Functional Analysis of a Peptide Sequence (Thr323 - Cys337) In ADAMTS13 Disintegrin-Like Domain Revealed Two Discontinuous Sequences Involved In the Interaction with VWF
Authors: Igari Atsuko , Moriki Takanori , Nakagawa Terumichi , Yamaguchi Yusuke , Murata Mitsuru 
Published: October 2019

Abstract
Abstract 2200
ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug.

Disclosures:
No relevant conflicts of interest to declare.