A Fluorescent Glycolipid-Binding Peptide Probe Traces Cholesterol Dependent Microdomain-Derived Trafficking Pathways

Abstract

BackgroundThe uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Aβ, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al [1] in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains.Methodology/Principal FindingsIn accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB). Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol.Conclusions/SignificanceThe current work presents the characterization and trafficking behavior of a novel sphingolipid-binding fluorescent probe, the SBD peptide. We show that SBD binding to membranes is dependent on the presence of cholesterol, sphingomyelin, and complex glycolipids. In addition, SBD targeting through the endolysosomal pathway in neurons is highly sensitive to cholesterol perturbations, making it a potentially useful tool for the analysis of sphingolipid trafficking in disease models that involve changes in cholesterol metabolism and storage.

Secretory sphingomyelinase in health and disease

“…Such acidic microenvironments have been linked to lipid microdomains (Steinert et al, 2008) as the site of optimal ASM action. The translocation of lysosomal V1 H + -ATPase to the cell membrane is a critical contribution to the formation of a local acid microenvironment to facilitate membrane ASM activation and, consequently, redox signalosome formation in coronary arterial endothelial cells (Xu et al, 2012).…”

Alternate raft pathways cooperate to mediate slow diffusion and efficient uptake of a sphingolipid tracer to degradative and recycling compartments

“…SBD, a fluorescently coupled peptide consisting of the V3-loop domain of the amyloid beta peptide (Aβ) , was shown to interact with a subset of glycosphingolipids, sphingomyelin and cholesterol in artificial membranes as well as with cholesterol-and sphingolipid-dependent microdomains in neurons Steinert et al, 2008). Here, we show that SBD uptake is dependent on several different mechanismsflotillin, cdc42, and dynamin -that have generally been viewed as independent and parallel pathways (Glebov et al, 2006;Mayor and Pagano, 2007;Sabharanjak et al, 2002).…”

“…mβCD, cholesterol, HEPES, PMSF, Chlorpromazine, and Dynasore were obtained from Sigma (St Louis, MO). Cholesterol depletion and overload was carried out as previously described Steinert et al, 2008). Chlorpromazine (5 μg/ml) was used to inhibit clathrin-dependent endocytosis by pretreating for 30 minutes at 37°C before cell labeling.…”

“…The fluorescently tagged SBD peptide acts as a sphingolipid-interacting tracer with potential applications in the study of neurodegenerative disease Steinert et al, 2008). For this reason, we examined the uptake characteristics and intracellular trafficking of the SBD marker in neuroblastoma SH-SY5Y cells.…”

“…Flotillin-2 forms hetero-and homotetrameric complexes with flotillin-1, and is required for the maintenance of flotillin-1, but has a slightly different distribution from flotillin-1 (Langhorst et al, 2005;Solis et al, 2007). A flotillin-2-GFP (GFP, green fluorescent protein) transfected fusion protein (Neumann-Giesen et al, 2004;Steinert et al, 2008) and endogenous flotillin-2 were compared to SBD as a second raft-associated marker. After 1 minute, SBD showed very little colocalization with flotillin-2-GFP (presumably because flotillin-2-GFP is systemically expressed, whereas SBD is added externally), but this rose to ~40% after only 10 minutes, similar to its degree of colocalization with the endogenous flotillin-2 protein ( Fig.…”