Abstract
Kallistatin is a heparin-binding serine proteinase inhibitor (serpin), which specifically inhibits human tissue kallikrein by forming a covalent complex. The inhibitory activity of kallistatin is blocked upon its binding to heparin. In this study we attempted to locate the heparin-binding site of kallistatin using synthetic peptides derived from its surface regions and by site-directed mutagenesis of basic residues in these surface regions. Two synthetic peptides, containing clusters of positive-charged residues, one derived from the F helix and the other from the region encompassing the H helix and C2 sheet of kallistatin, were used to assess their heparin binding activity. Competition assay analysis showed that the peptide derived from the H helix and C2 sheet displayed higher and specific heparin binding activity. The basic residues in both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2 sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of these mutants by heparin-affinity chromatography showed that the heparin binding capacity of the variant K187A/K188A was not altered, whereas the binding capacity of K307A/R308A and K312A/K313A mutants was markedly reduced. Titration analysis with heparin showed that the K312A/K313A mutant has the highest dissociation constant. Like kallistatin, the binding activity of K187A/K188A to tissue kallikrein was blocked by heparin, whereas K307A/R308A and K312A/K313A retained significant binding and inhibitory activities in the presence of heparin. These results indicate that the basic residues, particularly Lys(312)-Lys(313), in the region between the H helix and C2 sheet of kallistatin, comprise a major heparin-binding site responsible for its heparin-suppressed tissue kallikrein binding.
Highlights
Kallistatin is a specific serine proteinase inhibitor for human tissue kallikrein [1]
The mechanisms have been explained by using a ternary complex and an allosteric model (5, 6, 10 –13). Unlike most of these serpins, heparin suppresses the inhibitory activity of kallistatin toward tissue kallikrein, whereas it accelerates the interaction between kallistatin and chymotrypsin [1]
Our previous study suggested that replacing the residues in the hinge region of the reactive loop with bulky residues may hinder a partial insertion of the reactive loop into A -sheet and converting kallistatin from an inhibitor to a substrate for tissue kallikrein
Summary
Kallistatin is a specific serine proteinase inhibitor (serpin) for human tissue kallikrein [1]. High specificity and selectivity of kallistatin for tissue kallikrein is determined by its unique Phe at P1 position and probably by other subsite-binding residues in the reactive-center loop [1]. Other than the reactivecenter loop, heparin-binding sites of a serpin can regulate its inhibitory activity toward target serine proteinase. For most of the heparinbinding serpins, heparin accelerates the inhibitory activity toward their target serine proteinases [5,6,7,8,9]. The mechanisms have been explained by using a ternary complex and an allosteric model (5, 6, 10 –13) Unlike most of these serpins, heparin suppresses the inhibitory activity of kallistatin toward tissue kallikrein, whereas it accelerates the interaction between kallistatin and chymotrypsin [1]. We attempted to identify the heparinbinding sites in kallistatin by two approaches: 1) to design synthetic peptides derived from surface regions of kallistatin that contain a high density of basic residues and to assess their heparin binding capacity; 2) to create kallistatin mutants by substituting basic residues with alanine in the putative hepa-
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