A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): purification and characterization.
A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH2-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5'RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10(-3)mol/L and 4.09 × 10(-3)μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.
School of Life Science & Biotechnology, Dalian University of Technology, Dalian 116024, China.
Peng
P
Geng
School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China.
Lan
L
Lin
School of Life Science & Biotechnology, Dalian University of Technology, Dalian 116024, China.
Yuan
Y
Li
Dalian Institute of Technology, Liaoning Academy Agriculture Science, Dalian 116023, China.
Qi
Q
Fan
Shanxi Academy of Sericultural Science, Yuncheng 044000, China.
Naihong
N
Wang
School of Life Science & Biotechnology, Dalian University of Technology, Dalian 116024, China.
Lixin
L
Song
School of Life Science & Biotechnology, Dalian University of Technology, Dalian 116024, China. Electronic address: biolwl@dlut.edu.cn.
Wenli
W
Li
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002681:China; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D019476:Insect Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013927:Thrombosis / Q000378:metabolism / Q000517:prevention & control; D014357:Trypsin / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins; D005337:Fibrin; D005340:Fibrinogen; D001120:Arginine; C009727:benzoylarginine ethyl ester; D004950:Esterases; D010447:Peptide Hydrolases; C588098:cocoonase, Antheraea pernyi; D014357:Trypsin
Antheraea pernyi; Antithrombosis; Fibrin(ogen)olytic protease