Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

Kwang-Eun Kim,Won Gun Choi,Myeong-Gyun Kang,Jae Myoung Suh,Hyun-Woo Rhee,Jeesoo Kim,Isaac Park,Hyemi Shin,Jong-Seo Kim

doi:10.1038/s41467-021-25546-y

Abstract

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

Highlights

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology
To engineer a TurboID-based tool for the selective labeling of secretory proteins as they transit through the ER lumen, we first tested the functionality of two ER lumen-targeted TurboIDs, an ER lumen-localized TurboID (TurboID-V5-KDEL) and an ER membrane-anchored TurboID (Sec61b-V5-TurboID), in cultured cells
Immunofluorescence analysis of transfected cells with anti-V5 antibody and fluorescence-conjugated streptavidin confirmed expected patterns of ER localization for both TurboID-KDEL and Sec61bTurboID along with their biotinylated targets (Fig. 1b). These results are consistent with previous ER localization studies for APEX2KDEL and Sec61b-APEX27 and are in contrast with the cytosolic localization pattern of TurboID fused to a nuclear export signal (NES), TurboID-V5-NES, (Fig. 1b and Supplementary Fig. 1a)

Summary

Introduction

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. To control cells, a broad array of biotinylated proteins was detected in both cell lysates and culture supernatant of cells expressing TurboID-KDEL and Sec61bTurboID in a biotin treatment-dependent manner (Fig. 1c). We confirmed that Sec61bTurboID robustly labeled secretory proteins without selfsecretion in the HepG2 human liver cell line, whereas TurboID-KDEL was again found to be secreted into the culture supernatant (Supplementary Fig. 3).

Results

Conclusion