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N.A.G. Graça et al.
mutations, and were also effective at escaping binding of patients’ antibodies, albeit to a lesser degree. The RARAA mutant had the second lowest median reactivity with patients’ antibodies among this panel of alanine substitu- tions (27%). For both asparagine and alanine mutations, the more cumulative mutations were introduced, the higher was the effectiveness at escaping autoantibody binding.
Within the panels in which the R568 and R660 were also mutated, mutation of the arginines to alanines alone had a moderate effect on autoantibody resistance (median signal at 64% for mutant AFAYY) (Figure 2). The classic GoF molecule (KYKFF) also presented a moderate decrease in binding to patients’ antibodies when compared to the wild-type form and to other mutations introduced. The median reactivity of the GoF variant was 73%, a mild improvement compared to a similar conservative mutant (RYRFF: median reactivity: 92%). As can be deduced from the RARAA mutant, addition of a further arginine muta- tion with alanine (AARAA and RAAAA mutants) promot- ed a further, but more limited, reduction of median reac- tivity (21% and 24%, respectively). Further mutating the remaining arginines to lysines (i.e., AAKAA, KAAAA, KAKAA mutants) produced little to no variation in median reactivity (19%-25%), with the KARAA mutant showing a small increase when compared to KAKAA. The 5x ala
A
full-length mutant (AAAAA) had the greatest decrease in median reactivity among all full-length variants (median signal: 9%). Our findings show that cumulative replace- ment of aromatic residues in the spacer RFRYY epitope of ADAMTS13 with asparagine or alanine provides superior resistance towards immune TTP patients’ autoantibodies directed towards the spacer domain.
Autoantibody-resistant ADAMTS13 variants retain proteolytic activity
We subsequently assessed the impact of these muta- tions on the activity of ADAMTS13. All variants were tested in a FRETS-VWF73 assay at the same molar concen- tration and directly compared with the full-length wild- type ADAMTS13 (Online Supplementary Figure S4). The relative activities are shown in Figure 3. All full-length variants showed reduced activity in FRETS-VWF73 when compared to the full-length wild-type ADAMTS13. The degree of reduction varied depending on the mutations inserted. Conservative mutations retained the most activ- ity on average. The RYRYY mutant had 39% activity while RFRFF had 76%. The triple conservative RYRFF mutant retained 56% of activity. Semi-conservative leucine mutants also retain overall high activity, with sin- gle and double mutations retaining between 35% and 76% activity. The RLRLL mutant had a larger loss of activ-
Figure 3. FRETS-VWF73 activity data of mutant ADAMTS13 vari- ants. All mutant variants in this study were assessed for activity using a FRETS-VWF73 assay. Three independent assays were conducted, each one with its own calibration curve construct- ed using the recombinant full- length wild-type ADAMTS13 (an example of a calibration curve is shown in Online Supplementary Figure S4). In each assay, a lim- ited number of variants could be introduced. All values are nor- malized to the full-length wild- type molecule (100% activity). (A) The relative activities of the variants are shown. The full- length wild-type ADAMTS13 is represented in black, the con- servative variants in blue, the gain-of-function in purple, semi- conservative variants in green and non-conservative variants in red. (B) The relative activities of variants are shown. The full- length wild-type ADAMTS13 is represented in black, the aro- matic-alanine variants in light gray, the arginine-alanine vari- ants in dark gray, the full-length
B
AAAAA in golden, alanine/lysine hybrids in pink, the wild-type MDTCS in white and the MDTCS-AAAAA mutant in green.
the
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haematologica | 2020; 105(11)