Characterization of a novel variant of the second domain of bikunin with increased leukocyte elastase inhibitory activity


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.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Esmon, C.T. (2003) Inflammation and thrombosis. Journal of Thrombosis and Haemostasis, 1, 1343-1348.
[2] Mann, K.G. (1999) Biochemistry and physiology of blood coagulation. Journal of Thrombosis and Haemostasis, 82, 165-174.
[3] Gailani, D. and Renne, T. (2007) The intrinsic pathway of coagulation: A target for treating thromboembolic disease? Journal of Thrombosis and Haemostasis, 5, 1106-1112.
[4] Tucker, E.I., Gailani, D., Hurst, S., Cheng, Q., Hanson, S.R. and Gruber, A. (2008) Survival advantage of coagulation factor XI-deficient mice during peritoneal sepsis. Journal of Infectious Diseases, 198, 271-274.
[5] Martorell, L., Martinez-Gonzalez, J., Rodriguez, C., Gentile, M., Calvayrac, O. and Badimon, L. (2008) Thrombin and protease-activated receptors (PARs) in atherothrombosis. Journal of Thrombosis and Haemostasis, 99, 305-315.
[6] Opal, S.M. and Esmon, C.T. (2003) Bench-to-bedside review: Functional relationships between coagulation and the innate immune response and their respective roles in the pathogenesis of sepsis. Critical Care, 7, 23-38.
[7] Lee, W.L. and Downey, G.P. (2001) Leukocyte elastase: Physiological functions and role in acute lung injury. American Journal of Respiratory and Critical Care Medicine, 164, 896-904.
[8] Hirose, J., Ozawa, T., Miura, T., Isaji, M., Nagao, Y., Yamashiro, K., Nii, A., Kato, K. and Uemura, A. (1998) Human neutrophil elastase degrades inter-alpha-trypsin inhibitor to liberate urinary trypsin inhibitor related proteins. Biological and Pharmaceutical Bulletin, 21, 651-656.
[9] Pugia, M.J. and Lott, J.A. (2005) Pathophysiology and diagnostic value of urinary trypsin inhibitors. Clinical Chemistry and Laboratory Medicine, 43, 1-16.
[10] Fries, E. and Kaczmarczyk, A. (2003) Inter-alpha-inhibitor, hyaluronan and inflammation. Acta Biochimica Polonica, 50, 735-742.
[11] Fries, E. and Blom, A.M. (2000) Bikunin—Not just a plasma proteinase inhibitor. The International Journal of Biochemistry & Cell Biology, 32, 125-137.
[12] Xu, Y., Carr, P.D., Guss, J.M. and Ollis, D.M. (1998) The crystal structure of bikunin from the inter-alpha-inhibitor complex: A serine protease inhibitor with two Kunitz domains. Journal of Molecular Biology, 276, 955-966.
[13] Morishita, H., Yamakawa, T., Matsusue, T., Kusuyama, T., Sameshima-Aruga, R., Hirose, J., Nii, A., Miura, T., Isaji, M., Horisawa-Nakano, R., Nagase, Y., Kanamori, T., Nobuhara, M., Tanaka, R., Koyama, S. and Naotsuka, M. (1994) Novel factor Xa and plasma kallikrein inhibittory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. Thrombosis Research, 73, 193-204.
[14] Nii, A., Morishita, H., Yamakawa, T., Matsusue, T., Hirose, J., Miura, T., Isaji, M., Horisawa, Y., Sugihara, K., Kanamori, T. and Umeyama, H. (1994) Design of variants of the second domain of urinary trypsin inhibitor (R-020) with increased factor Xa inhibitory activity. Journal of Biochemistry, 115, 1107-1112.
[15] Creighton, T.E. (1992) Proteins: Structures and molecular properties. W. H. Freeman and Company, New York, 253-255.
[16] Rawlings, N.D. and Barrett, A.J. (1994) Families of serine peptidases. In: Barrett, A.J., Ed., Methods in Enzymology. Academic Press, San Diego, 19-61.
[17] Powers, J.C., Kam, C.M., Narasimhan, L., Oleksyszyn, J., Hernandez, M.A. and Ueda, T. (1989) Mechanism-based isocoumarin inhibitors for serine proteases: Use of active site structure and substrate specificity in inhibitor design. Journal of Cellular Biochemistry, 39, 33-46.
[18] Marquart, M., Walter, J., Deisenhofer, J., Bode, W. and Huber, R. (1983) The geometry of the reactive site and of the peptide groups in trypsin, trypsinogen and its complexes with inhibitors. Acta Crystallographica Section B, 39, 480-490.
[19] Miyata, T., Sakai, T., Sugimoto, M., Naka, H., Yamamoto, K., Yoshioka, A., Fukui, H., Mitsui, K., Kamiya, K., Umeyama, H. and Iwanaga, S. (1991) Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities. Biochemistry, 30, 11286-11291.
[20] Landt, O., Grunert, H.P. and Hahn, U. (1990) A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene, 96, 125-128.
[21] Messing, J. and Vieira, J. (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene, 19, 269-276.
[22] Navia, M.A., McKeever, B.M., Springer, J.P., Lin, T.Y., Williams, H.R., Fluder, E.M., Dorn, C.P. and Hoogsteen K. (1989) Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84A resolution. PNAS, 86, 7-11.
[23] Nakatani, K., Takeshita, S., Tsujimoto, H., Kawamura, Y. and Sekine, I. (2001) Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury. Journal of Leukocyte Biology, 69, 241-247.
[24] Navaneetham, D., Jin, L., Pandey, P., Strickler, J.E., Babine, R.E., Abdel-Meguid, S.S. and Walsh, P.N. (2005) Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2. Journal of Biological Chemistry, 280, 36165-36175.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.