Enzymatic activities and functional characterization of a novel recombinant snake venom proteinase from Agkistrodon acutus.
Snake venom from Agkistrodon acutus consists of a number of compounds which may potentially be used as drugs. However, it is hard to obtain enough pure protein for drug development. Recently, we reported expression and purification of a novel recombinant fibrinogenase which was named rFII. Here we reported for the first time the enzymatic activities and functional characterization of rFII. Circular dichroism spectra showed the gross conformation of FIIa and rFII to be notably similar. It is an alkaline proteinase and the amino acid sequence exhibits a high degree of sequence identity with other snake venom metalloproteinases. rFII also exhibits amidase activity against N-(p-Tosyl)-Gly-Pro-Lys-p-nitroanilide, which is specified synthetic substrate for plasmin. Functional characterization showed that rFII possesses both fibronectin and type IV collagen cleaving activities. In addition, rFII preferentially cleaved the Aalpha-chain of fibrinogen, followed by the Bbeta-chain and finally, the gamma(gamma) chain was affected. Furthermore, rFII was also capable of cleaving fibrin without plasminogen activation and suppressing ADP-induced platelet aggregation. The proteolytic activity of rFII was inhibited completely by PMSF and mostly by EDTA. The cations Ca(2+), Mg(2+), Na(+), K(+) didn't affect its proteolytic activity, while Cu(2+) and Zn(2+) slightly inhibited this activity. Study of hydrolysis of oxidized insulin B-chain reveals that rFII preferentially cleaved oxidized insulin B-chain at the site of Val(12)-Glu(13), Leu(15)-Tyr(16), and Phe(24)-Phe(25).
Jiang
Weijian
W
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, PR China.
Ma
Tao
T
Su
Xingwen
X
Qiu
Pengxin
P
Yan
Guangmei
G
D017836:Agkistrodon / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D024141:Collagen Type IV / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D045726:Metalloproteases / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014757:Viper Venoms / Q000235:genetics / Q000378:metabolism*
D002614:Chelating Agents; D024141:Collagen Type IV; D003435:Crotalid Venoms; D005353:Fibronectins; D007328:Insulin; D011994:Recombinant Proteins; D014757:Viper Venoms; D005340:Fibrinogen; D004492:Edetic Acid; D010450:Endopeptidases; D045726:Metalloproteases