WXXW基序对ADAMTS13分泌及功能的影响
摘要
ADAMTS13，又称为血管性血友病因子水解蛋白酶（VWF-CP），是存在于血浆中的金属蛋白酶，隶属于ADAMTSs（a disintegrin and metalloproteinase with thrombospondin motifs）金属蛋白酶超家族。人ADAMTS13基因于2001年首次被克隆报导，定位于9号染色体长臂端（9q34）。基因全长37kb，主要由29个外显子组成，其基因表达开放阅读框为4284bp，编码1427个氨基酸。ADAMTS13蛋白主要由9个结构功能域组成，从氨基端到羧基端依次为：信号肽、前导肽、金属蛋白酶结构域、去整合素结构域、凝血酶敏感蛋白1（TSP1）第1基序、富半胱氨酸区域、间隔区、凝血酶敏感蛋白1（TSP1）第2-8重复基序、补体结合区域（CUB）。ADAMTS13主要通过水解人血管性血友病因子（VWF），降解血浆中超大分子量VWF多聚体，使其对血小板的黏附与聚集能力降低，从而抑制血栓形成。ADAMTS13酶活性降低与血栓性血小板减少性紫癜（TTP）的发病密切相关。目前研究发现，ADAMTS13金属蛋白酶结构域识别并水解VWF蛋白A2区第1605酪氨酸残基和1606位蛋氨酸残基间肽键。而ADAMTS13金属蛋白酶结构域临近的羧基端结构域，包括去整合素结构域、凝血酶敏感蛋白1（TSP1）第1基序、富含半胱氨酸结构域、间隔区，确保ADAMTS13对VWF的正确识别及有效的水解。
本文围绕ADAMTS13的结构与功能进行研究，主要包括三个部分：1）构建了ADAMTS13的TSR1结构域中WXXW基序突变体（W387A），观察其对ADAMTS13分泌及酶活性的影响；2）构建了ADAMTS13-pEGFP-N1绿色荧光真核表达载体，为研究ADAMTS13细胞内合成及转运过程提供有力的研究工具；3）获得了多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株，以备进一步单克隆抗体分选及功能研究。
一、WXXW基序对ADAMTS13分泌及功能的影响
WXXW基序在凝血酶敏感蛋白1蛋白超家族中普遍存在，其中包括ADAMTSs金属蛋白酶家族。对于WXXW基序的研究主要集中在凝血酶敏感蛋白1蛋白上，此基序被认为是凝血酶敏感蛋白1蛋白结合并活化TGF-β （transforming growth factor-β）所必需的。在对霍乱毒素分泌蛋白EpsM的研究中发现，WXXW基序中芳香族氨基酸残基之间的相互作用可能与跨膜蛋白质的二聚化有关。WXXW基序在ADAMTSs金属蛋白酶家族中普遍存在，然而，其功能尚未可知。为了研究该基序的功能，我们构建了WXXW基序的突变体W387A的真核表达载体。
首先，我们用野生型ADAMTS13及W387A突变体的真核表达载体瞬时转染HeLa细胞，转染后48小时收集培养上清及细胞。将浓缩后培养上清及细胞裂解液行SDS-PAGE电泳及westblot，通过灰度值测定判断上清及细胞裂解液中蛋白的含量。用培养上清中蛋白含量除以培养上清及细胞裂解液中蛋白总量，计算蛋白的分泌量。实验结果表明：野生型ADAMTS13上清中蛋白含量为72.38%±10.70%，细胞裂解液中为27.62%±9.56%；而W387A突变体上清中蛋白含量为20.46%±5.85%，细胞裂解液中为79.54%±5.46%。结果表明，W387A突变抑制了ADAMTS13的分泌，使得大量的ADAMTS13蛋白积蓄在细胞内。蛋白细胞内定位实验结果显示，野生型蛋白细胞内分布呈指环状，而突变体蛋白细胞内分布弥散，提示突变体蛋白更多地滞留在内质网中。
其次，我们构建了稳定表达野生型ADAMTS13及W387A突变体蛋白的HeLa细胞株，并扩大培养，收集无血清培养上清，从而获得野生型ADAMTS13及W387A突变体蛋白，并进一步完成ADAMTS13功能检测。
第三，我们用非变性的人血浆来源VWF（pVWF）及经过预变性处理的pVWF包板，再分别加入野生型ADAMTS13或W387A突变体蛋白进行孵育，通过ELISA的方法，测定重组ADAMTS13蛋白与pVWF结合的量。结果表明，与野生型ADAMTS13相比，W387A突变体与变性的pVWF结合能力明显降低，而与非变性的pVWF的结合能力与野生型ADAMTS13没有明显区别。
第四，我们分别在变性条件下及高剪切力条件下，以pVWF为底物，检测了野生型ADAMTS13及W387A突变体对pVWF的水解能力。通过还原条件下的5%SDS-PAGE电泳及western blotting方法，分析176kDa分子量大小的酶切产物生成量，从而判断重组ADAMTS13酶活性的高低。我们发现，在变性条件下，加入野生型ADAMTS13的反应体系中，176kDa分子量大小的酶切产物生成量明显多于加入W387A突变体的反应体系。此外，在多聚体琼脂糖凝胶电泳分析中，我们发现在野生型ADAMTS13酶切体系中，大中分子量VWF多聚体降低水平明显高于W387A突变体酶切的反应体系，进一步证实了在变性条件下，W387A突变体对VWF的水解能力明显降低。然而在高剪切力条件下，无论是野生型ADAMTS13酶切体系还是W387A酶切体系，176kDa分子量大小的酶切产物生成量没有明显区别。而多聚体琼脂糖凝胶电泳分析也显示，两个体系中，大中分子量VWF多聚体减少的量基本一致。
第五，我们以以FRETS-VWF73为底物，测定野生型ADAMTS13及W387A突变体水解VWF的活性。以1：25稀释比例的正常人混合血浆中ADAMTS13活性为100%，计算重组ADAMTS13活性，结果以百分数表示。结果表明，野生型ADAMTS13活性约为97%±6.50%，明显高于W387A突变体活性（72%±5.50%）。此结果与变性条件下W387A突变体活性降低结果一致。
二、构建ADAMTS13-pEGFP-N1绿色荧光真核表达载体
既往研究表明，ADAMTS13与血栓性血小板减少性紫癜(TTP)的发病密切相关，ADAMTS13基因突变为遗传性TTP的基本病因。目前国际上报道的种ADAMTS13突变类型，绝大多数为错义突变。同时发现，大部分错义突变似乎都是通过影响蛋白的分泌而导致ADAMTS13缺失。为了进一步研究突变对ADAMTS13蛋白分泌的影响，我们构建了ADAMTS13-pEGFP-N1荧光表达载体，为深入研究ADAMTS13的突变体对其分泌的影响，以及进一步探讨ADAMTS13细胞内转运过程提供有力的研究工具。
首先，我们以野生型ADAMTS13通过PCR的方法获得目的基因片段，并在目的基因两端添加两个限制性内切酶位点。将目的基因连接至T载体后扩增，酶切后将酶切产物与pEGFP-N1荧光表达载体连接，获得ADAMTS13-pEGFP-N1真核表达载体。
其次，我们将该荧光表达载体转染HeLa细胞，48小时后收集细胞上清经超滤浓缩后6%SDS-PAGE电泳，以ADAMTS13-His蛋白为阳性对照western blotting鉴定。并将转染后细胞固定、破膜、染核后在荧光显微镜下观察绿色荧光蛋白的表达。结果显示：ADAMTS13-pEGFP蛋白在210kDa处显一蛋白条带，分子量略高于ADAMTS13-His。荧光显微镜下观察所见，转染pSecTag-ADAMTS13-His质粒的细胞内仅见DAPI蓝色荧光；转染pEGFP-N1空载体的细胞可见弥漫匀质绿色荧光；转染ADAMTS13-pEGFP-N1质粒的细胞内，可见多少不等的绿色荧光。表明带有绿色荧光标签的ADAMTS13蛋白成功表达。ADAMTS13-pEGFP-N1真核表达载体构建成功。
三、初步获得多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株，以备进一步分选及功能研究。
为了进一步方便ADAMTS13结构及功能的研究，并期待能筛选出具有一定功能活性的单克隆抗体，我们初步获得了多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株。
首先，我们构建了稳定表达截短型ADAMTS13-T7蛋白的HeLa细胞株，并扩大培养，收集无血清培养上清。截短型ADAMTS13-T7的真核表达载体为pSectag-2A，该载体在蛋白的羧基端添加了6个组氨酸His标签。通过Ni-NTA Agarose将浓缩后上清纯化，然后通过SDS-PAGE电泳后进行考马斯亮蓝染色和western blot鉴定，以全长ADAMTS13为对照。结果显示，浓缩后上清及纯化后蛋白均能在150kDa左右处显单一蛋白区带。
其次，我们用真核表达的截短型ADAMTS13-T7蛋白采用皮下多点注射免疫8周龄Balb/c雌性小鼠，并用纯蛋白液直接经尾静脉注射加强免疫。末次免疫后第3天眼球采血，分离上述小鼠抗血清，用ELISA方法测定免疫小鼠与ADAMTS13-T7蛋白的抗血清效价。用未免疫的正常小鼠血清作为阴性对照。用重组ADAMTS13-T7蛋白包板，ELISA检测免疫后balb/c雌性小鼠抗血清效价约为1:20000。取小鼠脾脏，制成单细胞悬液与SP2/0骨髓瘤细胞以10:1的比例混合，进行细胞的融合。将融合杂交瘤细胞稀释至96孔板后置于37℃、5%CO2培养箱中培养。两周后观察96孔板中是否有克隆，选生长良好的克隆孔检测，阳性克隆孔扩大培养并再次检测。初步检测结果显示已获得多株分泌抗重组ADAMTS13抗体的杂交瘤单克隆细胞株。
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-N1Vector
We 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 ADAMTS13
Monoclonal 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.
本文围绕ADAMTS13的结构与功能进行研究，主要包括三个部分：1）构建了ADAMTS13的TSR1结构域中WXXW基序突变体（W387A），观察其对ADAMTS13分泌及酶活性的影响；2）构建了ADAMTS13-pEGFP-N1绿色荧光真核表达载体，为研究ADAMTS13细胞内合成及转运过程提供有力的研究工具；3）获得了多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株，以备进一步单克隆抗体分选及功能研究。
一、WXXW基序对ADAMTS13分泌及功能的影响
WXXW基序在凝血酶敏感蛋白1蛋白超家族中普遍存在，其中包括ADAMTSs金属蛋白酶家族。对于WXXW基序的研究主要集中在凝血酶敏感蛋白1蛋白上，此基序被认为是凝血酶敏感蛋白1蛋白结合并活化TGF-β （transforming growth factor-β）所必需的。在对霍乱毒素分泌蛋白EpsM的研究中发现，WXXW基序中芳香族氨基酸残基之间的相互作用可能与跨膜蛋白质的二聚化有关。WXXW基序在ADAMTSs金属蛋白酶家族中普遍存在，然而，其功能尚未可知。为了研究该基序的功能，我们构建了WXXW基序的突变体W387A的真核表达载体。
首先，我们用野生型ADAMTS13及W387A突变体的真核表达载体瞬时转染HeLa细胞，转染后48小时收集培养上清及细胞。将浓缩后培养上清及细胞裂解液行SDS-PAGE电泳及westblot，通过灰度值测定判断上清及细胞裂解液中蛋白的含量。用培养上清中蛋白含量除以培养上清及细胞裂解液中蛋白总量，计算蛋白的分泌量。实验结果表明：野生型ADAMTS13上清中蛋白含量为72.38%±10.70%，细胞裂解液中为27.62%±9.56%；而W387A突变体上清中蛋白含量为20.46%±5.85%，细胞裂解液中为79.54%±5.46%。结果表明，W387A突变抑制了ADAMTS13的分泌，使得大量的ADAMTS13蛋白积蓄在细胞内。蛋白细胞内定位实验结果显示，野生型蛋白细胞内分布呈指环状，而突变体蛋白细胞内分布弥散，提示突变体蛋白更多地滞留在内质网中。
其次，我们构建了稳定表达野生型ADAMTS13及W387A突变体蛋白的HeLa细胞株，并扩大培养，收集无血清培养上清，从而获得野生型ADAMTS13及W387A突变体蛋白，并进一步完成ADAMTS13功能检测。
第三，我们用非变性的人血浆来源VWF（pVWF）及经过预变性处理的pVWF包板，再分别加入野生型ADAMTS13或W387A突变体蛋白进行孵育，通过ELISA的方法，测定重组ADAMTS13蛋白与pVWF结合的量。结果表明，与野生型ADAMTS13相比，W387A突变体与变性的pVWF结合能力明显降低，而与非变性的pVWF的结合能力与野生型ADAMTS13没有明显区别。
第四，我们分别在变性条件下及高剪切力条件下，以pVWF为底物，检测了野生型ADAMTS13及W387A突变体对pVWF的水解能力。通过还原条件下的5%SDS-PAGE电泳及western blotting方法，分析176kDa分子量大小的酶切产物生成量，从而判断重组ADAMTS13酶活性的高低。我们发现，在变性条件下，加入野生型ADAMTS13的反应体系中，176kDa分子量大小的酶切产物生成量明显多于加入W387A突变体的反应体系。此外，在多聚体琼脂糖凝胶电泳分析中，我们发现在野生型ADAMTS13酶切体系中，大中分子量VWF多聚体降低水平明显高于W387A突变体酶切的反应体系，进一步证实了在变性条件下，W387A突变体对VWF的水解能力明显降低。然而在高剪切力条件下，无论是野生型ADAMTS13酶切体系还是W387A酶切体系，176kDa分子量大小的酶切产物生成量没有明显区别。而多聚体琼脂糖凝胶电泳分析也显示，两个体系中，大中分子量VWF多聚体减少的量基本一致。
第五，我们以以FRETS-VWF73为底物，测定野生型ADAMTS13及W387A突变体水解VWF的活性。以1：25稀释比例的正常人混合血浆中ADAMTS13活性为100%，计算重组ADAMTS13活性，结果以百分数表示。结果表明，野生型ADAMTS13活性约为97%±6.50%，明显高于W387A突变体活性（72%±5.50%）。此结果与变性条件下W387A突变体活性降低结果一致。
二、构建ADAMTS13-pEGFP-N1绿色荧光真核表达载体
既往研究表明，ADAMTS13与血栓性血小板减少性紫癜(TTP)的发病密切相关，ADAMTS13基因突变为遗传性TTP的基本病因。目前国际上报道的种ADAMTS13突变类型，绝大多数为错义突变。同时发现，大部分错义突变似乎都是通过影响蛋白的分泌而导致ADAMTS13缺失。为了进一步研究突变对ADAMTS13蛋白分泌的影响，我们构建了ADAMTS13-pEGFP-N1荧光表达载体，为深入研究ADAMTS13的突变体对其分泌的影响，以及进一步探讨ADAMTS13细胞内转运过程提供有力的研究工具。
首先，我们以野生型ADAMTS13通过PCR的方法获得目的基因片段，并在目的基因两端添加两个限制性内切酶位点。将目的基因连接至T载体后扩增，酶切后将酶切产物与pEGFP-N1荧光表达载体连接，获得ADAMTS13-pEGFP-N1真核表达载体。
其次，我们将该荧光表达载体转染HeLa细胞，48小时后收集细胞上清经超滤浓缩后6%SDS-PAGE电泳，以ADAMTS13-His蛋白为阳性对照western blotting鉴定。并将转染后细胞固定、破膜、染核后在荧光显微镜下观察绿色荧光蛋白的表达。结果显示：ADAMTS13-pEGFP蛋白在210kDa处显一蛋白条带，分子量略高于ADAMTS13-His。荧光显微镜下观察所见，转染pSecTag-ADAMTS13-His质粒的细胞内仅见DAPI蓝色荧光；转染pEGFP-N1空载体的细胞可见弥漫匀质绿色荧光；转染ADAMTS13-pEGFP-N1质粒的细胞内，可见多少不等的绿色荧光。表明带有绿色荧光标签的ADAMTS13蛋白成功表达。ADAMTS13-pEGFP-N1真核表达载体构建成功。
三、初步获得多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株，以备进一步分选及功能研究。
为了进一步方便ADAMTS13结构及功能的研究，并期待能筛选出具有一定功能活性的单克隆抗体，我们初步获得了多株分泌抗ADAMTS13的单克隆抗体的杂交瘤细胞株。
首先，我们构建了稳定表达截短型ADAMTS13-T7蛋白的HeLa细胞株，并扩大培养，收集无血清培养上清。截短型ADAMTS13-T7的真核表达载体为pSectag-2A，该载体在蛋白的羧基端添加了6个组氨酸His标签。通过Ni-NTA Agarose将浓缩后上清纯化，然后通过SDS-PAGE电泳后进行考马斯亮蓝染色和western blot鉴定，以全长ADAMTS13为对照。结果显示，浓缩后上清及纯化后蛋白均能在150kDa左右处显单一蛋白区带。
其次，我们用真核表达的截短型ADAMTS13-T7蛋白采用皮下多点注射免疫8周龄Balb/c雌性小鼠，并用纯蛋白液直接经尾静脉注射加强免疫。末次免疫后第3天眼球采血，分离上述小鼠抗血清，用ELISA方法测定免疫小鼠与ADAMTS13-T7蛋白的抗血清效价。用未免疫的正常小鼠血清作为阴性对照。用重组ADAMTS13-T7蛋白包板，ELISA检测免疫后balb/c雌性小鼠抗血清效价约为1:20000。取小鼠脾脏，制成单细胞悬液与SP2/0骨髓瘤细胞以10:1的比例混合，进行细胞的融合。将融合杂交瘤细胞稀释至96孔板后置于37℃、5%CO2培养箱中培养。两周后观察96孔板中是否有克隆，选生长良好的克隆孔检测，阳性克隆孔扩大培养并再次检测。初步检测结果显示已获得多株分泌抗重组ADAMTS13抗体的杂交瘤单克隆细胞株。
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-N1Vector
We 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 ADAMTS13
Monoclonal 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.
引文
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[1] Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent onits conformation and requires calcium ion. Blood,1996,87(10):4235-4244.
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