Journal of Shanghai Jiao Tong University (Medical Science) >

2024 , Vol. 44 >Issue 12: 1504 - 1513

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.12.003

Basic research

Mechanistic studies on regulation of the activity of GPI-anchored serine protease testisin

  • Jiahui WAN ,
  • Aiwu ZHOU
Expand
  • Department of Pathophysiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
ZHOU Aiwu, E-mail: aiwu.zhou@googlemail.com.

Received date: 2024-04-12

  Accepted date: 2024-05-28

  Online published: 2024-12-28

Supported by

National Natural Science Foundation of China(32371306)

Fold

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

Cite this article

Jiahui WAN , Aiwu ZHOU . 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 . DOI: 10.3969/j.issn.1674-8115.2024.12.003

References

1 HEDSTROM L. Serine protease mechanism and specificity[J]. Chem Rev, 2002, 102(12): 4501-4524.
2 RAWLINGS N D, MORTON F R, KOK C Y, et al. MEROPS: the peptidase database[J]. Nucleic Acids Res, 2008, 36(Database issue): D320-D325.
3 ANTALIS T M, BUZZA M S, HODGE K M, et al. The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment[J]. Biochem J, 2010, 428(3): 325-346.
4 DI CERA E. Serine proteases [J]. IUBMB Life, 2009, 61(5): 510-515.
5 HEDSTROM L. Serine protease mechanism and specificity[J]. Chem Rev, 2002, 102(12): 4501-4524.
6 ANTALIS T M, BUGGE T H, WU Q Y. Membrane-anchored serine proteases in health and disease[J]. Prog Mol Biol Transl Sci, 2011, 99: 1-50.
7 VERGHESE G M, GUTKNECHT M F, CAUGHEY G H. Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor[J]. Am J Physiol Cell Physiol, 2006, 291(6): C1258-C1270.
8 HONDA A, YAMAGATA K, SUGIURA S, et al. A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein[J]. J Biol Chem, 2002, 277(19): 16976-16984.
9 HOOPER J D, NICOL D L, DICKINSON J L, et al. testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors[J]. Cancer Res, 1999, 59(13): 3199-3205.
10 TANG T, KMET M, CORRAL L, et al. testisin, a glycosyl-phosphatidylinositol-linked serine protease, promotes malignant transformation in vitro and in vivo[J]. Cancer Res, 2005, 65(3): 868-878.
11 YEOM S Y, JANG H L, LEE S J, et al. Interaction of testisin with maspin and its impact on invasion and cell death resistance of cervical cancer cells[J]. FEBS Lett, 2010, 584(8): 1469-1475.
12 AIMES R T, ZIJLSTRA A, HOOPER J D, et al. Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis[J]. Thromb Haemost, 2003, 89(3): 561-572.
13 DRIESBAUGH K H, BUZZA M S, MARTIN E W, et al. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin[J]. J Biol Chem, 2015, 290(6): 3529-3541.
14 PEROUTKA R J, BUZZA M S, MUKHOPADHYAY S, et al. testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis[J]. PLoS One, 2020, 15(6): e0234407.
15 L'ABBé D, BISSON L, GERVAIS C, et al. Transient gene expression in suspension HEK293-EBNA1 cells[J]. Methods Mol Biol, 2018, 1850: 1-16.
16 FUSEK M, LIN X L, TANG J. Enzymic properties of thermopsin[J]. J Biol Chem, 1990, 265(3): 1496-1501.
17 GOETTIG P, BRANDSTETTER H, MAGDOLEN V. Surface loops of trypsin-like serine proteases as determinants of function[J]. Biochimie, 2019, 166: 52-76.
18 CEDERHOLM-WILLIAMS S A. Concentration of plasminogen and antiplasmin in plasma and serum[J]. J Clin Pathol, 1981, 34(9): 979-981.
19 CESARMAN-MAUS G, HAJJAR K A. Molecular mechanisms of fibrinolysis[J]. Br J Haematol, 2005, 129(3): 307-321.
20 WU Q Y, PARRY G. Hepsin and prostate cancer[J]. Front Biosci, 2007, 12: 5052-5059.
21 DONG N Z, ZHOU T T, ZHANG Y, et al. Corin mutations K317E and S472G from preeclamptic patients alter zymogen activation and cell surface targeting.[Corrected][J]. J Biol Chem, 2014, 289(25): 17909-17916.
22 SHI Y E, TORRI J, YIEH L, et al. Identification and characterization of a novel matrix-degrading protease from hormone-dependent human breast cancer cells[J]. Cancer Res, 1993, 53(6): 1409-1415.
23 JIN X L, HIROSAKI T, LIN C Y, et al. Production of soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by trypsin[J]. J Cell Biochem, 2005, 95(3): 632-647.
24 BENAUD C, DICKSON R B, LIN C Y. Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor[J]. Eur J Biochem, 2001, 268(5): 1439-1447.
25 AFAR D E, VIVANCO I, HUBERT R S, et al. Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia[J]. Cancer Res, 2001, 61(4): 1686-1692.
26 LUCAS J M, TRUE L, HAWLEY S, et al. The androgen-regulated type Ⅱ serine protease TMPRSS2 is differentially expressed and mislocalized in prostate adenocarcinoma[J]. J Pathol, 2008, 215(2): 118-125.
27 TANABE L M, LIST K. The role of type Ⅱ transmembrane serine protease-mediated signaling in cancer[J]. FEBS J, 2017, 284(10): 1421-1436.
28 PAWAR N R, BUZZA M S, ANTALIS T M. Membrane-anchored serine proteases and protease-activated receptor-2-mediated signaling: co-conspirators in cancer progression[J]. Cancer Res, 2019, 79(2): 301-310.
29 TRUEBESTEIN L, TENNSTAEDT A, M?NIG T, et al. Substrate-induced remodeling of the active site regulates human HTRA1 activity[J]. Nat Struct Mol Biol, 2011, 18(3): 386-388.
30 LECHTENBERG B C, MURRAY-RUST T A, JOHNSON D J, et al. Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis[J]. Blood, 2013, 122(16): 2777-2783.
31 REYDA S, SOHN C, KLEBE G, et al. Reconstructing the binding site of factor Ⅹa in trypsin reveals ligand-induced structural plasticity[J]. J Mol Biol, 2003, 325(5): 963-977.
Options
Outlines

/

〈 〉