Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase.
Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase.
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DOI：
10.4049/jimmunol.144.6.2304
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发表时间：
1990-03
影响因子：
4.4
通讯作者：
L. Schwartz;T. Bradford;D. C. Lee;J. Chlebowski
中科院分区：
文献类型：
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作者：
L. Schwartz;T. Bradford;D. C. Lee;J. Chlebowski
人类胰蛋白酶（一种肥大细胞中性内切蛋白酶）的催化活性在酶处于其四聚体形式时表达，但在生理条件下伴随着涉及转化为单体形式的四级结构改变而丧失。在当前研究中注意到的CD光谱中的相关变化表明蛋白质二级结构的伴随改变。特别地，以228 nm为中心的负最小值的逐渐消失表明对β-折叠结构的影响，这对于单体-单体相互作用和/或催化活性的稳定可能是重要的。硫酸葡聚糖，像肝素一样，稳定类胰蛋白酶的催化活性和四级结构，并且在40摄氏度和超过40摄氏度的温度下也保持酶的天然二级结构。因此，硫酸葡聚糖稳定的类胰蛋白酶被用作免疫原，产生了三种识别该酶催化活性形式的鼠mAb（B2、C11和G4）。与塑料微量滴定威尔斯孔结合的无活性类胰蛋白酶不被任何新制备的抗体识别，而溶液中的无活性类胰蛋白酶被G4识别，当生物素化时，G4可用作类胰蛋白酶夹心ELISA中的检测抗体。每一种新制备的mAb都识别类胰蛋白酶的催化活性形式。因此，表位的改变，可能反映三级结构的改变以及二级和四级构象的变化，发生类胰蛋白酶失活。这些新产生的抗体的一个实用结果是通过连续层析用偶联至CH-琼脂糖和肝素-琼脂糖的B2亲和纯化至活性类胰蛋白酶的均一性。通过该技术纯化的类胰蛋白酶与对甲苯磺酰基-L-精氨酸甲酯的比活性为117 +/-9 U/mg，每分子活性酶（134，000 m.w.）用对硝基苯基-p ′-胍基苯甲酸酯滴定。目前研究中的光谱和免疫学数据与类胰蛋白酶二级和三级以及四级结构中与催化活性丧失相关的一致构象变化一致。硫酸葡聚糖未能逆转这些改变中的任何一个表明体内四聚体组装的途径比简单的亚基缔合更复杂。
The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.