Macromolecular interactions in vitro, comparing classical and novel approaches.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

Human Endogenous Retrovirus Type W Envelope from Multiple Sclerosis Demyelinating Lesions Shows Unique Solubility and Antigenic Characteristics.

In multiple sclerosis (MS), human endogenous retrovirus W family (HERV-W) envelope protein, pHERV-W ENV, limits remyelination and induces microglia-mediated neurodegeneration. To better understand its role, we examined the soluble pHERV-W antigen from MS brain lesions detected by specific antibodies. Physico-chemical and antigenic characteristics confirmed differences between pHERV-W ENV and syncytin-1. pHERV-W ENV monomers and trimers remained associated with membranes, while hexamers self-assembled from monomers into a soluble macrostructure involving sulfatides in MS brain. Extracellular hexamers are stabilized by internal hydrophobic bonds and external hydrophilic moieties. HERV-W studies in MS also suggest that this diffusible antigen may correspond to a previously described high-molecular-weight neurotoxic factor secreted by MS B-cells and thus represents a major agonist in MS pathogenesis. Adapted methods are now needed to identify encoding HERV provirus(es) in affected cells DNA. The properties and origin of MS brain pHERV-W ENV soluble antigen will allow a better understanding of the role of HERVs in MS pathogenesis. The present results anyhow pave the way to an accurate detection of the different forms of pHERV-W ENV antigen with appropriate conditions that remained unseen until now.

Chromatographic separation of Lewis a and Lewis x oligosaccharides as glycosynthons.

Lewis a and Lewis x oligosaccharides Gal beta 3(Fuc alpha 4)GlcNAc beta 3Gal beta 4Glc and Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4Glc are easily isolated as a mixture from biological fluids, including human milk. However, because they behave almost identically in most chromatographic systems, it is difficult to have each of them as a pure compound. Incidentally, we found that they were easily separated by HPLC as glycosynthons [Gal beta 3(Fuc alpha 4)GlcNAc beta 3Gal beta 4Glc-Glp-beta Ala-OBzl and Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4Glc-Glp-beta Ala-OBzl] after substitution of the terminal reducing sugar by a short peptide (pyroglutamyl-beta alanyl-O-benzyl ester) in a one-pot two-step reaction (Carbohydr. Lett. 1 (1995) 269; Bioconjug. Chem. 9 (1998) 268). Such glycosynthons are easily either converted back to native Lewis a and Lewis x oligosaccharides upon hydrazinolysis or used to synthesize glycoconjugates, such as glycoclusters, glycopeptides, glycooligonucleotides, glycosylated polymers or glycosylated matrices for therapeutic or analytical purposes.