Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.ADAMTS13 | von Willebrand factor | thrombotic thrombocytopenic purpura | hydrogen exchange | autoimmunity A cquired thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia and microangiopathic hemolytic anemia, resulting from disseminated microvascular thrombosis. Patients with TTP may suffer from widespread organ damage, resulting in death if not aggressively treated (1, 2). In most patients, thrombotic microangiopathy is caused by IgG-type autoantibodies against the plasma metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) (3-5). ADAMTS13 regulates the function of the multidomain von Willebrand factor (VWF) (∼600 to ∼20,000 kDa) by cleaving it at the central A2 domain to regulate platelet-induced blood clot formation (3, 4).Immunological studies show that many acquired TTP patients with low plasma ADAMTS13 activity (less than 10%) harbor IgG autoantibodies that bind ADAMTS13 especially at the Cysrich and/or the spacer domain (4, 6). Deletion of these domains or grouped mutations therein can eliminate the binding of polyclonal anti-ADAMTS13 IgGs derived from many patients (4, 7-11). However, the exact ADAMTS13 binding sites of these autoantibodies are not known. Current treatment...
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We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.
The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.
Acquired thrombotic thrombocytopenic purpura (TTP), a potentially fatal arterial thrombotic disorder, is primarily caused by autoantibodies that bind and inhibit plasma von Willebrand factor (VWF)-cleaving metalloprotease (ADAMTS13) activity. However, the mechanisms underlying autoantibody-mediated inhibition of ADAMTS13 activity and acquired TTP are not fully understood. By a hydrogen-deuterium exchange coupled with mass spectrometry (HX-MS) technique, we found that human monoclonal anti-ADAMTS13 antibodies, the single chain variable region fragments (scFvs)4-20, 4-16, and 3-1 that were, isolated by phage display from two patients with acquired TTP, predominantly bound to a discontinuous and conformational epitope in the spacer domain of ADAMTS13 with a subtle difference. The epitope for scFvs4-20 and 4-16 comprises five small flexible loops, including a previously described motif A (or exosite 3, R659-E664), motif B (exosite 4, L632-R639), and several other outlying residues (F592, Y658, and Y665), while scFv3-1 bound all other residues except for those in motif A. Site-directed mutagenesis and biochemical analysis demonstrated that both motifs A and B were found to be critical for recognition and proteolysis of VWF73 and multimeric VWF. Deletion of motif A or motif B in full-length ADAMTS13 abolished the binding of scFvs4-20 and 4-16 but not 3-1 (which did not bind motif A). Our findings demonstrate the powerful use of HX-MS for mapping antibody epitopes at nearly single amino acid resolution. This provides a new way to reveal mechanisms of autoantibody-mediated inhibition of plasma ADAMTS13 activity and acquired TTP. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
2214 Exosite binding plays a key role in cleavage of VWF by ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats, 13). Two exosites that are evolutionarily conserved from zebra fish to mammals have been identified in the spacer domain by sequence alignment. Previous studies have shown that exosite 3 in the spacer domain plays a critical role for substrate recognition (Blood 115:2300–10, 2010), and modification of this exosite generates ADAMTS13 variants with improved specific activity but reduced autoantibody binding (Blood 119:3836–43, 2012). In the present study, using a site-directed mutagenesis approach, we identified a novel exosite near exosite 3 in the spacer domain, termed exosite 4, a region between residues Glu634 and Arg639. A partial (ΔEx4a:deletion of Leu632-Asp635 or ΔEx4b:deletion of Arg636-Arg639) or complete deletion of the exosite (ΔEx4) significantly impaired proteolytic activity towards peptidyl VWF73 and multimeric VWF. Moreover, substitution of all surface exposed residues in Ex4A (LTED/AAAA) or Ex4b (RLPR/AAAA) with alanine had a similarly detrimental effect on proteolytic activity. Further studies demonstrated that the residues Asp635 and Arg636 in exosite 4 play a critical role for substrate recognition. We conclude that the region between residues Glu634 and Arg639 is a novel exosite necessary for recognition and cleavage of VWF. Disclosures: No relevant conflicts of interest to declare.
Acquired thrombotic thrombocytopenic purpura (aTTP), a potentially fatal syndrome, is primarily caused by autoantibodies against the metalloprotease ADAMTS13. Most patients with aTTP harbor an immunoglobulin (Ig) G isotype in blood that targets the spacer domain of ADAMTS13. The precise epitopes of the anti-ADAMTS13 IgGs and the mechanism underlying their inhibition activity are not fully understood. We hypothesized that inhibitory IgG autoantibodies from aTTP patients achieve their inhibitory function by binding to a discontinuous epitope in the spacer domain of ADAMTS13. To test this hypothesis, we determined the binding epitope of one out of >100 unique human monoclonal antibody (mAb) fragments (single-chain Fv, scFv) isolated by phage display from aTTP patients. We developed a novel hydrogen-deuterium exchange-mass spectrometry technology (HX-MS) to identify the antibody binding sites at single amino acid residue resolution. Human ADAMTS13 inhibitory scFv 4-20 was expressed in E. coli Top10 cells and purified to homogeneity by Ni-chelating affinity chromatography. In the HX-MS experiment, the mAb was coupled to affi-gel 10 resin and used to bind recombinant ADAMTS13-MDTCS fragment expressed in a stably transfected Drosophila schneider 2 (S2) cell line. After exchange with deuterium (D2O) oxide for various periods of time, the reaction was stopped, the protein was eluted, and digested to peptide fragments with pepsin, and the peptides with or without deuterium bound were resolved and identified by fast HPLC and mass spectrometry. We find that mAb scFv4-20 binds to amino acid residues Arg636, Leu637, Arg639, and Leu640 spanning from Leu632 to Leu640 (in exosite 4) in the spacer domain of ADAMTS13. This sequence is highly conserved in the ADAMTS13 spacer domains from zebrafish to mammals. In addition, mAb scFv4-20 binds Arg660, Tyr661, and Tyr665 in exosite 3, previously shown to play an important role in substrate recognition and anti-ADAMTS13 autoantibody-mediated inhibition, as well as Lys608, upstream exosites 3 and 4. Apparently, mAb scFv4-20 inhibits plasma ADAMTS13 activity (IC50 ∼0.40 nM) by binding these non-linear surface residues in the spacer domain (Fig. 1A). In agreement, site-directed mutagenesis shows that complete deletion (Δ632LTEDRLPR639) or partial deletion (Δ632LTED635 or Δ636RLPR639), or replacement of these residues with alanines (632LTED635/4A or 636RLPR639/4A) abolished or dramatically reduced mAb scFv4-20 binding. A deletion or alanine substitution of the surface residues on exosite 4 also abolished or reduced ADAMTS13 proteolytic activity toward a fluorescein-labeled VWF73 peptide and multimeric VWF (Fig. 1B), indicating that the ADAMTS13 epitope for mAb scFv4-20 is also part of ADAMTS13’s substrate recognition site. We conclude that anti-ADAMTS13 autoantibodies work by physically blocking the well-conserved VWF binding site on ADAMTS13. These results demonstrate the powerful use of HX-MS technology to determine both linear and non-linear antibody binding epitopes. The results provide valuable information concerning the mechanism of autoantibody-mediated aTTP that may be exploited to develop targeted therapy by reengineering ADAMTS13 to avoid autoantibody inhibition. Disclosures: No relevant conflicts of interest to declare.
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