title: Protein-glycolipid interactions studied in vitro using ESI-MS and nanodiscs: insights into the mechanisms and energetics of binding.
abstract: Electrospray ionization-mass spectrometry (ESI-MS) analysis combined with the use of nanodiscs (NDs) to solubilize glycolipids (GLs) has recently emerged as a promising analytical method for detecting protein-GL interactions in vitro and, when applied to libraries of GLs, ranking their affinities. However, there is uncertainty regarding the mechanism(s) of complex formation in solution and the extent to which the relative abundances of protein-glycolipid complexes observed by ESI-MS reflect the relative concentrations in solution. Here, we describe the results of a systematic ESI-MS study aimed at elucidating the processes that influence binding of water-soluble proteins to GLs incorporated into NDs and to exploit these insights to quantify the binding energetics. The interactions between the cholera toxin B subunit homopentamer (CTB5) and its native ganglioside receptor, β-D-Gal-(1 → 3)-β-D-GalNAc-(1 → 4)-[α-D-Neu5Ac-(2 → 3)]-β-D-Gal-(1 → 4)-β-D-Glc-ceramide (GM1), and between a recombinant fragment of family 51 carbohydrate-binding module (CBM), originating from S. pneumoniae, with a synthetic B type 2 neoglycolipid, α-D-Gal-(1 → 3)-[α-L-Fuc-(1 → 2)]-β-D-Gal-(1 → 4)-β-D-GlcNAc-1,2-di-O-dodecyl-sn-glycero (B2NGL) served as model protein-GL complexes for this study. The results of the ESI-MS measurements reveal that proteins bind reversibly to ND-bound GLs and that proteins possessing multiple ligand binding sites are able to interact with GLs originating from different NDs. Experimental evidence suggests that the diffusion of GLs between NDs is rapid and influences the nature of the protein-GL complexes that are detected. Using a newly developed ESI-MS assay, the proxy ligand method, the association constants for the CBM-B2NGL and CTB5-GM1 interactions were quantified and found to be slightly smaller than those for the corresponding oligosaccharides in solution.
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Ling
authors: L
authors: Han
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Elena N
authors: EN
authors: Kitova
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Jun
authors: J
authors: Li
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Sanaz
authors: S
authors: Nikjah
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Hong
authors: H
authors: Lin
authors: ‡Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6.
authors: Benjamin
authors: B
authors: Pluvinage
authors: ‡Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6.
authors: Alisdair B
authors: AB
authors: Boraston
authors: †Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: John S
authors: JS
authors: Klassen
mesh_terms: D001665:Binding Sites; D002772:Cholera Toxin / Q000737:chemistry*; D006017:Glycolipids / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014734:Vibrio cholerae / Q000737:chemistry
chemical_list: D006017:Glycolipids; D002772:Cholera Toxin