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Identification of elastase as a secretory protease from cultured rat microglia
K Nakajima1, M Shimojo, M Hamanoue
1Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.
Abstract:
In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.
Insights
Microglia secrete proteases, including elastase, which digest various proteins. This enzyme
Area of Science:
- Neuroscience
- Biochemistry
- Immunology
Background:
- Microglia are immune cells in the central nervous system.
- Their secretory products are crucial for brain function and disease.
- Protease activity in microglial secretions was previously uncharacterized.
Purpose of the Study:
- To identify and characterize proteases secreted by microglia.
- To investigate the de novo synthesis and regulation of these proteases.
Main Methods:
- Protease activity assays using myelin basic protein.
- Enzyme inhibition studies with specific protease inhibitors.
- Gel filtration and Western blotting for protein identification.
- [35S]methionine incorporation for synthesis analysis.
Main Results:
- Microglial conditioned medium exhibited protease activity, digesting various proteins.
- The major secreted protease displayed optimal activity at neutral pH and was identified as elastase or an elastase-like protease.
- Elastase was synthesized de novo by microglia and its secretion decreased in the presence of lipopolysaccharide.
Conclusions:
- Microglia actively secrete functional proteases, notably elastase.
- This finding highlights a novel role for microglia in extracellular matrix remodeling and protein degradation.
- The regulation of microglial elastase secretion has implications for neurological health and disease.