Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

Song-Yi Lee; Heegwang Roh; David Gonzalez-Perez; Mason R Mackey; Keun-Young Kim; Daniel Hoces; Colleen N McLaughlin; Stephen R Adams; Khanh Nguyen; David J Luginbuhl; Liqun Luo; Namrata D Udeshi; Steven A Carr; Rogelio A Hernández-López; Mark H Ellisman; Miguel Alcalde; Alice Y Ting

doi:10.1101/2024.10.29.620861

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

bioRxiv [Preprint]. 2024 Oct 29:2024.10.29.620861. doi: 10.1101/2024.10.29.620861.

Authors

Song-Yi Lee¹, Heegwang Roh², David Gonzalez-Perez³, Mason R Mackey⁴, Keun-Young Kim⁴, Daniel Hoces^{1

5}, Colleen N McLaughlin⁶, Stephen R Adams⁷, Khanh Nguyen⁸, David J Luginbuhl⁶, Liqun Luo⁶, Namrata D Udeshi⁸, Steven A Carr⁸, Rogelio A Hernández-López^{1

5

9}, Mark H Ellisman^{4

10}, Miguel Alcalde³, Alice Y Ting^{1

2

11

9}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA 94305, USA.
² Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
³ Department of Biocatalysis, Institute of Catalysis, ICP-CSIC, 28049, Madrid, Spain.
⁴ Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, USA.
⁵ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁶ Department of Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
⁷ Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁹ Chan Zuckerberg Biohub-San Francisco, San Francisco, CA 94158, USA.
¹⁰ Center for Research in Biological Systems and the National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA 92093, USA.
¹¹ Department of Biology, Stanford University, Stanford, CA 94305, USA.

Abstract

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O₂ instead of toxic H₂O₂, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

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Preprint

Abstract

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