Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering

Abstract

Lipid raft-specific glycosylation has been implicated in many biological processes, including intracellular trafficking, cell adhesion, signal transduction, and host–pathogen interactions. The major predicament in lipid raft-specific glycosylation research is the unavailability of tools for tracking and manipulating glycans on lipid rafts at the microstructural level. To overcome this challenge, we developed a multifunctional proximity labeling (MPL) platform that relies on cholera toxin B subunit to localize horseradish peroxidase on lipid rafts. In addition to the prevailing electron-rich amino acids, modified sialic acid was included in the horseradish peroxidase-mediated proximity labeling substrate via purposefully designed chemical transformation reactions. In combination with sialic acid editing, the self-renewal of lipid raft-specific sialic acid was visualized. The MPL method enabled tracking of lipid raft dynamics under methyl-β-cyclodextrin and mevinolin treatments; in particular, the alteration of lipid rafts markedly affected cell migration. Furthermore, we embedded functional molecules into the method and implemented raft-specific sialic acid gradient engineering. Our novel strategy presents opportunities for tailoring lipid raft-specific sialic acids, thereby regulating interactions associated with lipid raft regions (such as cell–virus and cell–microenvironment interactions), and can aid in the development of lipid raft-based therapeutic regimens for tumors.

Highly Sensitive Labeling, Clickable Functionalization, and Glycoengineering of the MUC1 Neighboring System

“…Biotin–phenol is a commonly used labeling molecule in such reactions to demonstrate the local labeling effect. Researchers have employed different approaches in the biotin–phenol labeling step, with some opting to remove unbound HRP in a process known as wash-type, , while others choose not to remove it, referred to as nonwash-type . However, the efficiency of labeling in wash-type and nonwash-type affinity-primed proximity labeling has not been thoroughly examined in prior studies.…”