Structural Mechanism Of Heparin Regulate The Activity Of Kallistatin

The Kallikrein-Kinin System is involved in many physiological processes including inflammation,cancer,blood pressure control and coagulation.Kallikrein cleaves Kininogen to release Kinin,which binds its receptors on the surface of vascular endothelial cells to stimlate vasodilation.Kallistatin(KAL)is a Kallikrein specific inhibitor and its interaction with Kallikrein is inhibited by heparin through an unclear mechanism.In this thesis I have addressed these Kallistatin mediated interactions by solving the structures of human kallistain and by kinetic study of heparin mediated kallikrein inhition.We have prepared recombinant human KAL from E coli and HEK293 expression systems and solved the crystal structures of glycosylation native KAL,Cleaved-Kallistatin and its complex with heparin complex at 3.0,1.9 and 1.8 A resolution respectively.Kallistatin is a member of the serpin superfamily and shares a conserved SERPIN fold which consists of a central beta-sheet and a surface exposed reactive central loop(RCL).The RCL of Kallistain is partially inserted into the central beta-sheet,however once the RCL is cleaved by protease,KAL undergoes typical serpin conformational changes where the RCL is fully incorporated into the central beta-sheet.KAL has a unique four-residue insertion(R306KRN309)between H ? helix(hH)and the strand 2 of ?-sheet C(s2C)which forms a unique 310 helix conformation.This helix is stablized by neibouring cation-? interactions from the adjacent residues(W254,R256,F277 and Y311).Notably,the crystal structure of heparin-KAL complex showed that heparin binds KAL near this unique 310 helix,where R259 of KAL forms three ionic bonds with the two sulfate groups of the heparin oligosaccharide,R300 forms two ionic bonds with sulfonic acid groups of the heparin oligosaccharide.Subsequent mutageneis and heparin bidnign assessment showed that K308,R308,K313,K313,R259,R300,R394 and H395 of KAL are involved in heparin binding and the most important residues are R256 and R394.Previous studies have shown that KAL can specifically inhibit kallikrein 1(KLK1)and heparin can completely inhibit the interaction between them,here we confirmed this.And also we found that KAL can efficiently inhibit the protease activity of kallikrein 7(KLK7) and most interestingly,heparin promotes the reaction rate of KAL with KLK7 by about 23 folds.This makes KAL the most potent KLK7 inhibitor currently known.To disect the protease-dependent regulation of KAL activity by heparin,we systematically analyzed the molecular properties of KLK1 and KLK7 through site-directed mutagenesis and enzyme kinetic studies.First,we found that the promotion of the interaction between KAL and KLK7 by heparin depends on the positively charged residues in the 70-80 loop region of the KLK7 molecule.Mutation of these amino acids leads to the loss of the promoting effect of heparin.We also found that the acceleration effect of heparin requires heparin with more that no less than 8 saccharide units.This suggests that heparin promotes the reaction between the two proteins though a template mechanism where a snglye heparin chain binds KAL and KLK7 simutaneously.Secondly,we found that KLK1 is an acidic protease with a largely negatively charged surface.When the 70-80 loop of KLK1 is replaced by that of KLK7,heparin can effectively promote the inhibition of KLK1 mutant by KAL.This suggests when heparin binds on KAL,it will expel KLK1 from docking on top of the top of KAL by "electrostatic repulsion" where heparin is also negatively charged.In summary,KALinhibits its target proteases though the classical SERPIN conformation changes.Heparin binds to a unique heparin binding site on KAL and regulates KAL's specificity towards targt proteases through template effect or simple electrostatic repulsion.In addition,these results also indicate that under physiological conditions,KAL anchored on the surface of endothelial cells where heparin or heparan sulphate is located is a sole inhibitor of KLK7.Altogether,this stsudy provides further knoweledge on the regulation of kallikrein-kinin system by heparin and sheds lights on the application of heparin and KAL in treating KLK7-overexpression related squamous lesions(Netherton Syndrome).