Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator
Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator
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DOI：
10.1055/a-1508-7919
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发表时间：
2021-05
影响因子：
6.7
通讯作者：
Tae Ito;Yuko Suzuki;H. Sano;N. Honkura;F. Castellino;T. Urano
中科院分区：
文献类型：
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作者：
Tae Ito;Yuko Suzuki;H. Sano;N. Honkura;F. Castellino;T. Urano
摘要背景组织型纤溶酶原激活物（tPA）和纤溶酶原激活物抑制剂1（派-1）之间的分子相互作用的细节仍然未知。方法和结果三种不同形式的高分子量复合物的证明。通过质谱法检测到其中两种形式。通过MALDI-TOF MS（基质辅助激光解吸电离-飞行时间质谱法）检测的高分子量为107，029 Da，其对应于完整tPA（65，320 Da）和完整派-1（42，416 Da）的分子量之和。较低分子量为104，367 Da，推测缺乏派-1的C末端诱饵肽（计算质量：3，804 Da），检测为3，808 Da片段。当通过SDS-PAGE（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳）分析复合物时，仅观察到单一条带。SDS和Triton X-100处理后，SDS-PAGE显示两种不同形式的复合物具有不同的迁移率。较高分子量条带在纤维蛋白自显影上显示出特异性tPA活性，而较低分子量条带则没有。然而，这两条带的肽序列分析出乎意料地揭示了两条带中C-末端切割肽的存在，并且其量在上带中较少。在上面的条带中，对应于其米氏中间体中两个分子之间的界面处的区域的序列被减少。因此，这两个条带对应于不同的非酰基-酶复合物，其中在所采用的条件下，仅上部条带释放游离tPA。结论在生理条件下，tPA-PAI-1以非酰化酶抑制剂复合物的形式存在。
Abstract Background Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown. Methods and Results Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl–enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed. Conclusion These data suggest that under physiological conditions a fraction of the tPA–PAI-1 population exists as nonacylated–enzyme inhibitor complex.