N-glycosylation is an essential process in eukaryotes and entails the post-translational modification of secretory proteins in the lumen of the endoplasmic reticulum (ER). A cascade of membrane-integral glycosyltransferases (ALG proteins) facilitate the biosynthesis of a high-mannose lipid-linked oligosaccharide (LLO), which is eventually transferred to target proteins by oligosaccharyltransferase. In eukaryotic cells, the initial assembly to a Man5- containing LLO is carried out in the cytoplasm, whereas the subsequent saccharides are added in the lumen of the ER. The involved enzymes have been identified and named ALG proteins, all of which are integral membrane proteins. Through in-vivo studies, the specific reactions were assigned to the individual ALG enzymes. However, their structure and reaction mechanism have remained elusive.
We have recombinantly expressed and purified several ALG proteins that catalyze the later, ER-internal, biosynthetic steps of LLO synthesis. We reconstituted them into lipidic nanodiscs and generated conformational antibody fragments (Fab’s) against them. Some of the selected Fab fragments thermostabilized their cognate ALG proteins. The resulting complex was of sufficient size to make it amenable for single particle cryo-EM studies. We have obtained a first structure of an ALG-Fab complex in nanodiscs at near atomic resolution. This provides unprecedented insight into the fold and potential reaction mechanism of this family of GT-C enzymes.