Abstract
The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an example for this kind of enzymatic reactions.
Keywords: Complement system, C3, Cathepsin L, Cleavage
Materials and Reagents
Equipment
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Procedure
Notes
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Acknowledgments
This protocol was developed by Claudia Kemper and validated by Martin Kolev. The protocol was originally published in Liszewski et al. (2013). This work was supported by the MRC Research Grant G1002165 (C.K.) the MRC Centre for Transplantation Grant MR/J006742/1, an EU-funded Innovative Medicines Initiative BTCURE (C.K.), and a Wellcome Trust Investigator Award (C.K).
References
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