In Vitro Glycosylation of the Membrane Protein γ-Sarcoglycan in Nanodiscs

Abstract

Membrane glycoproteins are proteins that reside in the membranes of cells and are post-translationally modified to have sugars attached to their amino acid side chains. Studies of this subset of proteins in their native states are becoming more important since they have been linked to numerous human diseases. However, these proteins are difficult to study due to their hydrophobic nature and their propensity to aggregate. Using membrane mimetics allows us to solubilize these proteins, which, in turn, allows us to perform glycosylation in vitro to study the effects of the modification on protein structure, dynamics, and interactions. Here, the membrane glycoprotein γsarcoglycan was incorporated into nanodiscs composed of long-chain lipids and membrane scaffold proteins to perform N-linked glycosylation in which an enzyme attaches a sugar to the asparagine side chain within the glycosylation site. We previously performed glycosylation of membrane proteins in vitro when the protein had been solubilized using different detergents and short-chain lipids. This work demonstrates successful glycosylation of a full-length membrane protein in nanodiscs providing a more biologically relevant sample to study the effects of the modification.