Directed evolution of LaccID for cell surface proximity labeling and electron microscopy.

Area of Science:

• Biochemistry
• Cell Biology
• Biotechnology

Background:

• Enzymes oxidizing aromatic substrates are used in proximity labeling and electron microscopy.
• Existing methods often require toxic hydrogen peroxide (H2O2).
• There is a need for safer and more versatile enzyme systems for cell-based applications.

Purpose of the Study:

• To introduce multicopper oxidases (laccases) as a new enzyme class for proximity labeling and electron microscopy in mammalian cells.
• To develop and characterize LaccID, a directed-evolution derived laccase for cell surface applications.
• To demonstrate LaccID's utility in mapping cellular proteomes and visualizing cellular structures.

Main Methods:

• Directed evolution of a fungal laccase to create LaccID.
• LaccID-based proximity labeling coupled with mass spectrometry.
• LaccID as a genetic tag for electron microscopy (EM) visualization.
• Testing LaccID activity in living and fixed mammalian cells and fly brains.

Main Results:

• LaccID catalyzes aromatic substrate oxidation using O2, avoiding toxic H2O2.
• LaccID exhibits selective activity at the plasma membrane of both living and fixed cells.
• LaccID successfully mapped the changing surface proteome of T cells interacting with tumor cells.
• LaccID enabled EM visualization of cell surface features in mammalian cells and fly brains.

Conclusions:

• LaccID represents a novel, non-toxic enzyme alternative for proximity labeling and EM.
• The enzyme's cell surface localization and O2-dependent activity offer advantages for biological research.
• LaccID has broad potential for future cell-based technologies and imaging applications.