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

Song-Yi Lee^#^{1

2

3}, Heegwang Roh^#^{4

5}, David Gonzalez-Perez⁶, Mason R Mackey^{7

8}, Daniel Hoces^{1

9}, Colleen N McLaughlin¹⁰, Chang Lin¹, Stephen R Adams¹¹, Khanh Nguyen¹², Keun-Young Kim⁷, David J Luginbuhl¹⁰, Liqun Luo¹⁰, Namrata D Udeshi¹², Steven A Carr¹², Rogelio A Hernández-López^{1

9

13}, Mark H Ellisman^{7

8}, Miguel Alcalde⁶, Alice Y Ting^{14

15

16

17

18}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, USA.
² Department of New Biology, DGIST, Daegu, Republic of Korea.
³ New Biology Research Center, DGIST, Daegu, Republic of Korea.
⁴ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁵ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
⁶ Department of Biocatalysis, Institute of Catalysis, ICP-CSIC, Madrid, Spain.
⁷ Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA.
⁸ Center for Research in Biological Systems and the National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA.
⁹ Department of Bioengineering, Stanford University, Stanford, CA, USA.
¹⁰ Department of Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
¹¹ Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
¹² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹³ Chan Zuckerberg Biohub - San Francisco, San Francisco, CA, USA.
¹⁴ Department of Genetics, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁵ Department of Chemistry, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁶ Chan Zuckerberg Biohub - San Francisco, San Francisco, CA, USA. ayting@stanford.edu.
¹⁷ Department of Biology, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁸ The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford, CA, USA. ayting@stanford.edu.

^# Contributed equally.

PMID: 40751001
DOI: 10.1038/s41589-025-01973-6

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

Song-Yi Lee et al. Nat Chem Biol. 2025.

. 2025 Aug 1.

doi: 10.1038/s41589-025-01973-6. Online ahead of print.

Authors

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, USA.
² Department of New Biology, DGIST, Daegu, Republic of Korea.
³ New Biology Research Center, DGIST, Daegu, Republic of Korea.
⁴ Department of Chemistry, Stanford University, Stanford, CA, USA.
⁵ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
⁶ Department of Biocatalysis, Institute of Catalysis, ICP-CSIC, Madrid, Spain.
⁷ Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA.
⁸ Center for Research in Biological Systems and the National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA, USA.
⁹ Department of Bioengineering, Stanford University, Stanford, CA, USA.
¹⁰ Department of Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA.
¹¹ Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
¹² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹³ Chan Zuckerberg Biohub - San Francisco, San Francisco, CA, USA.
¹⁴ Department of Genetics, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁵ Department of Chemistry, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁶ Chan Zuckerberg Biohub - San Francisco, San Francisco, CA, USA. ayting@stanford.edu.
¹⁷ Department of Biology, Stanford University, Stanford, CA, USA. ayting@stanford.edu.
¹⁸ The Phil & Penny Knight Initiative for Brain Resilience at the Wu Tsai Neurosciences Institute, Stanford, CA, USA. ayting@stanford.edu.

^# Contributed equally.

PMID: 40751001
DOI: 10.1038/s41589-025-01973-6

Abstract

Enzymes that oxidize aromatic substrates have been harnessed for cell-based technologies including proximity labeling and electron microscopy; however, they are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore multicopper oxidases (laccases) as a new enzyme class for proximity labeling and electron microscopy in mammalian cells. LaccID was generated through 11 rounds of directed evolution from an ancestral fungal laccase and catalyzes one-electron oxidation of diverse aromatic substrates using O₂ instead of toxic H₂O₂. Surprisingly, we found that LaccID is selectively active at the surface plasma membrane of both living and fixed cells. We use LaccID proximity labeling and mass spectrometry to map the changing surface proteome of T cells that engage with tumor cells through 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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Conflict of interest statement

Competing interests: A.Y.T. is a scientific advisor to Third Rock Ventures and Nereid Therapeutics. The remaining authors declare no competing interests.

Update of

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy.
Lee SY, Roh H, Gonzalez-Perez D, Mackey MR, Kim KY, Hoces D, McLaughlin CN, Adams SR, Nguyen K, Luginbuhl DJ, Luo L, Udeshi ND, Carr SA, Hernández-López RA, Ellisman MH, Alcalde M, Ting AY. Lee SY, et al. bioRxiv [Preprint]. 2024 Oct 29:2024.10.29.620861. doi: 10.1101/2024.10.29.620861. bioRxiv. 2024. Update in: Nat Chem Biol. 2025 Aug 1. doi: 10.1038/s41589-025-01973-6. PMID: 39554088 Free PMC article. Updated. Preprint.

References

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

References

Grants and funding

LinkOut - more resources

Research Materials