血管性血友病因子裂解酶CysR结构域生物学功能的研究

Biological Function of CysR Domain of ADAMTS13

目的:研究血管性血友病因子裂解酶(ADAMTS13)富含半胱氨酸(CysR)结构域在血管性血友病因子(vWF)裂解中的生物学功能,为探明血栓性血小板减少性紫癜(TTP)的发病机制提供实验证据。方法:利用点突变技术对ADAMTS13 CysR结构域中的EDGTLS氨基酸残基进行基因突变,制备质粒,表达并提纯蛋白。1%Sea Kem HGT琼脂糖凝胶分离后用Western blot方法观察野生型和突变型ADAMTS13在变性条件或剪切应力作用下对底物v WF的裂解活性。结果:成功构建了ADAMTS13突变体质粒(M1:Glu515Ala;M2:Asp516Ala;M3:Gly517Ala;M4:Thr518Ala;M5:Leu519Ala;M6:Ser520Al)。野生型和突变型ADAMTS13质粒在HEK293细胞中稳定表达,并提纯蛋白。在变性条件下,野生型ADAMTS13基本完全裂解了v WF多聚体,而突变体M1裂解v WF多聚体的活性显著降低(P <0.01)。在体外剪切应力作用下,与野生型ADAMTS13相比,突变体M1对v WF多聚体的裂解能力显著降低(P <0.01)。突变体M1对FRETS-vWF73的剪切能力与野生型ADAMTS13相比显著降低。与野生型ADAMTS13相比,突变体M1与v WF之间的结合力未见明显降低。结论:在变性条件和剪切应力作用下,ADAMTS13 CysR结构域在酶切v WF多聚体的过程中起着重要作用,其中Glu515可能是底物识别的重要作用位点。

Objective:To investigate the biological function of Cysteine rich(CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13(ADAMTS13) on cleavage of von Willebrand factor(vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura(TTP).Methods:The six amino acids(EDGTLS) in ADAMTS13 CysR domain were point mutated one by one,and the mutant ADAMTS13 proteins were expressed and purified.The cleavage products of vWF polymer by wild-type or mutant AD AMTS 13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot.Results:The mutant ADAMTS13 plasmids(M1:Glu515 Ala;M2:Asp516 Ala;M3:Gly517 Ala;M4:Thr518 Ala;M5:Leu519 Ala;M6:Ser520 Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified.Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition,while the cleavage activity of M1 mutant was significantly reduced in the same condition(P <0.01).The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress(P <0.01).The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13.However,the binding ability of M1 mutant to vWF was similar with that of wildtype ADAMTS13.Conclusion:The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress.The Glu515 amino acid residue might be an important site for substrate recognition.