Abstract
Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.
Highlights
Kallistatin is a serpin that inhibits tissue kallikrein by forming a covalent serpin-proteinase complex (1)
We investigated the P3-P2-P1 residues of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling
Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr99 and Trp219 of tissue kallikrein
Summary
Materials—Escherichia coli strain TOP10, the pTrc-His B expression vector, was purchased from Invitrogen (San Diego, CA); the restriction enzymes, T4 kinase, calf intestinal alkaline phosphatase, Klenow fragment, and isopropylthio--galactoside were from Life Technologies, Inc.; Taq polymerase was from PerkinElmer Life Sciences; nickel-nitrilotriacetic acid-agarose was from Qiagen (Santa Clarita, CA); the POROS® HE/1 column was from PerSeptive Biosystems (Cambridge, MA); heparin was from The Upjohn Co.; D-Val-Leu-Arg-methylcoumarinamide (MCA) and phenylmethylsulfonyl fluoride were from Enzyme System Products (Livermore, CA); human tissue kallikrein was purified as described previously (29); anti-kallistatin monoclonal antibody was generated as described previously (30). The effects of heparin on the association rate were demonstrated by preincubation of the recombinant kallistatins with 20 units/ml heparin at 37 °C for 5 min followed by kinetic assays Serine proteinases, their respective substrates, and reaction buffers used in the assays were as follows: human tissue kallikrein (3 nM), rat tissue kallikrein (3 nM), and activated protein C (8 nM) with 30 M Val-Leu-Arg-MCA in 20 mM sodium phosphate, pH 8.0; human plasma kallikrein (3.5 nM), trypsin (2 nM), and plasmin (10 units/ml) with 30 M Pro-Phe-Arg-MCA in 20 mM sodium phosphate, pH 8.0, 0.1 M NaCl; human thrombin (1 nM) with 30 M Phe-Pro-Arg-MCA in 20 mM sodium phosphate, pH 8.0, 0.1 M NaCl; bovine pancreatic chymotrypsin (2 nM) with 30 M N-succinyl-Ala-Ala-Pro-Phe-MCA in 20 mM sodium phosphate, pH 8.0, 0.1 M NaCl; human neutrophil cathepsin G (70 nM) with 1.0 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 50 mM TrisHCl, pH 7.5, 100 mM NaCl; and human neutrophil elastase (20 nM) with 1.0 mM Glu-Pro-Arg-p-nitroanilide in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl. All of the reaction buffers contained 0.1% bovine serum albumin. Inhibition stoichiometry was measured from titrations of human tissue kallikrein with kallistatins in 50 mM Tris, pH 8.0, 0.1 M NaCl, 0.1% bovine serum albumin at 37 °C for 24 h
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