ABSTRACT
A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.
Subject(s)
Crotalid Venoms/chemistry , Disintegrins/pharmacology , Viper Venoms/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Proliferation/drug effects , Cloning, Molecular , Crotalid Venoms/pharmacology , Disintegrins/biosynthesis , Disintegrins/isolation & purification , Endothelium, Vascular/cytology , Humans , Molecular Sequence Data , Platelet Aggregation Inhibitors/pharmacology , Sequence Alignment , Trimeresurus , Umbilical Veins/cytology , Viper Venoms/biosynthesis , Viper Venoms/isolation & purificationABSTRACT
Stejnitin, a novel class P-II snake venom metalloproteinase (SVMP) with a molecular weight of about 35kDa, was purified from Trimeresurus stejnegeri venom. The cDNA of stejnitin encoded a polypeptide of 295 amino acid residues which comprises a signal peptide, proprotein, metalloproteinase domain, spacer and disintegrin domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIa SVMPs which comprises metalloproteinase and disintegrin together. Results from DNA fragmentation and flow cytometry analysis also indicated that stejnitin is able to induce apoptosis of ECV304 cells (R=0.908, P=0.012).
Subject(s)
Crotalid Venoms/enzymology , Metalloproteases/chemistry , Amino Acid Sequence , Animals , Apoptosis/drug effects , Base Sequence , Cloning, Molecular , DNA Fragmentation , Flow Cytometry , Humans , Metalloproteases/genetics , Metalloproteases/isolation & purification , Molecular Sequence Data , Peptide Mapping , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/isolation & purification , Sequence AlignmentABSTRACT
Using new flight hardware, a Chinese mission of space protein crystallization has been performed aboard the Chinese spacecraft SZ-3. Preliminary analyses of the experimental results have shown that a few proteins produced better crystals in space. At least, the crystals of cytochrome b5 mutant could diffract X-ray beyond the highest resolution reported so far for the same kind of crystals. In addition, some rules derived from our numerical studies of the liquid/liquid diffusion protein crystallization were proved by the crystallization of lysozyme as model protein in this space experiment, which also clearly showed the advantages and disadvantages of the gelation of the protein solution used in microgravity growth of protein crystals.