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Purification and Characterization of a C-type Lectin-related Protein from Gloydius Ussuriensis Venom

Author: CuiYan
Tutor: SunDeJun
School: Jilin University
Course: Biomedical Engineering
Keywords: Gloydius ussuriensis Snake venom C-type lectin-related protein (CLRPs) Separation and purification Procoagulant activity
CLC: Q51
Type: Master's thesis
Year: 2010
Downloads: 99
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Abstract

Gloydius ussuriensis are mainly distributed in the northeast of China, partly in the northern part of North Korea and Russian. Snake venom contains complicated chemical components, such as protein and peptide, amino acid, neutral lipid, monosaccharide, phosphate, biogenic amine, nucleoside and trace metal ions. Among of them, protein and peptide are the principal components of venom,which accounts for approximately 85-90 percent of the dry weight of the snake venom.C-type lectin-related proteins (CLRPs) are vital components of C-type lectin from snake venom. CLRPs contain not only heterodimer that is formed by a andβsubunits covalently, but also a number of oligo heterodimeric which are composed by multiple heterodimer with high molecular weight. The a and P subunits have analogical structure which both are made up by 123 to 135 amino acids that owned 40%homology, so they have analogical molecular weight, approximately 15kD. Functionally, C-type lectin-related proteins play multiple roles, for example they can act on platelets and most coagulation factors, thus regulate the blood coagulation system, revealed anti-coagulation, induction or inhibition of platelet aggregation.Nowadays as the C-type lectin-related proteins from snake venom have the conspicuous features on spatial structure and the biological function, the C-type lectin-related proteins have become a research hotspot in recent years. A number of C-type lectin-related proteins from different sources of snake venom have been found in the domestic and foreign studies,. while there have rarely reportes about C-type lectin-related protein from Gloydius ussuriensis venom.Objective:In this study, IDA-Sepharose FF affinity chromatography and Sephadex G-100, Sephadex G-50 gel filtration chromatography and other methods have been used to isolate a new C-type lectin-related protein from Gloydius ussurensis venom. By SDS-PAGE and determination of coagulation activity of fibrinogen, hydrolyzing activity and many other biological assay, we detected and assessed the protein’s structural features and biological functions.Content:Utilization of IDA-Sepharose FF affinity chromatography and Sephadex G-100,Sephadex G-50 gel-filtration chromatography to separate and purify a novel protein from Gloydius ussuriensis venom. We detected and assessed the protein’s structural features and biological functions by SDS-PAGE and determination of coagulation activity of fibrinogen, procoagulant activity and many other biological assays. According to the experimental results we preliminary classify the protein as C-type lectin-related protein.In the purification process,we used the IDA-Sepharose FF affinity chromatography for preliminary separation. In this study we pioneered a new approach that utilized IDA-Sepharose FF affinity chromatography as an independent mean to purify the proteins containing metal ligand specificly and the results achieve more satisfactory. At the same time we have tried a metal ion as the eluent, using uniform concentration and gradient concentration elution and other IDA-Sepharose FF affinity chromatography conditions for exploration. This provided a new way of affinity chromatography design.And then by Sephadex G-100, Sephadex G-50 gel filtration chromatography for further purification, reducing and non-reducing SDS-PAGE showed a single band The calculated molecular weight of reducing conditions is about 15.7kD. Although the reduction and non-reducing conditions of electrophoresis patterns showed a single band, the composition of C-type lectin-related proteins have very similar molecular weight that means it was difficult to judge whether it was the homodimer or heterodimer by electrophoretic which required further experimental work to confirm.Using Coomassie brilliant blue staining, we measured the protein concentration of the obtained solution that contain C-type lectin-related proteins,the concentration of the protein solution is 1.025mg/ml.In fibrinogen coagulation activity tests, we found Gloydius ussuriensis C-type lectin-related protein can promote the coagulation of fibrinogen. MnCl2 and CaCl2 can significantly reduced fibrinogen clotting time. These results indicates Gloydius ussuriensis C-type lectin-related proteins contain metal ion binding sites.,and that the site may be associated with the activity of the protein. While the reducing agent (DTT) significantly prolonged the clotting time of fibrinogen, suggesting that the hydrolytic activity of protein is related to the disulfide bonds.In addition, in the assay of hydrolytic activity of fibrinogen we found Gloydius ussuriensis C-type lectin-related protein first hydrolyzed the a-chain and then degradated theβ-chain, but not y-chain. This activity is also completely inhibited by DTT. This proved that the hydrolytic activity is related with disulfide bonds once again.The in vitro coagulation activity assay of Gloydius ussuriensis venom C-type lectin-related protein revealed that the protein had a direct procoagulant activity, And the protein also had procoagulant effect on the platelet rich plasma and platelet-poor plasma. Combination the result of the effect of Gloydius ussuriensis C-type lectin-related protein on fibrinogen, we can conclude that procoagulant activity is related with the activity of hydrolytic fibrinogen.In the phospholipid hydrolytic activity assay,Gloydius ussuriensis C-type lectin-related protein showed weak phospholipid hydrolysis activity. Along with the increase of sample concentration, phospholipid hydrolytic activity is increased.In addition,Gloydius ussuriensis venom C-type lectin-related protein showed no hemorrhagic activity, edema activity and fibrinolytic hydrolysis activity.Conclusion:In this study,a novel C-type lectin-related protein was isolated from Gloydius ussuriensis snake venom. The protein was consisted of two subunits combinded by disulfide bonds between chains, the molecular weight of which is about 15.7kD. The results on biological activity of protein demonstrate that this protein posess direct procoagulant activity which related to the agglomeration of fibrinogen. Furthermore this protein has weak phospholipid hydrolytic activity, whereas little hemorrhagic activity, edema activity and fibrin hydrolysis activity.Comparing with other sources of snake venom C-type lectin-related protein,Gloydius ussuriensis venom C-type lectin-related protein has a direct procoagulant activity, which was first time identified in the C-type lectin-related protein family.Therefore,this result provides a good material for revealing the structure and function mechanism of the C-type lectin-related protein family. Since the protein has a good procoagulant activity, it can be expected to be developed as a hemostatic agent, which will promote the industry of procoagulant drugs Relevant experimental material is applied for patent.

Purification and Characterization of a C-type Lectin-related Protein from Gloydius Ussuriensis Venom

Abstract

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