Identification of a major heparin-binding site in kallistatin
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Data
2001-01-12
Autores
Chen, V. C.
Chao, L.
Pimenta, Daniel Carvalho [UNIFESP]
Bledsoe, G.
Juliano, Luiz [UNIFESP]
Chao, J.
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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 Il 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 Ca 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/308A 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.
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Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 276, n. 2, p. 1276-1284, 2001.