Development of fluorescent cholesterol derivatives for the exogenous introduction of proteins to the plasma membrane

The exogenous introduction of fluorescentlipoproteins into cell membranes is a method for visualizingthe cellular traffic of membrane associated proteins, and alsofor altering the cell surface in a controlled manner. In order toachieve the cell membrane anchoring of proteins and theirsubsequent fluorescence based detection, a cholesterolderivative was designed. The headgroup of the novelcholesterol anchor contains a fluorescent reporter and a thiolreactive maleimide for protein conjugation. Protein conjuga-tion was demonstrated by the addition of a green fluorescentmaleimido anchor to the C-terminus of a Cys extended redfluorescent protein, mCherry. The resulting dual fluorescentcholesteryl lipoprotein was successfully separated from themicellar associates of the surplus fluorescent lipid anchor without denaturing the protein, and the lipoprotein containing only thecovalently linked, stoichiometric fluorescent lipid was efficiently delivered to the plasma membrane of live cells. It wasdemonstrated that the membrane fluorescence could be directly assigned to the protein−anchor conjugate, because no excess offluorescent lipid species were present during the imaging experiment and the protein and anchor fluorescence colocalized in thecell membrane. Molecular dynamics simulations and subsequent trajectory analysis suggest also the spontaneous and stablemembrane association of the cholesterol anchor. Thus, the method could be beneficially applied for studying membraneassociated proteins and for preparing mimetics of glycosylphosphatidylinositol (GPI)-anchored proteins to target cholesterol-richmembrane microdomains. ■ INTRODUCTIONLipidation plays an important role in the localization andfunction of proteins. Four common modifications of proteinswith lipid moieties are myristoylation, palmitoylation, prenyla-tion, and the attachment of glycosylphosphatidylinositol (GPI)anchors. The amidation of the protein C-terminus with aGPI glycolipid results in proteins tethered to the extracellularleaflet of the cell membrane. This modification is widespreadthroughout eukaryotes, and more than 150 human GPI-anchored proteins (GPI-APs) are known with variousfunctions: enzymes, receptors, complement regulation proteins,antigens, or adhesion molecules. Beyond the normalphysiological functions, GPI-APs are associated with a rangeof diseases including paroxysmal nocturnal hemoglobinuria,prion diseases, carcinogenesis, and sleeping sickness. Theimportant functional role of the GPI-APs is further evidencedby the embryonic lethality of the GPI-deficient mice. TheGPI glycolipids have the conserved Man(α1−2)Man(α1−6)Man(α1−4)GlcN(α1−6)myoIno glycan core and variationsarise in the substitution pattern and in the lipid composi-tion. The lipid part of the mammalian GPIs isphosphatidylinositol with stearyl chains; therefore, the GPI-APs are able to temporarily associate with sphingolipidandcholesterol-rich membrane microdomains, i.e., lipid rafts.This clustering is mainly due to the favorable hydrophobicinteractions between the saturated acyl chains of the GPIanchor and the lipid constituents of the rafts. It wasevidenced that the lipid raft association of the GPI-APs couldbe abrogated by cholesterol depletion of the cell membrane andby replacing the GPI moiety with a transmembrane anchor. Received: April 24, 2013Revised: September 4, 2013Published: September 10, 2013Article