Glycan-Protein Interactions Determine Kinetics ofN-Glycan Remodeling

Abstract

A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure context of N-glycan substrates affected N-glycan remodeling in a site-specific way. We propose that differential, tertiary structure context dependent N-glycan reactivities lead to different glycan structures in the same protein through kinetically controlled processing pathways.

Glycoengineering Chinese hamster ovary cells: a short history

“…While these point mutation strategies are highly specific to individual molecules and to distinctive protein–carbohydrate environments, modifications to the protein–carbohydrate interface were also found to be relevant to glycan processing in protein disulfide isomerase, confirming the efficacy and wider applicability of modifying the primary sequence for glycoengineering [ 113 ]. Recently, the same group used molecular dynamics simulations to predict glycan accessibility for processing enzymes and confirmed in vitro how N-glycan accessibility and tertiary structure modifications affect processing enzyme kinetics at different glycosylation sites in protein disulfide isomerase [ 114 ]. The reader is encouraged to read these last two studies to gain a deeper understanding of how protein backbone engineering may be used to direct glycosylation pathways in a broader context.…”