Andrew L Darrow, Jian-Shen Qi
1Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development LLC, Welsh and McKean Roads, Spring House, PA 19477-0776, USA.
Abstract:
We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids 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 beta-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.
Insights
We discovered a new serine protease, EOS, highly expressed in macrophages. This finding may illuminate macrophage roles in inflammatory diseases and wound healing.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Serine proteases are crucial enzymes involved in various physiological and pathological processes.
- Macrophages play significant roles in immunity, inflammation, and tissue repair.
Purpose of the Study:
- To identify and characterize a novel serine protease.
- To investigate the expression pattern and cellular localization of the novel protease.
- To explore the potential role of this protease in macrophage-associated functions and diseases.
Main Methods:
- Sequence identity searches and protein analysis to identify the novel protease (EOS).
- Enzyme activity assays to determine substrate specificity.
- Immunohistochemistry and in situ hybridization for tissue distribution and cellular localization.
- Double immunofluorescence and cell culture studies (U937 cells treated with PMA) to confirm macrophage association and activation.
Main Results:
- A novel serine protease, EOS, was identified, sharing 44% identity with beta-tryptase and belonging to the S1 trypsin-like serine-protease family.
- EOS exhibits amidolytic activity, cleaving substrates before arginine residues.
- EOS is highly expressed in the spleen and moderately in the intestine, colon, lung, and brain, primarily associated with macrophages.
- EOS expression and mRNA levels significantly increase in activated macrophages (PMA-treated U937 cells).
Conclusions:
- EOS is a novel serine protease localized to macrophages.
- The up-regulation of EOS in activated macrophages suggests its involvement in macrophage-mediated functions.
- Further research into EOS could elucidate its role in pathological conditions involving macrophages, such as inflammation and atherosclerosis.