Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes

Abstract

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-L-SerN 3 ) or carbonyl (C-L-SerN 3 ) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.

Subcellular imaging of lipids and sugars using genetically encoded proximity sensors

“…One concern with any metabolic labeling approach is that tagged molecules might not fully mimic native compounds, which is especially relevant for lipids with minimal headgroups. For several azido lipids, previous studies suggest that compounds localize across a range of cells as expected 4,5 , though it is challenging to preclude all differences between labeled and unlabeled species. An additional limitation common to bioorthogonal approaches is the time required for conjugation and washing out of unreacted fluorogens; these might make the analysis of rapid redistributions of the underlying labeled species, like those relevant for lipid signaling 50 , challenging.…”

“…Investigating membrane chemical heterogeneity remains a challenge due to the paucity of tools for imaging lipids in live cells. Bioorthogonal ‘click’ chemistry has emerged as one tool for fluorescence labeling of specific lipid classes – including phosphatidylcholine (PC) 4 , phosphatidylserine (PS) 5 , phosphatidylinositol (PI) 6 , phosphatidic acid 7 , and glycosphingolipids 8 – that can be metabolically tagged with azide-bearing head group components (Fig. 1a).…”

Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism

“…The resulting tagged lipid products can be conveniently derivatized via click chemistry to append various reporter tags of use for detecting and tracking lipid biosynthesis. A common lipid metabolic labeling approach has focused on labeling specific phospholipid structures using clickable probe analogs of substrates that map onto the headgroups of target glycerophospholipids [14] including phosphatidylcholine (PC), [15] phosphatidic acid (PA), [16] phosphatidylserine (PS), [17] phosphatidylinositol (PI) [18] and glycophosphatidylinositol (GPI) anchors. [19] This bioorthogonal strategy also extends to labeling ceramide or other sphingolipids.…”

Reactive Oxygen Species-Responsive Sequentially Targeted AIE Fluorescent Probe for Precisely Identifying the Atherosclerotic Plaques

“…32 However, protein implementation has also many issues in clinical applications because of the cost of production, endogenous competition, and the restrained diffusion to the targeted sites. 33 A viable alternative to large proteins is the use of smaller peptides to target PS with high affinity and specificity. A PS-specific peptide with an amino acid sequence of CLIKKPF (CF) has been demonstrated to bind to membranes containing externalized PS of apoptotic cells, which have high affinity and specificity for PS.…”

“…Phosphatidylserine (PS) is an attractive target for imaging apoptosis because it is transferred onto the exterior surface of the apoptotic cells, it represents a high-capacity target, and PS externalization is an early marker of apoptosis. − PS targeting with fluorescent annexin V has been widely employed for noninvasive imaging of apoptosis . However, protein implementation has also many issues in clinical applications because of the cost of production, endogenous competition, and the restrained diffusion to the targeted sites . A viable alternative to large proteins is the use of smaller peptides to target PS with high affinity and specificity.…”