Luminescence detection of peptide:N-glycanase activity using engineered split inteins

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Peptide:N-glycanase (PNGase) is a crucial enzyme involved in protein deglycosylation.
• Detecting PNGase activity in live cells is essential for understanding its biological roles.
• Existing methods for PNGase detection often lack sensitivity or are not suitable for real-time cellular analysis.

Purpose of the Study:

• To develop a novel, sensitive, and live-cell compatible assay for detecting PNGase activity.
• To leverage split intein technology for protein trans-splicing and reporter gene activation.
• To enable real-time monitoring of PNGase enzymatic function within living cells.

Main Methods:

• A split intein system was engineered, with the N-intein peptide fused to a PNGase substrate.
• The C-intein peptide was fused to a reporter protein (luciferase).
• PNGase activity was detected by observing the protein trans-splicing reaction between the N- and C-intein fragments, leading to active luciferase generation.

Main Results:

• The developed split intein-based method successfully detected PNGase activity in live cells.
• PNGase cleaved the linkage between N,N'-diacetylchitobiose and the Asn side-chain of the N-intein peptide.
• The resulting fragments rapidly underwent protein trans-splicing with the C-intein, generating a functional luciferase enzyme.

Conclusions:

• The split intein-based approach provides a robust platform for detecting PNGase activity in live cellular environments.
• This method offers a sensitive and real-time readout of PNGase function.
• The developed system has potential applications in studying PNGase biology and screening for PNGase inhibitors.