Proximity Enzymatic Glyco-Remodeling Enables Direct And Highly Efficient Lipid Raft Imaging On Live Cells

Lipid rafts are highly ordered membrane domains(<200 nm)on cell membranes that are rich in cholesterol and sphingolipids.Lipid rafts selectively enrich specific types of lipids and proteins to be closely related to various physiological functions such as immune signaling,host-pathogen interaction,cancer occurrence and development,and cardiovascular disease.Therefore,in situ imaging and long-term tracking of lipid rafts will help to further deepen the understanding of lipid raft selective recruitment mechanisms and physiological functions.At present,lipid raft imaging technology mainly uses fluorescence imaging,but in these methods,fluorescent molecules are labeled on probes that specifically distinguish lipid rafts,rather than straightforward lighting up the exact components residing in rafts,so direct visualization and dynamic tracking of raft component cannot be achieved.And these rapid intracellular entry of recognition probes or fluorescent lipids,just as in the case of cholera toxin B subunit(CTx B),the probes may lose identification capability at an uncertain time point and lead to doubtful results.Therefore,the development of methods that can directly label and track lipid molecules for a long time is of great significance for the research of lipid rafts.In this paper,inspired by the notably high level of protein(lipid)glycosylation on mammalian cell membrane,we propose a proximity enzymatic glyco-remodeling(PEGR)solution by utilizing raft-resident glycans(carbohydrate chains covalently linked to protein or lipids)as the labeling and imaging sites by taking lipid rafts of MCF-7 cell membrane as research model.This method combines lipid rafts recognition,glycan remodeling enzyme inhibition-activation switching and adjacent glycan remodeling,and combines with bio-orthogonal labeling to achieve direct labeling of lipid raft regions.Using gold nanoparticles(Au NP)as the loading unit,the lipid raft recognition molecules CTx B and the glycan remodeling molecules galactose oxidase(GO)were integrated as the probe GO-Au NP-CTx B.Through the binding between CTx B and GM1,the probe whose GO activity is inhibited by potassium ferrocyanide is specifically introduced into the lipid raft region without performing the glycan remodeling operation.After washing off the unbound probe,potassium ferricyanide is added to reactivate the GO activity.At this time,the probe performs a glycan remodeling operation on the adjacent area(oxidized terminal galactose/Nacetylgalactosamine),and then uses hydrazide-FITC or hydrazide-AF647 to perform bioorthogonal labeling under the catalysis of aniline,and finally imaged by confocal microscope.Because the probe size is smaller than the lipid raft,and the probe anchoring and remodeling events are strictly separated,the direct labeling restricted to the lipid raft is successfully achieved.Moreover,due to the strong loading capacity of Au NP,the circulating oxidation ability of GO,and the high expression of glycan conjugates in the lipid raft region,the method successfully transformed one,lipid raft recognition event to multiple,lipid raft-confined glyco-remodeling operations,and achieved efficient,direct labeling and imaging of components in the lipid raft area.This direct and efficient lipid raft labeling function not only improves the sensitivity of raft imaging,but also enables in-situ and long-term tracking of lipid raft components.Proximity enzymatic glyco-remodeling technology provides a new perspective for lipid raft imaging.When combined with other techniques such as mass spectrometry(MS),the method may be further exploited for colocalization analysis of glycoproteins/glycolipids surrounding GM1 and also profiling of raftophilic elements.Thus,the proposed method will facilitate the revelation of sorting mechanisms and biological functions of raft domains.