The Structural-functional Research Of AhV_TL-I And Four Kinds Of PLA₂

Abundant protease have been isolated and characterized from snake venoms, such as snake venom thrombin-like enzyme and snake venom phospholipase A2(PLA2). Thrombin-like enzymes form snake venoms belong to a class of serine proteases that can cause blood clotting in vitro, but render the blood uncoagulable when acting in vivo apparently by depleting the circulating fibrinogen. PLA2is one of the major components of snake venom that hydrolyzes glycerophospholipids at the sn-2position of the glycerol backbone releasing lysophospholipids and fatty acids. Besides the enzymatic activity, snake thrombin-like enzyme and snake venom PLA2can also induce a large variety of pharmacological and toxic effects, such as myotoxicity, neurotoxicity, hemolysis, bleeding and effects on platelet aggregation.In vascular smooth muscle cells, Ca2+release via IP3receptors (IP3Rs) and ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) Ca2+store contributes significantly to the regulation of cellular events such as gene regulation, growth and cellular contraction. Ca2+release from SR increses cytoplasmic [Ca2+], and then activates myofilaments so as to generate muscle contraction, which is defined as excitation-contraction coupling.A snake venom thrombin-like enzyme from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aa and Bβ chains of bovine fibrinogen. AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined and the molecular mass was confirmed to29389.533Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of1.75A. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the99-loop and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC50of147nmol·L-1. Besides, the vasoconstrictor effect of AhV_TL-I was also independent of the enzymatic activity. The results of [Ca2+]i measurement showed that the vasoconstrictor effect of AhV_TL-I was attributed to Ca2+releasing from Ca2+store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). From A. halys pallas venom, we also purified two snake venom PLA2s, which named as AhV_bPA and AhV_aPA. Meanwhile, another two PLA2s, PA2-Vb and CTs-R6were isolated from Trimeresurus stejnegeri venom. The two acidic PLA2s, AhV_aPA and PA2-Vb, have highly enzymatic activity. The basic PLA2, AhV_bPA, show a lower enzymatic activity than the acidic isoforms. While, CTs-R6has no hydrolyses activity for it is a Asn49type PLA2. The structure of CTs-R6shows that it has no Ca2+-binding site. The structural analysis between AhV_aPA and AhV_bPA indicated the asymmetric positively charge distribution in i-face of AhV_bPA may attribute to the difference in enzymatic activity. The dimerization of PLA2is related to the Ca2+-loop and C-loop in AhV_bPA and is related to i-face in PA2-Vb. A glycerophosphate was mapped in the structure of AhV_bPA, which offers new insights into the binding of substrate in PLA2. The tension measurement of mouse thoracic aortic rings indicated that AhV_bPA, AhV_aPA and PA2-Vb could remarkably induce a contractile response of the vessel rings. The vasoconstrictor effects of these three PLA2are attributed to Ca2+release from Ca2+store. pBPB, which can completely inhibit the enzymatic activity of PLA2, did not significantly reduce the contractile response on vessel rings induced by the three PLA2s. The vasoconstrictor effects were strongly reduced by preincubation with hepain, indicating that the basic amino acid residues in the C-terminal of PLA2may be involved in the binding of these PLA2s to the molecular receptor to induce the vasoconstrictor effect. Further studies showed that Ca2+release from SR was related to the activation of IP3Rs but not RyRs.It is hypothesized that AhV_TL-I and the three snake venom PLA2s can bind to certain receptors in the membrance of smooth muscle cells. The released messengers and the physical connection between receptors and RyRs (or IP3Rs) active RyRs (or IP3Rs) and induce the Ca2+-release from SR. These results offer new insights into the functions of snake venom thrombin-like enzyme and PLA2and provide a novel pathogenesis of A. halys pallas venom and T. stejnegeri venom.