The WXXW motif in the TSR1 of ADAMTS13 is important for its secretion and proteolytic activity

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• 作者：Jing Ling ; Jian Su ; Zhenni Ma ; Changgeng Ruan ; ^{changgengruan@hotmail.com}
• 关键词：ADAMTSs ; a disintegrin and metalloproteinase with thrombospondin motifs ; pVWF ; Plasma-derived human VWF ; RIPA ; radioimmune precipitation assay buffer ; PMSF ; phenylmethylsulfonyl fluoride ; PBS ; phosphate-buffered saline ; BSA ; bovine serum albumin ; TSR ; thr
• 刊名：Thrombosis Research
• 出版年：2013
• 出版时间：June, 2013
• 年：2013
• 卷：131
• 期：6
• 页码：529-534
• 全文大小：402 K

文摘

Introduction

ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) is a metalloprotease enzyme that regulates the size and activity of the von Willebrand factor (VWF). ADAMTS13, like many other ADAMTSs, has a WXXW motif in its thrombospondin type 1 repeat domain (TSR1). However, the function of the WXXW motif in ADAMTSs is unclear.

Materials and Methods

The constructs of wild-type (WT) and WXXW mutant (W387A) ADAMTS13 was generated by PCR site-directed mutagenesis. The secretion of the protein was quantified with western blotting methods. The binding affinity of the WT or W387A mutant ADAMTS13 with Plasma-derived human VWF (pVWF) was investigated by using enzyme linked immunosorbent assay. The Cleaving activity of the WT or W387A mutant ADAMTS13 against full length pVWF was measured under denatured conditions or shear stress. The proteolytic activity was also validated with the FRETS-VWF73 assay.

Results

The W387A mutant was secreted less efficiently and had a reduced binding affinity for pre-denatured pVWF in comparison to WT ADAMTS13. However, both the WT and mutant ADAMTS13 interacted equally with native pVWF. The W387A mutant showed less cleaving activity against VWF under denaturing conditions, and the same result was observed when the fluorescence resonance energy transfer substrate VWF73 (FRETS-VWF73) was used as the substrate. However, under high shear stress conditions the mutant and WT ADAMTS13 were equally able to cleave VWF.

Conclusion

The WXXW motif is important for the secretion of ADAMTS13 and that it modulates the proteolytic cleavage of VWF by ADAMTS13 under denaturing conditions.