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ADAMTS13 variants to escape autoantibodies in iTTP
latively mutated to either an asparagine or alanine. These mutations likely disrupt the hydrophobic cluster centered in F592 and formed together with Y661, Y665 and other neighboring residues.33 The greatest impact was achieved with cumulative mutations of all three aromatic residues, alone or together with mutations of the arginines. The highest resistance to autoantibody binding was observed for the full-length 5x ala mutant (AAAAA), followed by the triple asparagine RNRNN mutant (Figure 2).
Protein surfaces enriched in tyrosine and arginine are usually involved in protein-protein interactions. Arginine salt-bridges are common players in these interactions.34 Tyrosine itself is a highly promiscuous residue participat- ing in many types of interactions.35 This epitope contains
two arginine residues, R568 and R660, which are likely involved in arginine-salt bridges for antibody binding (Figure 2), and also in intramolecular hydrogen bonds and cation-π interactions with Y661 and Y665 (Online Supplementary Figure S5). Paratopes of the antibodies are considered to have a core enriched in aromatic residues, particularly tyrosine.36,37 The primary sequence of a large collection of anti-ADAMTS13 autoantibodies revealed that most autoantibodies targeting ADAMTS13 – encod- ed by heavy chain variable genes VH1-69 and VH1-311-13 – have a complementary determining region 3 (CDR3) con- sisting of multiple residues with either aromatic or nega- tively charged side chains.11,12 Exchanging a tyrosine for a phenylalanine (and vice versa) within exosite-3 conserves
Figure 6. Autoantibody-resistant ADAMTS13 variants are capable of cleaving high-molecular weight multimers of von Willebrand factor. Selected variants with dif- ferent levels of autoantibody reactivity/FRETS-VWF73 activity were tested for their proteolytic activity against recombinant multimeric von Willebrand factor (VWF) (1.9 nM ADAMTS13 for 40 nM VWF) in a static assay in which urea was used to denature VWF multimers. The full-length wild-type ADAMTS13 variant was used as a control for each individual reaction, with similar outcomes for all reactions. An example is shown in each panel for comparison. High-molecular weight VWF multi- mers are cleaved in a time-dependent manner, with accumulation of satellite bands in lower molecular weight regions. A lane containing 40 nM multimeric VWF with- out ADAMTS13 was included in the gels for comparison. All full-length variants displayed a reduction in proteolytic activity compared to the wild-type molecule. However, residual proteolytic activity was retained by several of them (including one of the most autoantibody-resistant variants, RARAA). The wild-type MDTCS and MDTCS-AAAAA were also tested, and while the wild-type MDTCS retained similar activity levels compared to the wild-type molecule, MDTCS-AAAAA displayed a reduc- tion to the same level as its full-length counterpart, variant AAAAA.
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