Multiple asparagine deamidation of <i>Bacillus anthracis</i> protective antigen causes charge isoforms whose complexity correlates with reduced biological activity

Powell, Bradford S.; Enama, Jeffrey T.; Ribot, Wilson J.; Webster, W. Maurice; Little, Stephen F.; Hoover, Timothy A.; Adamovicz, Jeffrey J.; Andrews, Gerard P.

doi:10.1002/prot.21432

Cited by 24 publications

(45 citation statements)

References 81 publications

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“…These studies have reported loss of stability, activity of proteins, and also increased aggregation properties as shown in crystallins, 7,8,11,12,23,24,[51][52][53] superoxide dismutase, 49 and immunoglobins. 14,34,54 Reported properties of mutC suggest that this protein has retained the secondary and tertiary structure with a partial loss in activity but with a significant improvement in the ability to withstand multiple heat cycles.…”

Section: Discussionmentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

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At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75 C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90 C. MutC has retained more than 50% activity even after incubation at 100 C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.

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Section: Discussionmentioning

confidence: 99%

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

2014

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“…While deamidation of Asn residues in rPA has been demonstrated, little is known about the effect that deamidation might have on the immunogenicity of the protein. Previously, Ribot et al (19) examined the immunogenicity and protective efficacy of isoforms of rPA that differed in deamidation levels (18,19). The two isoforms compared in that study exhibited comparable immunogenicity and protective immunity; however, the difference in the extent of deamidation between the two isoforms used in that study was minimal, as judged by the incremental difference in charge between the two isoforms (18,19).…”

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confidence: 97%

“…Previously, others have demonstrated that certain Asn residues within rPA deamidate on a time scale relevant to vaccine dating periods (17,18). In a comprehensive study, Powell et al (18) detected measurable deamidation of 7 of the 68 Asn residues of rPA that had been subjected to conditions conducive to deamidation.…”

mentioning

confidence: 99%

“…In a comprehensive study, Powell et al (18) detected measurable deamidation of 7 of the 68 Asn residues of rPA that had been subjected to conditions conducive to deamidation.…”

mentioning

confidence: 99%

“…Previously, Ribot et al (19) examined the immunogenicity and protective efficacy of isoforms of rPA that differed in deamidation levels (18,19). The two isoforms compared in that study exhibited comparable immunogenicity and protective immunity; however, the difference in the extent of deamidation between the two isoforms used in that study was minimal, as judged by the incremental difference in charge between the two isoforms (18,19). Thus, this difference in deamidation most likely did not adequately mimic the extent of deamidation that might be expected upon long-term vaccine storage.…”

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confidence: 99%

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Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

et al. 2013

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c Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)-the major component of new-generation anthrax vaccines-affects vaccine immunogenicity, we created a "genetically deamidated" form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

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Didehydroaspartate Modification in Methyl‐Coenzyme M Reductase Catalyzing Methane Formation

2016

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All methanogenic and methanotrophic archaea known to date contain methyl‐coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl‐coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post‐translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high‐resolution X‐ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine‐tuning the active site to increase the catalytic efficiency.

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Multiple asparagine deamidation of Bacillus anthracis protective antigen causes charge isoforms whose complexity correlates with reduced biological activity

Cited by 24 publications

References 81 publications

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase

Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

Didehydroaspartate Modification in Methyl‐Coenzyme M Reductase Catalyzing Methane Formation

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