ABSTRACT
The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.
Share and Cite:
Nakamura, M. , Takeuchi, T. , Maeda, Y. , Hosaka, Y. and Furusako, S. (2012) Characterization of a novel variant of the second domain of bikunin with increased leukocyte elastase inhibitory activity.
Journal of Biophysical Chemistry,
3, 132-141. doi:
10.4236/jbpc.2012.32015.