Conformational implications of asparagine-linked glycosylation.

Abstract

The effects of cotranslational protein modification on the process of protein folding are poorly understood. Time-resolved fluorescence energy transfer has been used to assess the impact of glycosylation on the conformational dynamics of flexible oligopeptides. The peptide sequences examined are selected from glycoproteins of known three-dimensional structure. The energy transfer modulation associated with N-linked glycosylation is consistent with the glycopeptides sampling different conformational profiles in water. Results show that glycosylation causes the modified peptides to adopt a different ensemble of conformations, and for some peptides this change may lead to conformations that are more compact and better approximate the conformation of these peptides in the final folded protein. This result further implies that cotranslational glycosylation can trigger the timely formation of structural nucleation elements and thus assist in the complex process of protein folding.The synthesis of many proteins takes place on membraneassociated ribosomes and follows the secretory pathway (1). These proteins are subject to a wide array of enzyme-catalyzed covalent chemical modifications. Early protein modification reactions, such as glycosylation, carboxylation, and hydroxylation, occur on protein substrates that are either only locally folded or in the form of "collapsed intermediates" (2). Because these protein processing events occur cotranslationally, questions arise as to the impact of each modification on the local peptide conformation and on the progress and efficiency of subsequent protein-folding events. Numerous observations attest to the importance of asparagine glycosylation for the appropriate folding and assembly of intact proteins. For example, the biosynthesis of proteins in the presence of N-linked glycosylation inhibitors, such as tunicamycin, often results in extensive aggregation associated with incomplete or incorrect folding (3-6).Previous studies on the conformational effects of protein glycosylation have principally relied upon NMR (7-9) and CD spectroscopy (10, 11). However, these methods present some limitations when considering the conformational dynamics of flexible peptides. Specifically, the time frame of NMR measurements is such that conformational averaging could obscure specific effects of glycosylation on a polypeptide framework. In addition, the molecular weights of the peptides and glycopeptides under evaluation are such that rotational correlation times would greatly affect the nuclear Overhauser effect (NOE) measurements, making the comparative studies difficult (12). Although CD spectroscopy operates on a faster time scale, the technique is mostly applicable to peptides with strong spectroscopic signals, such as a-helical motifs (10). In contrast, fluorescence energy transfer (FET) studies allow the assessment of specific conformational features, through the measurement of a single interprobe distance, on the same rapid time scale over which conformational fluctuations occu...

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a Broadly Neutralizing Human Antibody

“…Indeed, N417S/S419N escape variants could still be neutralized by HC33.1, albeit at higher antibody concentrations, and HC33.1 retained significant binding to recombinant E1E2 bearing these mutations (28). Apart from affecting interactions with HC33.1 directly, a glycan at Asn-419 may influence the structure or conformational dynamics of the E2 412-423 epitope, as noted in studies of peptide dynamics (60), or interact with other regions of E2. Another possibility is that the side chain change of Ser-419 to Asn has a direct effect on HC33.1 recognition; this would be supported by the hydrogen bond of Ser-419 with Asn-54 on the HC33.1 H chain, as well as some predicted loss of affinity upon in silico mutation (Table 2).…”

Identification of the N-Linked Glycosylation Sites on the High Density Lipoprotein (HDL) Receptor SR-BI and Assessment of Their Effects on HDL Binding and Selective Lipid Uptake

“…To date, the functional role of the extensive N-linked glycosylation of SR-BI has not been explored. Oligosaccharides in glycoproteins can serve a variety of functions, including facilitating protein folding, protecting against proteolysis, directly participating in intermolecular interactions, directing intracellular trafficking and secretion, and influencing cell surface expression and activity (27)(28)(29). In some cases it has not been possible to attribute a specific function to a given N-linked glycan.…”

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

“…Glycosylation event could serve to funnel the nascent polypeptide structure through a particular pathway for folding. In the absence of glycosylation, specific folded intermediates would be inaccessible, and the outcome would be a delinquent protein product (Imperiali and Rickert, 1995). Furthermore, the potential of N-linked oligosaccharides for structural variation is not confined to their chain-terminating sugars.…”

“…Blocking the N-linked glycosylation often results in a damaged protein lacking functional activity (Riederer and Hinnen, 1991;Copeland et al, 1988). In particular asparagine glycosylation is very important for the appropriate folding and assembly of intact proteins (Imperiali and Rickert, 1995).…”