Exploring the CD38-associated surfaceome via nanobody-targeted TurboID

Area of Science:

• Cell Biology
• Proteomics
• Cancer Research

Background:

• CD38 is a cell-surface glycoprotein with poorly understood molecular functions.
• Understanding the CD38-proximal surfaceome is crucial for elucidating its biological roles.
• Current methods lack the ability to comprehensively map proteins in close proximity to CD38 on live cells.

Purpose of the Study:

• To develop and validate a novel proximity labeling method to characterize the CD38-proximal surfaceome.
• To identify proteins associated with CD38 in different cell types, including lung cancer cells.
• To investigate the functional role of CD38 in cell migration and tumor-endothelial interactions.

Main Methods:

• Development of nanobody-targeted TurboID (NBID) using protein-of-interest (POI)-specific nanobody-TurboID chimeras (NTCs).
• Application of NBID to A549 lung cancer cells and THP-1 monocytic leukemia cells.
• Validation using EGFR-targeting NTCs and combination with SILAC (stable isotope labeling by amino acids in cell culture).
• Total internal reflection fluorescence microscopy and functional assays for cell migration.

Main Results:

• NBID successfully identified known EGFR-associated proteins, validating the approach.
• Enrichment of proteins involved in adhesion, ECM organization, and lipid raft domains around CD38.
• Identification of CD38-associated cadherin adhesion complexes (CDH2, DSG2) at the tumor-endothelial interface.
• Demonstration of CD38's contribution to transendothelial migration of A549 cells.

Conclusions:

• NBID is a robust platform for studying surface protein environments and intercellular interactions in native conditions.
• A CD38-associated membrane adhesion network was revealed, involving cadherins.
• CD38 plays a significant role in tumor cell migration and tumor-endothelial interactions.