Application of directed evolution and back-to-consensus algorithms to human alpha1-antitrypsin leads to diminished...

Primarily known as an elastase inhibitor, human alpha1-antitrypsin also exerts anti-inflammatory and immunomodulatory effects, both in vitro and in vivo. While the anti-protease mechanism of alpha1-antitrypsin is attributed to a particular protein domain coined the reactive center loop, anti-inflammatory and immunomodulatory loci within the molecule remain to be identified. In the present study, directed evolution and back-to-consensus algorithms were applied to human alpha1-antitrypsin. Six unique functional candidate sites were identified on the surface of the molecule; in manipulating these sites by point mutations, a recombinant mutant form of alpha1-antitrypsin was produced, depicting a requirement for sites outside the reactive center loop as essential for protease inhibition, and displaying enhanced anti-inflammatory activities. Taken together, outcomes of the present study establish a potential use for directed evolution in advancing our understanding of site-specific protein functions, offering a platform for development of context- and disease-specific alpha1-antitrypsin-based therapeutics.