Effect of ADAMTS13 spacer domain mutations on biological function of enzyme
10.13481/j.1671-587X.20240403
- VernacularTitle:血管性血友病因子裂解酶间隔区结构域突变对酶生物学功能的影响
- Author:
Meng WANG
1
;
Hao WU
;
Hua LI
;
Yihong ZHAO
;
Shengyu JIN
Author Information
1. 延边大学附属医院血液内科,吉林延吉 133000
- Keywords:
Von Willebrand factor lyase;
ADAM metalloproteinase with thrombospondin type 1 motifs 13;
Spacer domain;
Von Willebrand factor;
Thrombotic thrombocytopenic purpura
- From:
Journal of Jilin University(Medicine Edition)
2024;50(4):900-907
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To discuss the biological function of the spacer domain of ADAM metalloproteinase with thrombospondin type 1 motifs 13(ADAMTS13)in the cleaving process of von Willebrand factor(vWF),and to clarify the role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura(TTP).Methods:The point mutation method was introduced sequentially into the amino acid residues TEDRLPR of the ADAMTS13 spacer domain(mutants M1-M7)by site-directed mutagenesis.The constructed ADAMTS13 and its mutants plasmids were transfected into the human embryonic kidney HEK293 cells,and the recombinant proteins were purified after stable expression.The cleavage capabilities of both wild type and mutant ADAMTS13 were observed under denaturation conditions,shear stress,and after treatment with ADAMTS13 antibodies.Results:The fluorescence resonance energy transfer(FRET)assay results showed that compared with wild type ADAMTS13,the cleavage abilities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)on the FRET-vWF73 were decreased(P＜0.05).Under denaturation conditions,the wild-type ADAMTS13 could cleave the vWF multimers;compared with wild-type ADAMTS13,the cleavage activities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)were significantly decreased(P＜0.01).Under in vitro shear stress,compared with wild type ADAMTS13,the abilities of ADAMTS13 mutant M4(R635A)and mutant M7(R638A)to cleave vWF multimers were significantly decreased(P＜0.01).Compared with wild type ADAMTS13,the binding affinity between vWF and ADAMTS13 mutant M4(R635A)and mutant M7(R638A)had no significant difference(P＞0.05),indicating there were multiple binding sites between C-terminal of ADAMTS13 and vWF.The ADAMTS13 antibodies were able to inhibit the cleavage ability of both wild-type and mutant ADAMTS13 to some extent.Conclusion:The activity of ADAMTS13 after spacer domain mutation is decreased.The ADAMTS13 mutant M4(R635A)and mutant M7(R638A)may be the important action sites for AD AMTS 13 in substrate recognition.
