Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B

João, Heidi C.; Scragg, Ian G.; Dwek, Raymond A.

doi:10.1016/0014-5793(92)80709-p

Cited by 96 publications

(65 citation statements)

References 12 publications

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“…Asparagine-linked protein glycosylation may serve many diverse roles. Some proteins require N-linked oligosaccharides to maintain proper function (7,29) or to be correctly targeted (1). In other cases, the presence or absence of the oligosaccharide seems to make no difference to the function of the native protein (30).…”

Section: Resultsmentioning

confidence: 99%

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Conformational implications of asparagine-linked glycosylation.

Imperiali

Rickert

1995

Proc. Natl. Acad. Sci. U.S.A.

157

103

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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...

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Section: Resultsmentioning

confidence: 99%

“…Previous studies on the conformational effects of protein glycosylation have principally relied upon NMR (7)(8)(9) and CD spectroscopy (10,11). However, these methods present some limitations when considering the conformational dynamics of flexible peptides.…”

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confidence: 99%

Conformational implications of asparagine-linked glycosylation.

Imperiali

Rickert

1995

Proc. Natl. Acad. Sci. U.S.A.

157

103

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“…Bovine RNases A and B share identical amino acid compositions (15) and sequences, including a single Asn 67 -Gly 68 residue pair that was shown to undergo selective deamidation in RNase A (19). The protein moieties of RNases A and B have essentially identical structures as revealed by crystallography (20) and by NMR, both for shorter (21) and longer (22) glycoforms. The global conformational stability of RNase B, as measured by thermal denaturation, is slightly higher than that of RNase A (23).…”

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confidence: 98%

Glycosylation and Specific Deamidation of Ribonuclease B Affect the Formation of Three-dimensional Domain-swapped Oligomers

Gotte

Libonati

Laurents

2003

Journal of Biological Chemistry

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RNase A oligomerizes via the three-dimensional domain-swapping mechanism to form a variety of oligomers, including two dimers. One, called the N-dimer, forms by swapping of the N termini of the protein; the other, called the C-dimer, forms by swapping of the C termini. RNase B is identical in protein sequence and conformation to RNase A, but its Asn 34 bears an oligosaccharide chain that might affect oligomerization. The ability of RNase B to oligomerize under two sets of conditions has been examined. The amount of oligomers formed via lyophilization was somewhat lower for RNase B than RNase A, and RNase B oligomerized more rapidly in 40% ethanol solution at high temperature than RNase A. The ratio of the N-dimer to C-dimer formed increased with the size of the carbohydrate chain under both sets of conditions. These results suggest that the oligosaccharide chain either favors productive collisions or stabilizes the oligomers, especially the N-dimer. Endoglycosidase H treatment of RNase B partially restored RNase A-like oligomerization. Derivatives of RNase A conjugated at the amine groups to polyethylene glycol chains showed a greatly reduced capacity for oligomerization, suggesting that oligomerization can be impeded sterically. Commercial preparations of RNase B eluted as two main peaks by cation exchange chromatography. Using chromatography, mass spectroscopy, and two-dimensional NMR, the major peak was identified as RNase B selectively deamidated at Asn 67 . This deamidated protein showed a >4°C drop in thermal stability, disruption of the native structure of residues 67-69, and a decreased ability to oligomerize compared with unmodified RNase B.In their classic work, Crestfield et al.(1) discovered and characterized the ability of bovine ribonuclease A to dimerize by exchanging segments of secondary structure when lyophilized from 50% acetic acid. Since then, many proteins have been found to form oligomers by the same mechanism, known as three-dimensional domain swapping (Ref. 2; see Ref. 3 for an excellent recent review). This process of oligomerization is being studied as a mechanism for the formation of oligomeric proteins by evolution. Moreover, it has been proposed that three-dimensional domain swapping might lead to inappropriate formation of large aggregates of cross-␤-structure, known as amyloid, which have been linked to Ͼ23 diseases, including Alzheimer's, Parkinson's, and prion-induced diseases (4). This hypothesis is supported by recent findings that the amyloidogenic protein cystatin C (5) and the human prion protein (6) dimerize via three-dimensional domain swapping between monomers.Modern studies on the oligomerization of bovine RNase A show that this normally monomeric protein can form a variety of dimers, trimers, and larger oligomers (7) via three-dimensional domain swapping of the N-terminal ␣-helix, the C-terminal ␤-strand, or both (8 -11). The dimers, trimers, and tetramers each form at least two conformational isomers, which can be separated by cation exchange chromatography as a less...

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“…Asparagine-linked protein glycosylation may serve many diverse roles. Some proteins require N-linked oligosaccharides to maintain proper function (Joao et al, 1992;Rudd et al, 1994) or to be correctly targeted (Pfeffer and Rothman, 1987). N-linked glycosylation occurs cotranslationally (Bergman and Kuehl, 1978;Kiely and Schimke, 1976) and has the potential to affect the course of protein folding.…”

Section: Discussionmentioning

confidence: 99%

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

Gambino

Ruiu

Pagano

et al. 1997

Journal of Lipid Mediators and Cell Signalling

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We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the Abbre6iations: apo H, apolipoprotein H; SDS -PAGE, sodium dodecyl sulfate polyacrylamide gel

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Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B

Cited by 96 publications

References 12 publications

Conformational implications of asparagine-linked glycosylation.

Conformational implications of asparagine-linked glycosylation.

Glycosylation and Specific Deamidation of Ribonuclease B Affect the Formation of Three-dimensional Domain-swapped Oligomers

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

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