Determination of Glycolipid–Protein Interaction Specificity

Abstract

Glycolipids are found on all eukaryotic cells. Their expression varies among tissues, with the highest density found in the brain, where glycolipids are the most abundant of all glycoconjugate classes. In addition to playing roles in membrane structure, glycolipids also act as cell surface recognition molecules, mediating cell-cell interactions, as well as binding certain pathogens and toxins. Because of their amphipathic nature, underivatized glycolipids are amenable to immobilization on hydrophobic surfaces, where they can be probed with lectins, antibodies, pathogens, toxins, and intact cells to reveal their binding specificities and affinities. Three particularly useful methods to probe specific glycolipid-mediated recognition events are micro-well adsorption (ELISA), thin layer chromatography overlay, and surface plasmon resonance (SPR) spectroscopy.

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

“…A single chain variable fragment (scFv, MW 26,539 Da) of the monoclonal antibody Se155-4, which served as a reference protein (P ref ) [31,32] for direct ESI-MS binding measurements, was produced using recombinant technology as described elsewhere [33]. To prepare stock solutions of CTB 5 and Stx1B 5 , each protein was dialyzed against 200 mM ammonium acetate (pH 6.8) using 0.5 mL Amicon microconcentrators (EMD Millipore, Billerica, MA, USA) with a 30 kDa MW cutoff. A similar procedure, using microconcentrators with a 10 kDa MW cutoff, was applied to scFv.…”

“…With the exception of GM3, which does not form micelles [28,29], the concentrations of gangliosides were above the critical micelle concentration [29,30]. The ganglioside interactions identified for CTB 5 and Stx1B 5 by CaR-ESI-MS, together with binding data acquired for CTB 5 and gangliosides solubilized in NDs or PDs, as well as affinity data measured for CTB 5 and the ganglioside oligosaccharides, were used to assess the reliability of using ESI-MS (and CaR-ESI-MS) and glycomicelles for detecting protein-GL interactions in aqueous solutions.…”

“…Glycolipids are amphipathic molecules consisting of a hydrophobic lipid moiety, which inserts into the cell membrane, and a hydrophilic mono-, oligo-, or polysaccharide head group that is exposed to the aqueous environment. Glycolipids are readily immobilized on hydrophobic surfaces and, thus, their interactions with water-soluble proteins can be studied using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) spectroscopy, and thin layer chromatography (TLC) [4][5][6]. In addition, microarrays prepared using naturally-occurring GLs or synthetic GLs (neoGLs), which enable GL-based glycan screening, have been successfully used for the discovery of protein-GL interactions [7][8][9].…”

“…CTB 5 exhibits broad specificity for gangliosides; S t x 1 B 5 i s k n o w n t o b i n d g l o b o s i d e s ( n e u t r a l glycosphingolipids) such as Gb3 and Gb4, but does not recognize gangliosides [26,27]. The CaR-ESI-MS assay, which is outlined in Figure 1, was used to analyze aqueous solutions of CTB 5 or Stx1B 5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. With the exception of GM3, which does not form micelles [28,29], the concentrations of gangliosides were above the critical micelle concentration [29,30].…”

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

“…An ELISA assay was used to compare the binding of MAG to the Leg form of GD1a and to the gangliosides GM1a, GD1a, GT1b and GD1b [16]. Sets of four wells were coated with 5, 25, 50 or 100 pmol of each glycolipid, a chimeric protein containing the first three Ig-like domains of 6 MAG conjugated to human Fc (MAG 3 Fc) was added and after incubation then washing, the amount bound was determined with an anti-human Fc antibody conjugated to alkaline phosphatase.…”