posted on 2020-02-11, 22:29authored byLing Han, Pavel I. Kitov, Jianing Li, Elena N. Kitova, John S. Klassen
Interactions between
glycosphingolipids (GSLs) on the surfaces
of cells and glycan-binding proteins (GBPs) mediate a wide variety
of essential and pathological processes. Despite the biological importance
of these interactions, the GSL ligands of most GBPs remain to be identified
and the mechanisms controlling recognition of GSLs are incompletely
understood. Recently, it was suggested that, when present together
with high affinity ligands, low affinity GSL ligands can contribute
significantly to the binding of GBPs with multiple binding sites through
a process called heteromultivalent binding. Here, with goal of directly
establishing the existence of heteromultivalent GSL interactions and
elucidating the mechanism underlying their formation, we investigated
cholera toxin B subunit homopentamer (CTB5) binding to
ganglioside mixtures in model membranes (nanodiscs) using native mass
spectrometry (MS) and competitive ligand binding. Electrospray ionization
(ESI)-MS analysis revealed that the presence of the high affinity
ligand GM1 (at substoichiometric amounts relative to binding sites)
in the nanodisc promotes GD1b binding to CTB5; no GD1b
binding was detected in the absence of GM1. No direct ESI-MS evidence
of CTB5 binding to the other five gangliosides tested,
alone or present together with GM1 in the nanodiscs, was observed.
Affinity measurements, carried out using the proxy ligand ESI-MS binding
assay, confirmed that GD1b binding to CTB5 is dramatically
enhanced (>1000-times higher affinity compared to the GD1b oligosaccharide
affinity) when present with GM1. NDs containing GM1 and GM2, GD1a,
or GT1b also exhibited enhanced CTB5 binding, however,
the effect was smaller. The results of molecular dynamics simulations
performed on ganglioside-containing nanodiscs suggest that the participation
of low affinity ligands in heteromultivalent binding with GM1 may
be regulated by the positions of the internal Gal-linked Neu5Ac residues
of the gangliosides relative to the membrane surface.