Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

Capasso, Sante; Salvadori, Severo

doi:10.1034/j.1399-3011.1999.00111.x

Cited by 55 publications

(53 citation statements)

References 23 publications

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“…Deamidation can be acid-or base-catalyzed (39,40), and the conversion of Peak Y to Peak X is found to be accelerated at a higher pH. The fact that the acceleration is only 3.2-fold and not 10-fold at pH 9.2 where [OH Ϫ ] is 10-fold higher relative to pH 8.2 is probably due the limited mobility of Asn 67 and Gly 68 within the folded structure (41,42).…”

Section: Discussionmentioning

confidence: 96%

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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Section: Discussionmentioning

confidence: 96%

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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“…Studies of b-turn model peptides 26,27 and some proteins 21,28,29 have demonstrated that b-turn secondary structures can stabilize Asn against deamidation, especially when Asn is located at the third position in a b-turn structure (except for type II turns). Such stabilization probably arises from structural constraints and reduced flexibility, which hinder formation of the favored conformation required for cyclic-imide formation.…”

Section: Crystal Structure Analysismentioning

confidence: 99%

Asparagine deamidation in recombinant human lymphotoxin: Hindrance by three-dimensional structures

Xie

Shahrokh²,

Kadkhodayan³

et al. 2003

Journal of Pharmaceutical Sciences

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“…For example, it has been shown recently that deamidation of glutamate dehydrogenase produced in Escherichia coli can be substantially reduced by temperature decreases during growth and during protein isolation (63). Additional studies have indicated the pH effect on the deamidation rate (77). Indeed, preliminary studies in our laboratory clearly indicate a decrease in the deamidation rate upon PA storage (as deduced from the reduction in the appearance of acidic isoforms) at pH 7.4 and at lower temperature concomitant with an improved biological stability of the protein.…”

Section: Pa Proteolytic Cleavage By Furin-asnmentioning

confidence: 99%

Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen

Zomber

Reuveny

Garti³

et al. 2005

Journal of Biological Chemistry

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Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t1 ⁄ 2 ≈ 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: The Gram-positive spore-forming bacterium Bacillus anthracis, the causative agent of anthrax, produces a bipartite A/B-type toxin. The B subunit is the 83-kDa protective antigen (PA) 3 receptor-binding moiety (named for its use as a vaccine), and the two catalytic A subunit moieties are edema factor (EF; 89 kDa) and lethal factor (LF; 90 kDa) (1). EF is a Ca 2ϩ -and calmodulin-dependent adenylate cyclase (2). LF is a Zn 2ϩ protease that cleaves and inactivates mitogen-activated protein kinase kinase-1 and -2 (3, 4). Following PA binding to cell receptors (5-8), it is cleaved by a furin family protease (9) to a 20-kDa N-terminal fragment with no known further function and to a 63-kDa fragment that forms ring-shaped heptamers on the cell surface (10). The heptamers then bind up to three molecules of EF or LF (11,12). Following binding, the heptamer-LF/EF complex undergoes endocytosis, and LF or EF is released to the cytosol (13-15). The crystal structure (16) and functional studies (11,12,(17)(18)(19)(20) have demonstrated that the PA polypeptide is folded into four distinct domains with well defined functions. Domain I (residues 1-258) prevents premature PA polymerization and harbors the furin cleavage site, which is located in an unstructured flexible loop; domain II (residues 259 -487) is involved in heptamerization and is in the membrane insertion loop; domain III (residues 488 -595) is also involved in heptamerization (18) PA has been reported previously to be a thermally unstable protein, losing its in vitro cytotoxic activity upon storage at 37°C within 48 h (32) or within few minutes above 40°C as a result of aggregation (33). Protein loss of function under mild storage conditions is generally attributed to a variety of nonenzymatic modifications such as deamidation, isomerization, oxidation, and alternative disulfide pairings (34, 35). Spontaneous deamidation, which occurs mainly at Asn side chains and at a much slower rate at Gln residues (36), is a major and well documented degradation pathway in proteins. Deamidation rates depend on pH, temperature, primary sequence ("nearest neighbor" effect), and protein conformation (37-40). Deamidation proceeds by nucleophilic attack on the side chain carbonyl carbon of Asn by the nitrogen of the adjacent peptide bond, resulting in the formation of an unstable five-member succinimide ring, which is hydrolyzed to produce m...

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Effect of the three‐dimensional structure on the deamidation reaction of ribonuclease A

Cited by 55 publications

References 23 publications

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

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

Asparagine deamidation in recombinant human lymphotoxin: Hindrance by three-dimensional structures

Effects of Spontaneous Deamidation on the Cytotoxic Activity of the Bacillus anthracis Protective Antigen

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