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N.A.G. Graça et al.
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Figure 7. Models of three-dimensional structure and surface charge of ADAMTS13 spacer domain variants included in this study. (A-F) Three-dimensional structure model of the spacer domain of: (A) wild-type ADAMTS13 (RFRYY); (B) triple-alanine mutant variant (RARAA); (C). 5x alanine mutant variant (AAAAA); (D) gain-of-func- tion variant (KYKFF); (E) triple-asparagine non-conservative mutant variant (RNRNN); and (F) semi-conservative variant (RLRLL). Negative charges are represented in red, neutral charges in white and positive charges in blue.
the aromatic ring and the capacity for establishing these intra- and other inter-molecular interactions. Replacement of arginines by lysines conserves the cationic charge in these positions. Our data clearly show that non-conserva- tive arginine and alanine mutations in the ADAMTS13 spacer RFRYY epitope provide superior autoantibody resistance, likely because these mutations promote disrup- tion of these intramolecular interactions and can potential- ly change the position of the loops of the epitope. We con- structed three-dimensional models of selected epitope variants and these revealed reduction of total surface charge (Figure 7) and/or epitope surface size (Online Supplementary Figure S6).
The concept of ADAMTS13 variants that resist autoan- tibodies and remain active has been explored previously. However, the majority of previous studies designed with this intention were conducted with truncated ADAMTS13 variants,23,38 with the exception of the GoF26 and a few ala- nine variants.22 We present here for the first time a compre- hensive study in which, in the context of the full length ADAMTS13 molecule, the spacer domain was modified with different types of mutations. We also demonstrated quantitatively that several full-length ADAMTS13 mutants escape the majority of autoantibodies from most patients. Therefore, we sought to assess whether these variants con- served proteolytic activity. The activities of ADAMTS13 variants have been measured in several forms of in vitro and in vivo assays. Evidence strongly suggests that the C-termi- nal TSP2-8/CUB1-2 domains of ADAMTS13 are important for full in vivo proteolytic activity39,40 because these domains allow ADAMTS13 to anchor itself onto the D4-CK domains of full-length VWF under shear flow condi- tions.41,42 The static FRETS-VWF73 assay is commonly used to measure the activity of ADAMTS13 for the diagnosis of
TTP.31 In minimal peptide assays, however, it appears that the C-terminal domains of ADAMTS13 and the spacer domain itself are less relevant for maintaining activity.43 In FRETS-VWF73 the activity of the MDTCS fragment is higher than that of full-length ADAMTS1332,44 due to the lack of the terminal TSP2-8 and CUB1-2 domains, elimi- nating their auto-inhibitory properties.44 Our data are in agreement with these earlier findings: both the wild-type MDTCS and the MDTCS-AAAAA variant had higher activity in the FRETS-VWF73 assay compared to their full- length counterparts. In agreement with previous studies most alanine changes induced loss of activity.22,23,25 Our data suggest that both aromatic residues and arginine residues have similar importance for activity. We observed a clear inverse correlation between resistance to autoantibodies and proteolytic activity of the variants (Figure 4). Selected mutants tested in our VWF multimer assay also had lower activity than the wild-type molecule, with the exception of wild-type MDTCS. This highlights the functional impor- tance of the spacer exosite-3 in our VWF multimer assay. Among the selected full-length autoantibody-resistant mutants, the RARAA mutant demonstrated residual activ- ity in conditions of sub-physiological ADAMTS13 concen- trations (Figure 6). This variant was tested in FRETS- VWF73 together with patients’ autoantibodies (Figure 5) exhibiting full resistance to the inhibitory action of poly- clonal autoantibodies from patients.
Surprisingly, the GoF variant described previously showed less activity than that of wild-type ADAMTS13. The GoF variant was originally characterized in a COS-7 cell background26 and in all subsequent in vitro32,44,45 and in vivo studies46,47 it was reported to be more active than wild-type ADAMTS13. We are the first group observing a loss of function of this molecule. The reasons for this are not clear.
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haematologica | 2020; 105(11)