Recombinant-Chemosynthetic Biosensors for Probing Cell Surface Signaling of Red Blood Cells and Other Cells

The complex signaling mechanisms in red blood cells (RBCs) enable them to adapt to physiological stresses such as exposure to low O₂ levels, metabolic demands, oxidative stress, and shear stress. Since Ca²⁺ is a crucial determinant of RBC fate, various ion channels, pumps, and exchangers regulate the delicate balance of Ca²⁺ influx and efflux in RBCs. Elevated intracellular Ca²⁺ can activate processes such as membrane phospholipid scrambling and alter RBC deformability, which is essential for effective capillary transit. However, the dynamic information about Ca²⁺ regulation in RBCs is limited. Although static mapping and bioanalytical methods have been utilized, the absence of a nucleus and the presence of hemoglobin create challenges for real-time probing of RBC signaling, necessitating innovative approaches. This work introduces a synthetic chemistry-recombinant protein-based strategy to assemble sensors at genetically intact healthy human RBC surfaces for measuring dynamic signaling. Using this approach, we measured autocrine regulation of RBC Ca²⁺ influx in response to low O₂ tension-induced ATP release. The study also explores the utilization of synthetic glycosylphosphatidylinositol (GPI) anchor mimics and sortagging for targeting sensors to the surfaces of primary as well as immortalized cells. This demonstrated the wide applicability of this approach to probe dynamic signaling in intact cells.