GPI锚定丝氨酸蛋白酶testisin的活化机制研究

doi:10.3969/j.issn.1674-8115.2024.12.003

摘要/Abstract

摘要：

目的·探讨睾丸素（testisin）酶原在体外的活化方式及活性调控机制，为进一步研究testisin在生物体内的生理功能奠定基础。方法·通过全基因合成，构建鼠源testisin（mTN）蛋白酶原的真核表达质粒，并将其转染至真核HEK293S细胞中；通过镍离子亲和层析等方法获得纯化重组testisin蛋白；将酶原激活位点和活性中心位点进行定点突变，利用酶活性检测等手段分析testisin的活化机制，通过改变酶原的孵育条件确定蛋白的活化速率；利用Edman降解法及定点突变确定蛋白酶的自剪切位点；通过检测蛋白对底物的分解速率分析蛋白酶的活性调控机制。结果·重组mTN酶原在真核HEK293S细胞中高效表达，通过一步纯化可以获得纯度较高且性质均一的重组蛋白。该蛋白在酸性条件下稳定，在中性或碱性环境下会发生自激活，其激活速率受到酸碱度和温度等因素的影响，且这种自激活依赖于该酶激活位点Arg⁴⁶和酶活性中心Ser²⁴⁰的完整性。重组mTN酶原的活化常伴随分子表面170/175肽环的自剪切，但该自剪切并不影响其蛋白酶的活性。活化的mTN最适反应pH值为8.0，且在其最适pH值附近较为稳定；最适反应温度为50 ℃，且蛋白适宜保存在低于30 ℃的环境中；Zn²⁺和Ca²⁺对mTN的活性有明显的抑制作用。结论·成功建立了一种有效制备重组mTN酶原的方法，发现重组mTN在体外可以发生自激活和自剪切。

关键词: testisin, 自激活, 丝氨酸蛋白酶, 自剪切

Abstract:

Objective ·To explore the activation methods and activity regulation mechanisms of the testisin zymogen in vitro, laying a foundation for further research on the physiological functions of testisin in organisms. Methods ·The eukaryotic expression plasmid for the mouse-derived testisin (mTN) zymogen was constructed through full-gene synthesis and subsequently transfected into eukaryotic HEK293S cells for expression. Purified recombinant testisin protein was obtained through methods such as nickel ion affinity chromatography. Site-directed mutagenesis was performed on the activation site and active center of the zymogen, and the activation mechanism of testisin was analyzed through enzymatic activity assays. Additionally, the activation rate of the protein was determined by altering the incubation conditions of the zymogen. The self-cleavage site of the protease was identified by using Edman degradation and site-directed mutagenesis. Finally, the regulatory mechanism of protease activity was examined by analyzing the proteolytic rate of the protein towards its substrates. Results ·The recombinant mTN zymogen was expressed in eukaryotic HEK293S cells, and high-purity, uniform recombinant protein was obtained through a two-step purification process. This protein was found to remain stable under acidic conditions but underwent self-activation in neutral or alkaline environments. The rate of activation was influenced by factors such as pH value and temperature. The self-activation process required on the integrity of the enzyme's activation site, Arg⁴⁶, and its active center, Ser²⁴⁰. Additionally, the self-activation of the recombinant mTN zymogen was often accompanied by the self-cleavage at the 170/175 loop on the molecular surface, but this self-cleavage did not affect the activity of testisin. Furthermore, the optimal reaction pH for activated mTN was 8.0, with the protein remaining relatively stable near this pH. The optimal reaction temperature was 50 ℃, while the protein was best stored at temperatures below 30 ℃. Zn²⁺and Ca²⁺ were found to significantly inhibit mTN activity. Conclusion ·An effective method for the preparation of recombinant mTN zymogen is successfully established, and it is discovered that the recombinant mTN undergoes self-activation and self-cleavage in vitro.

Key words: testisin, self-activation, serine protease, self-cleavage

中图分类号:

万家惠, 周爱武. GPI锚定丝氨酸蛋白酶testisin的活化机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(12): 1504-1513.

WAN Jiahui, ZHOU Aiwu. Mechanistic studies on regulation of the activity of GPI-anchored serine protease testisin[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1504-1513.

图/表 5

表1 影响mTN活化速率的孵育条件

Tab 1 Incubation conditions affecting the activation rate of mTN

Group	mTN concentration/(mg·mL^-1)	pH	Salt concentration/(mol·L^-1)	Temperature/℃
1	0.1	pH7.4	0.1	20
2	0.2	pH7.4	0.1	20
3	0.1	pH6.0	0.1	20
4	0.1	pH7.4	1.0	20
5	0.1	pH7.4	0.1	37

图1 mTN在体外的自激活验证Note：A. SDS-PAGE of the preliminary activation tests of mTN under reducing conditions. B. SDS-PAGE of mTN obtained by Q anion exchange chromatography under reducing conditions. C.Schematic diagram of the molecular structure of mTN with two mutation sites prominently identified. D. Activation status of mTN and the two variants was detected every 24 h. The relative activity of mTN was derived from slope of the linear plot of absorbance at 405 nm vers incubation time during activity assay. Western blotting analysis revealed that a band of approximately 5 000 corresponded to the zymogen, while a band of approximately 1 500 corresponded to the degraded C-terminal fragment.

Fig 1 Self-activation verification of mTN in vitro

图2 mTN酶原自激活速率的影响因素Note： Every 24 h, an equal amount of mTN under different incubation conditions was diluted into a substrate S2238 solution to detect the activation status of the protein. Western blotting was performed to assess protein cleavage under reducing conditions.

Fig 2 Influencing factors of mTN self-activation rate

图3 N-端测序结果和mTN-3A、GGA突变体的自激活验证Note：A. The results for the N-terminal sequence of the light chain peptide of mTN (the first five amino acids). The red arrow indicates the measured amino acid signals. The Y-axis represents signal intensity, and the X-axis represents the testing time. Au—Absorbance unit.1 Au=1 000 mAu. B. The comparison result of the catalytic domain sequences of mTN with other serine proteases (partial). The five amino acids indicated by the black triangle corresponded to the first five amino acids of the N-terminal sequencing; the six amino acids indicated by the blue triangle corresponded to amino acids 211?216 (KKPDFR) of the recombinant protein, where the cleavage site was thought to be located. C.The enzymatic activity trend and Western blotting result of the 3A mutant. D. The enzymatic activity trend and Western blotting result of the GGA mutant. E. Predicted structure of the mTN zymogen generated by Alphafold 2.

Fig 3 N-terminal sequencing results and validation of self-activation in mTN-3A & GGA mutants

图4 酸碱度、温度和金属离子对mTN活性的影响Note：A. The optimal pH value for mTN reaction. B. pH value stability of mTN. C. The optimal temperature for mTN reaction. D. Thermal stability of mTN. E. The effect of Zn2+ and Ca2+ on the activity of mTN. F. The effect of Na+ on the activity of mTN.

Fig 4 Effect of pH value, temperature and metal ions on mTN activity

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