Identification of epitopes on ADAMTS13 recognized by a panel of monoclonal antibodies with functional or non-functional effects on catalytic activity.
BACKGROUND
von Willebrand factor (VWF) cleavage by ADAMTS13 is mediated by multi-step interactions between their multi-domain structures. To clarify the relationship between inhibitory effects of monoclonal antibodies and epitopes on each ADAMTS13 domain, we analyzed how each ADAMTS13 domain contributes to catalyze VWF using a mouse anti-ADAMTS13 monoclonal antibody panel.


METHODS
FRETS-VWF73 assay was used to examine the effects of 14 anti-ADAMTS13 monoclonal antibodies on the catalytic activity of plasma ADAMTS13. Epitope mapping was performed using phage surface display. Libraries expressing peptide fragments of ADAMTS13 were screened with the monoclonal antibodies.


RESULTS
Eleven epitopes of 14 monoclonal antibodies were successfully defined. Three monoclonal antibodies recognizing metalloprotease or disintegrin-like domains strongly inhibited the catalytic activity and their epitopes were on Gln159-Asp166, Tyr 305-Glu327, and Asn308-Glu376. Five monoclonal antibodies recognizing TSP1-3 to -7 repeats showed weak inhibitory effects, and their epitopes were on Pro744-Ala806, Pro856-Cys864, Gln892-Gly940, Cys1007-Cys1072, and Gln1163-Asn1185. Four monoclonal antibodies recognizing the TSP1-1, TSP1-2, CUB1 or CUB2 domains had no inhibitory effects, and their epitopes, except that for TSP1-1, were Pro682-Cys742, Thr1200-Cys1213, and Gln1409-Glu1414. Two monoclonal antibodies recognizing cysteine-rich and spacer domains showed moderate inhibitory effects, but their epitopes were not determined.


CONCLUSIONS
We revealed the epitopes of 11 monoclonal anti-ADAMTS13 antibodies on each of the domains and clarified their association with inhibitory effects on VWF catalysis under static conditions. Catalytic activity correlated strongly with the epitopes on metalloprotease and disintegrin-like domains, weakly with those on TSP1-3 to -7 repeats, and negatively with those on TSP1-1, -2, and CUB domains.
Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan.
Atsuko
A
Igari
Terumichi
T
Nakagawa
Takanori
T
Moriki
Yusuke
Y
Yamaguchi
Masanori
M
Matsumoto
Yoshihiro
Y
Fujimura
Kenji
K
Soejima
Mitsuru
M
Murata
D051722:ADAM Proteins / Q000276:immunology*; D000071120:ADAMTS13 Protein; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D051379:Mice; D014841:von Willebrand Factor / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; C029417:Von Willebrand antigen; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human