Mechanism of Asparagine Deamidation During Human Senile Cataractogenesis

Takemoto, L.; Fujii, Nobutaka; Boyle, D

doi:10.1006/exer.2001.0983

Cited by 51 publications

(72 citation statements)

References 11 publications

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“…Reported studies investigated the properties of deamidated properties or considered N to D substitution as a missense mutation. 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: 97%

“…5,6 Deamidation results in an addition of negative charge to the residue resulting in alterations in protein interactions, which could affect the activity or refolding. 7,8 Interest in deamidation has gained importance since the extent of deamidation was associated with aging, 9,10 cataract formation, 9,11,12 immune recognition, 13 shelf-life of protein pharmaceuticals such as antibodies, 9,13,14 and so on. Deamidation of asparagines is initiated by the cyclization reaction, which occurs between the carboxyl group of the asparagine and amide nitrogen of carboxyl side amino acid (n11) followed by deamination resulting in the formation of succinimide intermediate.…”

Section: Introductionmentioning

confidence: 99%

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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: 97%

Section: Introductionmentioning

confidence: 99%

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

2014

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“…Within chaperones, deamidation of crystallin, a member of the Hsp27 family, occurs during aging and increases during cataract formation. 25,26 Interestingly, crystallin deamidation is in part catalyzed by tissue transglutaminase, an enzyme known to be involved in mouse MDB formation. 24,27 Notably, protein deamidation may lead to protein aggregation as noted for the spontaneous deamidation of the amylin peptide that induces the formation of amyloidlike aggregates.…”

Section: Discussionmentioning

confidence: 99%

“Toxic memory” via chaperone modification is a potential mechanism for rapid mallory-denk body reinduction

Strnad¹,

Guo²,

So³

et al. 2008

Hepatology

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The cytoplasmic hepatocyte inclusions, Mallory-Denk bodies (MDBs), are characteristic of several liver disorders, including alcoholic and nonalcoholic steatohepatitis. In mice, MDBs can be induced by long-term feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 3 to 4 months or rapidly reformed in DDC-induced then recovered mice by DDC refeeding or exposure to a wide range of toxins for only 5 to 7 days. The molecular basis for such a rapid reinduction of MDBs is unknown. We hypothesized that protein changes retained after DDC priming contribute to the rapid MDB reappearance and associate with MDB formation in general terms. Two-dimensional differential-in-gel-electrophoresis coupled with mass spectrometry were used to characterize protein changes in livers from the various treatment groups. The alterations were assessed by real-time reverse-transcription polymerase chain reaction and confirmed by immunoblotting. DDC treatment led to pronounced charged isoform changes in several chaperone families, including Hsp25, 60, 70, GRP58, GRP75, and GRP78, which lasted at least for 1 month after discontinuation of DDC feeding, whereas changes in other proteins normalized during recovery. DDC feeding also resulted in altered expression of Hsp72, GRP75, and Hsp25 and in functional impairment of Hsp60 and Hsp70 as determined using a protein complex formation and release assay. The priming toward rapid MDB reinduction lasts for at least 3 months after DDC discontinuation, but becomes weaker after prolonged recovery. MDB reinduction parallels the rapid increase in p62 and Hsp25 levels as well as keratin 8 cross-linking that is normally associated with MDB formation. Conclusion: Persistent posttranslational modifications in chaperone proteins, coupled with protein cross-linking and altered chaperone expression and function likely contribute to the "toxic memory" of DDC-primed mice. We hypothesize that similar changes are important contributors to inclusion body formation in several diseases. (HEPATOLOGY 2008;48:931-942.)

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“…This modification is so ubiquitous in longerlived tissues that it is considered one of the factors limiting protein lifetimes (Robinson and Robinson 2004). Deamidation is one of the most common post-translational modification in the eye lens, occurring early in life and building up with age (Hains and Truscott 2010;Lampi et al 2014;Takemoto et al 2001). There are some sites of deamidation that occur more frequently in cataract-affected lenses compared to healthy lenses of the same age (Hooi et al 2012).…”

Section: Deamidationmentioning

confidence: 99%

“…Figure 9 shows the deamidation pathway for asparagine. Both asparagine and aspartic acid are capable of ring closing to form a succinimide intermediate, with the intermediate ring opening in both directions to subsequently produce the aspartate and isoaspartate residues (Lampi et al 2014;Takemoto et al 2001). The nature of the residue adjacent to the Asn/Gln is an important factor in both the rate of modification and propensity for deamidation.…”

Section: Deamidationmentioning

confidence: 99%

Biophysical chemistry of the ageing eye lens

Ray

2015

Biophys Rev

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This review examines both recent and historical literature related to the biophysical chemistry of the proteins in the ageing eye, with a particular focus on cataract development. The lens is a vital component of the eye, acting as an optical focusing device to form clear images on the retina. The lens maintains the necessary high transparency and refractive index by expressing crystallin proteins in high concentration and eliminating all large cellular structures that may cause light scattering. This has the consequence of eliminating lens fibre cell metabolism and results in mature lens fibre cells having no mechanism for protein expression and a complete absence of protein recycling or turnover. As a result, the crystallins are some of the oldest proteins in the human body. Lack of protein repair or recycling means the lens tends to accumulate damage with age in the form of protein posttranslational modifications. The crystallins can be subject to a wide range of age-related changes, including isomerisation, deamidation and racemisation. Many of these modification are highly correlated with cataract formation and represent a biochemical mechanism for age-related blindness.

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Mechanism of Asparagine Deamidation During Human Senile Cataractogenesis

Cited by 51 publications

References 11 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

“Toxic memory” via chaperone modification is a potential mechanism for rapid mallory-denk body reinduction

Biophysical chemistry of the ageing eye lens

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