Photoaffinity Labeling Methods to Explore Internalization Mechanisms of Arginine-Rich Cell-Penetrating Peptides

Abstract

Peptides with membrane permeability have been used extensively as carriers for intracellular delivery of bioactive molecules, and endocytosis plays an important role in the cellular uptake of these peptides. Elucidating the mechanisms of uptake will contribute to the development of more sophisticated delivery systems. In this chapter, we introduce a novel approach for identifying receptors involved in the cellular uptake of oligoarginines, a representative class of cell-penetrating peptides (CPPs). Photoaffinity probes with oligoarginines together with trifluoromethyl diazirines as photoreactive moieties and biotin as an isolation tag were designed. We identified C-X-C chemokine receptor type 4 (CXCR4) as a receptor for dodecaarginine (R12) uptake via macropinocytosis, a typical uptake pathway for arginine-rich CPPs. Interestingly, CXCR4 does not play a significant role in the uptake of octaarginine (R8) and HIV-1 TAT peptides, suggesting that more than one form of macropinocytosis is involved in arginine-rich CPP uptake. The cytoplasmic protein LanCL1 was also identified because of its ability to promote R8 cellular uptake. A novel probe with a diazobenzene linker, which allowed chemoselective cleavage of the photocrosslinked proteins from affinity beads, facilitated isolation of the target proteins. Using this probe, syndecan-4 was identified as a receptor involved in R8 uptake via clathrin-mediated endocytosis.