Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions

Nonaka, Yasuyuki; Aizawa, Tomoyasu; Akieda, Daisuke; Yasui, Masanori; Watanabe, Masahiro; Watanabe, Nobuhisa; Tanaka, Isao; Kamiya, Masakatsu; Mizuguchi, Mineyuki; Demura, Makoto; Kawano, Keiichi

doi:10.1002/prot.21927

Cited by 8 publications

(3 citation statements)

References 42 publications

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“…Equine b-casein and equine a-la undergo spontaneous deamidation under physiological conditions at Asn 135 -Gly 136 and Asn 45 -Gly 46 , respectively (Girardet et al, 2006(Girardet et al, , 2004 which has been reported also for canine milk Lyz (Nonaka et al, 2008) and human Lf (Belizy et al, 2001) but not, to our knowledge, for bovine or human b-casein. Recent research has demonstrated that temperature may be an important factor controlling the spontaneous deamidation process and at 10 C the phenomenon is strongly reduced (Matéos et al, 2009a(Matéos et al, , 2009b.…”

Section: B-caseinmentioning

confidence: 79%

Equine milk proteins: Chemistry, structure and nutritional significance

Uniacke‐Lowe

Huppertz

Fox

2010

International Dairy Journal

186

170

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Section: B-caseinmentioning

confidence: 79%

Equine milk proteins: Chemistry, structure and nutritional significance

Uniacke‐Lowe

Huppertz

Fox

2010

International Dairy Journal

186

170

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“…The protein unfolds, exposing regions that are normally protected, which then immediately aggregate to decrease surface exposure of these hydrophobic residues. Previous reports (48) have shown that secondary structure formation prevents Asn from deamidation due to restrained flexibility of local structure, which might provide an explanation for the low levels of deamidation observed only at solvent-exposed Asn residues in the 65C/pH 8.5 stressed sample. In contrast, many Asn residues are extensively deamidated in the 90C stressed sample because of the most extreme disruption of protein secondary and tertiary structures.…”

Section: Discussionmentioning

confidence: 96%

Chemical Modifications in Therapeutic Protein Aggregates Generated under Different Stress Conditions

Luo

Joubert

Stevenson

et al. 2011

Journal of Biological Chemistry

182

168

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In this study, we characterized the chemical modifications in the monoclonal antibody (IgG 2 ) aggregates generated under various conditions, including mechanical, chemical, and thermal stress treatment, to provide insight into the mechanism of protein aggregation and the types of aggregate produced by the different stresses. In a separate study, additional biophysical characterization was performed to arrange these aggregates into a classification system (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118 -25133). Here, we report that different aggregates possessed different types and levels of chemical modification. For chemically treated samples, metal-catalyzed oxidation using copper showed site-specific oxidation of Met 246 , His 304 , and His 427 in the Fc portion of the antibody, which might be attributed to a putative copper-binding site. For the hydrogen peroxidetreated sample, in contrast, four solvent-exposed Met residues in the Fc portion were completely oxidized. Met and/or Trp oxidation was observed in the mechanically stressed samples, which is in agreement with the proposed model of protein interaction at the air-liquid interface. Heat treatment resulted in significant deamidation but almost no oxidation, which is consistent with thermally induced aggregates being generated by a different pathway, primarily by perturbing conformational stability. These results demonstrate that chemical modifications are present in protein aggregates; furthermore, the type, locations, and severity of the modifications depend on the specific conditions that generated the aggregates.

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“…These structural changes may lead to exposure of new residues, making additional sites susceptible to deamidation, as has been shown for ribonuclease A in vitro (Zabrouskov et al 2006). However, since deamidation can occur even under mild conditions in vitro (Nonaka et al 2008), the use of high pH and high temperature may artifactually introduce deamidation. The purpose of the experiments in this study was to determine which physiological deamidation sites have the greatest affect on structure and stability as a first step in determining the overall relevance of deamidation during aging and cataracts.…”

mentioning

confidence: 99%

Deamidation destabilizes and triggers aggregation of a lens protein, βA3‐crystallin

et al. 2008

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Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the bA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of bA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of bA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the bB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type bA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other b-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.Keywords: lens; bA3-crystallin; deamidation; protein aggregation; cataract; sedimentation velocity Cataract is the most common cause of preventable blindness in the world (Resnikoff et al. 2004). Age-related cataract is a protein aggregation disease associated with the insolubization of modified proteins in the lens (Harrington et al. 2004). The lens contains a high concentration of proteins that are mostly structural proteins called crystallins. To enable transparency, crystallins form hetero-oligomers that assemble into ordered structures (Delaye and Tardieu 1983). Modifications that disrupt this order most likely contribute to aggregation and precipitation.The proteins from older lenses contain many modifications including truncation, methylation, oxidation, disulfide bond formation, glycation, racemization, and deamidation (Groenen et

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Spontaneous asparaginyl deamidation of canine milk lysozyme under mild conditions

Cited by 8 publications

References 42 publications

Equine milk proteins: Chemistry, structure and nutritional significance

Equine milk proteins: Chemistry, structure and nutritional significance

Chemical Modifications in Therapeutic Protein Aggregates Generated under Different Stress Conditions

Deamidation destabilizes and triggers aggregation of a lens protein, βA3‐crystallin

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