Biological Function Of Spacer Domain Of ADAMTS13

Objective: A disintegrin and metalloprotease with thrombospondin type 1 repeats-13(ADAMTS13)plays an important role in the pathogenesis of thrombotic thrombocytopenic purpura(TTP).The abnormal function of ADAMTS13 may lead to the occurrence of TTP,but the mechanism of ADAMTS13 in TTP is still unclear.Therefore,this experiment studied the biological function of Spacer domain in ADAMTS13 during the cleavage of von Willebrand factor(v WF),so as to provide a theoretical basis for the study of the pathogenesis of TTP.Methods: 1.Mutation of TEDRLPR amino acid residues in ADAMTS13 Spacer domain one by one using point mutation technology.The mutant plasmids were prepared,and the successfully constructed ADAMTS13 and its mutant plasmids were transfected into HEK293 cells,western blot was used to check the protein expression,and G418 was used to screen and collect the recombinant protein supernatant of ADAMTS13 and its mutants stably expressed in cells.The purified recombinant protein was finally obtained through Q-fast flow ion binding column,Ni-NTA affinity chromatography column and molecular sieve.2.The recombinant protein was separated on 1% Sea Kem HGT agarose gel,and then the wild-type and mutant ADAMTS13 were observed by western blotting under the denaturing conditions of 1.5M urea or guanidine-HCl or under the shear stress generated by the shaker or after adding ADAMTS13 antibody.Observe the cleavage activity on the substrate v WF.Results: 1.ADAMTS13 mutant plasmids were successfully constructed(M1: Thr632A;M2: Glu633A;M3: Asp634A;M4: Arg635A;M5: Leu636A;M6: Pro637A;M7: Arg638A).2.Wild-type and mutant ADAMTS13 plasmids were stably expressed in HEK293 cells,and the protein was purified.3.The splicing ability of FRETS-v WF73 by mutants M4 and M7 was significantly lower than that of wild-type ADAMTS13(P<0.05).4.Under denaturing conditions,wild-type ADAMTS13 could basically completely cleave v WF multimers,and mutants M4 and M7 had significantly lower cleavage activities of v WF multimers(P<0.01)5.Under the action of shear stress in vitro,the ability of mutant M4 and M7 to cleave v WF multimers was significantly lower than that of wild-type ADAMTS13(P < 0.01).6.Compared with wild-type ADAMTS13,the binding force between mutant M4 and M7 to v WF was not significantly reduced,indicating that there are multiple binding sites between the C-terminus of ADAMTS13 and v WF.7.After adding ADAMTS13 antibody,the function of wild-type and mutant ADAMTS13 was inhibited to a certain extent.Conclusions:1.After the mutation of the Spacer region of ADAMTS13,its activity decreased.2.M4(Arg635)and M7(Arg638)may be the important role sites of ADAMTS13 in substrate recognition.