Identification of N-linked glycosylation and putative O-fucosylation, C-mannosylation sites in plasma derived ADAMTS13.

BACKGROUND: Acquired deficiency of ADAMTS13 causes a rare and life-threatening disorder called thrombotic thrombocytopenic purpura (TTP). Several studies have shown that aberrant glycosylation can play an important role in the pathogenesis of autoimmune diseases.N-linked glycosylation and putative O-fucosylation sites have been predicted or identified in recombinant ADAMTS13. However, it is not known which of these sites are glycosylated in plasma derived ADAMTS13. OBJECTIVES: Here we investigated the presence of putative O-fucosylation, C-mannosylation and N-linked glycosylation sites on plasma derived ADAMTS13. METHODS/RESULTS: Sites of N-linked glycosylation were determined by the use of peptide N-glycosidase-F (PNGase F), which removes the entire carbohydrate from the side chain of asparagines. Nine of the 10 predicted N-linked glycosylation sites were identified in or near the metalloproteinase,spacer, thrombospondin type 1 repeat (TSR1) and the CUB domain of plasma ADAMTS13. Moreover, six putative O-fucosylated sites were identified in the TSR domains of plasma ADAMTS13 by performing searches of the tandem mass spectrometry (MS/MS) data for loss of hexose (162 Da), deoxyhexose (146 Da), or hexose deoxyhexose(308 Da). The use of electron transfer dissociation (ETD) allowed for unambiguous identification of the modified sites. In addition to putative O-fucosylation and N-linked glycosylation, two putative C-mannosylation sites were identified within the TSR1 and TSR4 domains of ADAMTS13. CONCLUSIONS: Our data identify several glycosylation sites on plasma derived ADAMTS13. We anticipate that our findings may be relevant for the initiation of autoimmune reactivity against ADAMTS13 in patients with acquired TTP.

Humoral immune response to ADAMTS13 in acquired thrombotic thrombocytopenic purpura.

The apparently spontaneous development of autoantibodies to ADAMTS13 in previously healthy individuals is a major cause of thrombotic thrombocytopenic purpura (TTP). Epitope mapping studies have shown that in most patients antibodies directed towards the spacer domain of ADAMTS13 are present. A single antigenic surface comprising Arg(660) , Tyr(661) and Tyr(665) that contributes to the productive binding of ADAMTS13 to unfolded von Willebrand factor is targeted by anti-spacer domain antibodies. Antibodies directed to the carboxyl-terminal CUB1-2 and TSP2-8 domains have also been observed in the plasma of patients with acquired TTP. As yet it has not been established whether this class of antibodies modulates ADAMTS13 activity. Inspection of the primary sequence of human monoclonal anti-ADAMTS13 antibodies suggests that the variable heavy chain germline gene segment VH1-69 is frequently incorporated. We suggest a model in which 'shape complementarity' between the spacer domain and residues encoded by the VH1-69 gene segment explain the preferential use of this variable heavy chain gene segment. Finally, a model is presented for the development of anti-ADAMTS13 antibodies in previously healthy individuals that incorporates the recent identification of HLA DRB1*11 as a risk factor for acquired TTP.