The WXXW Motif In The TSR1of ADAMTS13is Important For Its Secretion And Proteolytic Activity

ADAMTS13belongs to a family of proteases called ADAMTSs (a disintegrin andmetalloproteinase with thrombospondin motifs). ADAMTS13consists of1,427amino acid residues andis identified as a von Willebrand factor (VWF) cleaving protease. ADAMTS13cleaves the bondbetween the Tyr1605and Met1606residues in the central A2domain of VWF to decrease the activityof VWF. VWF is a carrier protein for factor VIII and, upon binding to platelets and the extracellularmatrix, promotes platelet aggregation or platelet adhesion to areas of vascular damage. A deficiency ofADAMTS13or the presence of autoantibodies against ADAMTS13causes congenital or acquiredidiopathic thrombotic thrombocytopenic purpura.ADAMTS13consists of a signal peptide, a propeptide, a metalloproteinase domain, adisintegrin-like domain, a central TSP1repeat, a cys-rich domain, a spacer domain, seven C-terminalTSP1repeats, and two CUB domains. With regard to the cleaving activity of ADAMTS13, previousstudies have demonstrated that the metalloprotease domain of ADAMTS13recognizes and cleaves theTyr1605–Met1606bond in the central A2domain of VWF. The proteolytic efficiency and specificitycan be enhanced by the participation of other domains of ADAMTS13such as the disintegrin domain,the first TSP1repeat, or the spacer domain. These additional domains might increase the bindingaffinity of ADAMTS13for VWF, and the first TSP1repeat appears to bind the more proximal VWFsegment Gln1624–Val1630from the scissile bond.As ADAMTS13is such an important factor in the pathophysiological process of thrombosis, ourstudy is focused on ADAMTS13, its structure, and its cleaving activity of VWF.Part I: The WXXW motif in the TSR1of ADAMTS13is important for its secretion andproteolytic activity.There is a WXXW motif in the first TSP1repeat of ADAMTS13, which is frequently found inproteins of the thrombospondin type1repeat (TSR) super family. Most studies of the WXXW motif areperformed with thrombospondin1. In thrombospondin1, WXXW is essential for the binding andactivation of latent transforming growth factor-β. WXXW is also the structural determinant for theadsorptivity of fibrinogen and the core consensus motif of five peptides, which could mimic thedrug-binding activity of P-glycoprotein. Moreover, a study of the cholera toxin secretion protein EpsMshowed that the WXXW motif is involved in the dimerization of transmembrane proteins and that suchdimerization is dependent on aromatic-aromatic interactions. However, the function of the WXXWmotif in ADAMTSs is unclear.We generated wild-type and WXXW mutant (W387A) constructs of ADAMTS13by PCRsite-directed mutagenesis and expressed these constructs in HeLa cells. To examine whether the WXXW motif is necessary for the secretion of ADAMTS13, we constructed a W387A mutant andexpressed the WT and mutant ADAMTS13in HeLa cells. After the transfections, equivalent fractionsof the media and cell lysates were subjected to SDS-PAGE followed by western blotting analysis. Ourresults showed that the percentage of protein secretion for the WT ADAMTS13was72.38%±10.70%,while the secretion of the W387A mutant was reduced dramatically to20.46%±5.85%. Thepercentages of the protein that remained in the WT and W387A mutant transfected cell lysates were27.62%±9.56%and79.54%±5.46%, respectively. These results indicate that the W387A mutationcauses the defective secretion of ADAMTS13and that the mutant form of ADAMTS13is accumulatedinside cells.To determine whether the W387A mutation affects the binding of ADAMTS13to VWF, humanplasma-derived VWF (native or denatured,7.5 g/ml)was coated onto a microtiter plate and incubatedwith His-tagged WT and W387A ADAMTS13proteins, which had been expressed in HeLa cells. AnHRP-labeled mouse anti-His monoclonal antibody was used to detect the binding. Our results showedthat, in comparison to the WT protein, the W387A mutant binding to the pre-denatured VWF wasreduced significantly. However, no significant difference was observed in the binding of the WT andW387A mutant proteins to native VWF.To investigate the enzymatic activity of the WT and W387A mutant ADAMTS13proteins, weused human plasma-derived VWF as the substrate under denatured condition or shear stress. Thecleaved products were detected by SDS-PAGE under reducing conditions and agarose gelelectrophoresis to visualize the VWF multimers. Under static/denaturing condition, the amount of the176kDa VWF cleaved product in the WT ADAMTS13reactions was increased significantly; however,the W387A mutant reactions produced much less of the176kDa VWF cleaved product. Consistent withthese results, the amounts of the high and intermediate molecular weight multimers of VWF weredramatically reduced when WT ADAMTS13was used to digest the VWF multimers, in comparison toW378A mutant digestions. However, under shear stress, no obvious differences in the amounts of176kDa cleaved products were detected between the WT and W378A mutant groups. In addition, the WTADAMTS13showed no detectable proteolysis of the VWF without guanidine chloride. A multimeranalysis also showed equal reductions in the high and intermediate molecular weight multimers of VWFafter WT or W378A mutant ADAMTS13digestion. Experimental reactions without ADAMTS13, orwith ADAMTS13in the presence of20mM EDTA, showed no detectable proteolysis of VWF, whichindicates that the proteolytic degradation of VWF by ADAMTS13is specific.The FRETS-VWF73assay was also used to validate the proteolytic activity of the WT and mutantADAMTS13proteins. The percentage of recombinant ADAMTS13activity was defined as the valuedivided by that of the normal human plasma diluted at1:25, and multiplied by100%. The W387Amutant activity was approximately72%±5.50%, which was much less than that of the WTADAMTS13(97%±6.50%). This result was consistent with the full-length VWF cleaving activity ofADAMTS13under static/denaturing condition. Part II: Construction of ADAMTS13-pEGFP-N1VectorWe generated a pEGFP-N1Vector of von Willebrand factor cleaving protease (ADAMTS13, adisintegrin and metalloprotease with a thromboSpondin type1motifs13), for the further studies on itssynthesization and secretion. Human full-length cDNA sequence of ADAMTS13was acquired bypolymerase chain reaction with Phusion High-Fidelity (NEB). Then the PCR products were doubledigested with EcoRⅠ a ndXho Ⅰ. The products of digestion were purified and ligated to the pEGFP-N1vector. DNA sequence analysis showed that ADAMTS13was ligated to the pEGFP-N1vector correctly.After transient expression in Hela cells, the green fluorescence was visible under fluorescencemicroscope, and the proteins were identified with SDS-PAGE and western blotting. The results showedthat the ADAMTS13-pEGFP-N1vector was generated correctly, and it could be widely used in furtherresearch on the mechanism of the synthesis and secretion of ADAMTS13.Part III: Preparation of monoclonal antibodies against ADAMTS13Monoclonal antibodies (mAb) are used extensively in basic biomedical research, in diagnosis ofdisease, and in treatment of illnesses. Antibodies are important tools used by many investigators in theirresearch and have led to many medical advances. For the further studies on ADAMTS13, we preparedseveral monoclonal antibodies against ADAMTS13.Production of monoclonal antibodies involves in vivo or in vitro procedures. At first, thegeneration of mAb-producing cells requires the use of animals, usually mice. We expressed theADAMTS13-T7constructs in HeLa cells, and then the recombined protein ADAMTS13-T7waspurified by Ni-NTA agarose. After purification, the purified protein was used to immunize three femaleBalb/c mice of8weeks old as the antigen. These mice were immunized every4weeks. When asufficient antibody titer was reached in serum, immunized mice were euthanized and the spleens wereremoved to use as a source of cells for fusion with myeloma cells. After the immunized mice wereeuthanized, blood samples were obtained from mice for measurement of serum antibodies. Serumantibody titer was determined with enzyme-linked immunosorbent assay (ELISA). The result showedthat the titer was about1:20000. A week before cell fusion, myeloma cells are grown in8-azaguanine.Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells.The cells were then distributed to96well plates containing feeder cells derived from saline peritonealwashes of mice, and were cultured in HAT selection medium after cell fusion. Only fused cells willgrow in the special selection medium. Then the small clusters of hybridoma cells from the96wellplates were grown in tissue culture followed by selection for antigen binding. Cloning by “limitingdilution” at this time ensured that a majority of wells each contained at most a single clone. After twocycles of cloning by limiting dilution, several hybrid cells that will produce the antibodies aregenerated.