Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom.
A snake venom thrombin-like enzyme (SVTLE) 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 Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da 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 of 1.75 Å. 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 the 99-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 EC(50) of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca(2+)](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca(2+) releasing from Ca(2+) store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026, People's Republic of China.
Fuxing
F
Zeng
Bing
B
Shen
Zhongliang
Z
Zhu
Ping
P
Zhang
Yonghua
Y
Ji
Liwen
L
Niu
Xu
X
Li
Maikun
M
Teng
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism / Q000494:pharmacology; D020134:Catalytic Domain; D002417:Cattle; D002478:Cells, Cultured; D003435:Crotalid Venoms / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005342:Fibrinolysis / Q000187:drug effects; D006031:Glycosylation; D006868:Hydrolysis; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects; D014757:Viper Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D011480:Protease Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D014757:Viper Venoms; D001120:Arginine; C009727:benzoylarginine ethyl ester; D002265:Carboxylic Ester Hydrolases; C019763:benzoylarginine ethyl esterase; C583442:AhV_TL-I protein, Agkistrodon halys; D012697:Serine Endopeptidases; D013917:Thrombin