Conformational influences of glycosylation of a peptide: A possible model for the effect of glycosylation on the rate of protein folding

Abstract

Improved strategies for synthesis make it possible to expand the range of glycopeptides available for detailed conformational studies. The glycopeptide 1 was synthesized using a new solid phase synthesis of carbohydrates and a convergent coupling to peptide followed by deprotection. Its conformational properties were subjected to NMR analysis and compared with a control peptide 2 prepared by conventional solid phase methods. Whereas peptide 2 fails to manifest any appreciable secondary structure, the glycopeptide 1 does show considerable conformational bias suggestive of an equilibrium between an ordered and a random state. The implications of this ordering effect for the larger issue of protein folding are considered.Although glycosylation of many natural products is a widespread phenomenon, the consequences of this modification on the molecular properties of proteins are not well understood. An intriguing possibility is that glycosylation of the protein effects the conformation of the nascent chain and influences its rate of folding (1-3). In spite of this and many other outstanding issues pertaining to glycosylated species, studies have been relatively few due to the limited availability of suitable material and model compounds. Some relief is now being realized with improvements in methodology for synthesis of oligosaccharides and glycopeptides (4-11). For instance, access to the glycopeptide 1 studied here (vide infra) is a consequence of these advances (12). Its pentapeptide segment was designed to incorporate the Asn-Xxx-Thr consensus sequence for N-glycosylation, and a trisaccharide was linked through the side chain amide of the asparagine residue to the peptide.A detailed picture of the organization of a pendant oligosaccharide on a fully folded protein in solution has been obtained from the recent study of the glycosylated CD2 protein (13). However, in trying to determine the extent to which glycosylation influences early stages of the protein folding process, glycopeptides provide more appropriate models since the conformation of the system is not dominated by the overall fold of the ultimately organized protein structure. Some recent studies on glycopeptides have provided insights into potential conformational consequences of glycosylation for both .In the case of O-linked glycopeptides (14,15,20), NMR studies have shown that the peptide backbone responds to glycosylation, as evidenced by changes in sequential amideamide nuclear Overhauser effect (NOE) interactions. The response is further modulated by whether the sugar component is a mono-or disaccharide. Direct evidence for local interaction between sugar and peptide backbone has been indicated by the existence of an NOE crosspeak between the amide proton of the N-acetylglucosamine attached to the O-linked residue and the backbone amide of the threonine to which the sugar was attached (14). In the case of N-linked species in aqueous solution, fluorescence energy transfer and NMR studies support the notion of a change in the distribution of ...

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

“…This observation substantiates the general importance of this residue to structural integrity, even though it is not always necessary to preserve a folded structure, as it is for hCD2ad (17,19). Furthermore, the proximal N-linked GlcNAc of model synthetic N-linked glycopeptides is the most influential for rigidifying and biasing conformational space, including promoting ␤-turn and disulfide bond formation (5,8,(28)(29)(30), consistent with GlcNAc a playing a general kinetic role in glycoprotein folding.…”

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

“…However, in our model, we found that exogenous heparin had no effect on shedding, suggesting that it is unlikely that syndecans are binding to MMPs and directly inhibiting their activity. Another possible mechanism could be related to a change in protein folding and stabilization, which can be altered by changes in glycosylation (30,40). Removal of heparan sulfate could alter syndecan-1 core protein conformation and expose cryptic sites that are susceptible to protease cleavage.…”

The interplay of glycosylation and disulfide formation influences fibrillization in a prion protein fragment

“…The evidence showing that a reducing environment can promote the generation of PrP Sc -like species (6,(16)(17)(18) suggests that factors affecting the stability of the disulfide bridge could also have an effect in preventing or promoting the PrP C to PrP Sc transition. Previous glycopeptide studies reveal that N-linked glycosylation can impact the conformation of protein fragments (28)(29)(30). In particular, we have shown that glycosylation can alter the thermodynamics of disulfide bond formation, favoring the oxidized form (31).…”