The Structure And Function Study Of Angiotensinogen

As a hormone precursor of all angiotensins,angiotensinogen(ANT)plays a key role in renin-angiotensin system(RAS)which is related with homeostasis balance and blood pressure.The action of renin on angiotensinogen is the initiative and rate-limiting process of the RAS.Angiotensinogen is a member of serine protease inhibitors(SERPINs),and its genes exist throughtout all vertebrates.Angiotensinogen proteins of higher vertebrates are non-inhibitory,but in ancient vertebrate lampreys,angiotensinogen is not only a hormone carrier in the regulation of blood pressure,but also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system.These detailed mechanisms on evolution characteristics are unclear.Here we have studied the characteristics of structure and function of angiotensinogen from several vertebrates at different evolution stages.We expressed and purified the angiotensinogen proteins,and determined the crystal structures of cleaved lamprey ANT(l-ANT)at 2.7? resolution and zebrafish ANT(z-ANT)at 2.16? resolution.Then we characterised the features of lamprey ANT in heparin binding and protease inhibition.Also we try to explain the mechanism of hormone release from ANT,especially the role of an important disulfide bond.The results show that lamprey ANT undergoes a typical stressed-to-relaxed conformational transformation during thrombin cleavage.Heparin binds lamprey ANT tightly at a widely positively charged surface region around helix D,and promotes thrombin inhibition by lamprey ANT with a conserved bridge mechanism.Lamprey ANT has a flexible N-terminal angiotensin peptide,but zebrafish,mouse and human ANT possess the partly buried N-terminal peptide hormone which is stabilized by a key disulfide bond.These two cystines are conservative in all vertebrate angiotensinogen proteins except for lampreys,and they could form disulfide linkage at least in bony fish like zebrafish.It is speculated that zebrafish ANT need rearrange its conformation to make the renin cleavage site accessible for proteolysis,similar to interaction between human ANT(h ANT)and human renin which was reported to relate with a redox swich of the key disulfide linkage during the conformational rearrangement of h ANT.Furthermore,we have found that the ratio of the reduced unbridged form and the oxidized sulphydryl-bridged form of angiotensinogen is different in human plasma and other animal plasma.In summary,the structures and functions of angiotensinogen are finely turned for various functions during the evolution.Our study improves understanding in the regulation of thrombin activity in the primitive coagulation system and explains the mechanism of redox-oxidation transition related angiotensin I release.This provides basis for further research in clinical diagnosis and treatment of hypertension related diseases.