Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

Abstract

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.

Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif

“…Previously, SBD peptide was suggested to bind to gangliosides in the presence of sphingomyelin and cholesterol using surface plasmon resonance and fluorescence correlation spectroscopic assays [ 37 , 38 ]. GT1b was found to have a marginally higher binding at neutral pH than other common brain gangliosides, such as GM1, GD1a, and GQ1b [ 20 , 38 ]. Our simulations employed the same 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin (18:0) (SM) and cholesterol (CHOL) composition used in these experimental studies.…”

“…It has been proposed that toxin-membrane interactions are mediated by a discrete sphingolipid binding motif rich in aromatic amino acids containing one or more key basic residues [ 19 , 20 ]. This loosely-defined domain was suggested to exist in Aβ [ 21 ], HIV gp120 [ 22 ], prion [ 23 ], Shiga toxin [ 24 ], as well as more recently in α-synuclein [ 25 ].…”

“…In a recent series of studies, a fluorescently-tagged variant of Aβ 1–25 , referred as to Sphingolipid Binding Domain (SBD) peptide, was constructed and characterized as a ganglioside and sphingolipid domain tracer in cellular and artificial membranes [ 37 , 38 , 39 , 40 ]. A mutation of K16E was also examined for sphingolipid binding [ 20 ]. Surprisingly, this Aβ 1–25 variant showed a similar ganglioside preference as the Aβ 1–40 peptide [ 28 , 41 ], even in the absence of the sequence from 26–40.…”

Recent applications of fluorescence correlation spectroscopy in live systems

“…FCS has been also used to study membrane-induced aggregation of peptides [124,125] (number 3 in Fig. 1).…”

The substrate matters in the Raman spectroscopy analysis of cells

“…Therefore the understanding of the behaviour of such living cells is a very important issue in bioelectronics. Several optical bio-compatible methods are known for the investigation of cellular behaviour in culture: infrared spectroscopy 3 , surface plasmon resonance 4 , optical coherence tomography 5 , and bioluminescence imaging 6 . In addition, there is progress in non-optical methods such as electron tomography 7 .…”