Exploring the CD38-Associated Surfaceome via Nanobody-Targeted TurboID

Cluster of differentiation (CD38) is a cell-surface glycoprotein whose molecular functions are closely linked to its surrounding protein environment. To date, omics-level understanding of CD38-associated surface proteins remains lacking. To investigate the CD38-proximal surfaceome in live adherent cell culture, we developed nanobody-targeted TurboID (NBID), a proximity labeling approach using protein of interest-specific nanobody-TurboID chimeras, and applied this approach to characterize CD38-proximal proteome on the cell surface. The approach was validated using epidermal growth factor receptor-targeting NTC construct, which recovered multiple known epidermal growth factor receptor-associated proteins. CD38-specific NTC was applied to A549 lung cancer cells and Tohoku Hospital Pediatrics-1 monocytic leukemia cells, and enriched multiple proteins involved in adhesion, ECM organization, and lipid raft-associated membrane domains. In A549 cells, proteins related to Wnt signaling were additionally enriched. NAD⁺ treatment did not significantly alter the CD38-proximal surfaceome in A549 cells. Total internal reflection fluorescence microscopy further demonstrated coclustering of several enriched candidates with CD38 on membrane protrusions. Functional assays showed that CD38 contributes to transendothelial migration of A549 cells. By combining NBID with stable isotope labeling by amino acids in cell culture, we further identified CD38-associated cadherin adhesion complexes involving N-cadherin and desmoglein-2 at the tumor-endothelial interface. Together, these results establish NBID as a robust platform for investigating surface protein environments and intercellular protein neighborhoods under native conditions. Furthermore, our findings reveal a CD38-associated membrane adhesion network and provide new insight into the role of CD38 in tumor cell migration and tumor-endothelial interactions.