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Published on: May 25, 2018
Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease
Flávio Antônio de Oliveira-Simões1, Isabela Victorino da Silva Amatto2, Camila Langer Marciano2
1Pharmaceutical Sciences Program, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Abstract:
Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.
Insights
Researchers characterized the biochemical properties of the Transmembrane Serine Protease 2 (TMPRSS2) serine protease domain. Optimal activity was found with specific metal ions, providing insights into TMPRSS2 function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Transmembrane Serine Protease 2 (TMPRSS2) is a type II transmembrane serine protease implicated in oncogenesis and SARS-CoV-2 spike protein priming.
- TMPRSS2 possesses a self-activating serine protease domain crucial for its function.
Purpose of the Study:
- To biochemically characterize the serine protease domain of TMPRSS2 (rTMPRSS2_SP).
- To determine optimal conditions for rTMPRSS2_SP enzymatic activity and assess its stability.
Main Methods:
- Expression and purification of rTMPRSS2_SP in *Komagataella phaffii* using affinity and size exclusion chromatography.
- Utilized fluorescence resonance energy transfer (FRET) peptides, specifically Abz-QARK-(Dnp)-NH2, to assay enzymatic activity.
- Investigated the effects of metal ions, surfactants, and reducing agents on protease activity and determined long-term stability.
Main Results:
- Successfully expressed and purified the rTMPRSS2_SP.
- Identified Ca2+ and Na+ as enhancers of enzymatic activity, while ionic surfactants and reducing agents inhibited it.
- Characterized the stability of rTMPRSS2_SP for potential long-term storage.
Conclusions:
- This study provides the first comprehensive biochemical characterization of the TMPRSS2 serine protease domain.
- The findings offer valuable insights into the enzymatic properties and optimal conditions for TMPRSS2 activity.
- Understanding TMPRSS2's biochemical characteristics is crucial for its role in viral entry and potential therapeutic targeting.
