Structural Insight into the Stabilizing Effect of O-Glycosylation

Abstract

Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far, the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide are valuable steps toward being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule's physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.

Just a spoonful of sugar: Short glycans affect protein properties and functions

“…Importantly, increased conformational rigidity, due to specific interactions between the O‐glycan and key amino acid residues in the protein sequence, seems to contribute significantly to the increased stability of certain glycoforms. This is supported by recent results from NMR studies on various homogeneous O‐glycoforms of CBM and by proteolytic stability assays and molecular dynamic simulation on a model of intrinsically disordered proteins . Taken together, these findings suggest a coevolution of the glycan protective motif with the adjacent amino acid composition.…”

A molecular switch orchestrates enzyme specificity and secretory granule morphology

“…Glycosylation of proteins is known to influence their structure 44 – 46 . In particular, the high levels of O-glycosylation typically seen in the serine and threonine-rich repetitive regions of mucins are known to confer extended, rod-like structures to these regions 44 , 45 , 47 . MUC2, a highly O-glycosylated intestinal mucin, has been shown to dimerize via a C-terminal cysteine knot domain, and then trimerize via N-terminal von Willebrand domains in a pH and Ca 2+ -dependent manner 17 , 48 , 49 .…”

The impact ofO-glycan chemistry on the stability of intrinsically disordered proteins

“…Small differences in protein backbone flexibility and concomitant large differences in resistance to proteolysis were also recently found for a structured protein with a single attached glycan, α-mannose or α-glucose. 24 Chaffey et al suggested that a chain of specific interactions between O -mannosyl and side chains of close residues may be propagating stiffening along the protein backbone. The similar behavior observed with IDPs suggests that the effects of α-mannose on protein stiffening may not be exclusive to a specific protein fold.…”