Deamidation Levels Research Articles

Post-Translational Modifications in the Nuclear Region of Young, Aged, and Cataract Human Lenses

The urea-soluble proteins from the nucleus of two young, two aged, and two early-stage nuclear cataract lenses were subjected to tryptic digestion and analysis by 2D LC-MS/MS. Several novel post-translational modifications were identified. Deamidation was, by far, the most common modification. A number of differences were found in cataract compared to normal lenses, most notably an increase in the number of oxidized tryptophan residues. Semiquantitative analysis revealed that there appeared to be a trend toward increased levels of deamidation with age; however, there was no apparent increase upon the onset of nuclear cataract. This is in contrast to Trp oxidation, where an increase in the extent of modification was apparent in cataract lenses when compared to aged normal lenses. These findings suggest Trp oxidation may be involved in nuclear cataract development.

Improvement of Digestibility, Reduction in Allergenicity, and Induction of Oral Tolerance of Wheat Gliadin by Deamidation

Wheat gliadin was deamidated by using a cation-exchange resin in the presence or absence of added cysteine, with the change in digestibility being measured. The allergenicity of the gliadin was evaluated by using sera from patients RAST-positive to wheat. Gliadin-specific IgE was measured after the gliadin had been orally administered to rats. The addition of cysteine before the treatment with a cation exchanger effectively increased the deamidation level of gliadin. Deamidated gliadin showed higher solubility than the undeamidated form. There was no difference in the peptic digestibility of the gliadin, whereas deamidation enhanced the pancreatic digestibility in vitro and the digestibility in the mouse stomach in vivo. Deamidation of gliadin reduced its reactivity toward the sera of patients with wheat allergy. Rats administered with deamidated gliadin showed suppressed elevation of the gliadin-specific IgE level.

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

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 (t(1/2) approximately 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: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: implications for the study of amyloid formation.

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.

Preparation of phytate-removed deamidated soybean globulins by ion exchangers and characterization of their calcium-binding ability.

Phytate-removed deamidated soybean globulins were prepared using ion-exchange resins to provide them with a functional property to enhance calcium absorption in the body. The phosphorus level was reduced from 45 to 20 micromol/g using 0.05 g/mL of AE-4, an anion-exchange resin with a (2-hydroxyethyl)dimethylammonium group, in 0.2% soybean globulin solution for 1 h at 4 degrees C, and 90-92% of the phosphorus in defatted soybeans could be removed. As for deamidation, CE-4, a cation-exchange resin of the carboxylate type, showed a much higher deamidation activity than CE-1 and CE-2, cation-exchange resins of the sulfonate type. No peptide bond hydrolysis was observed for any cation-exchange resin treated at 4 degrees C. There was no significant difference in the amount of acid amide deamidated at temperatures between 4 and 50 degrees C. The deamidation level was able to increase to 73% using 0.10 g/mL of CE-4 in a 0.2% soybean globulin solution for 6 h at 4 degrees C. The amount of calcium bound to the soybean globulins decreased with removal of the phytate but increased with deamidation.

Effects of Dissociation, Deamidation, and Reducing Treatment on Structural and Surface Active Properties of Soy Glycinin

Different soluble protein derivatives were prepared from soy glycinin by means of progressive dissociation, deamidation, and later reducing thermal treatment. Dissociation was achieved at 4 °C in low ionic strength or mild acid conditions (0.1 M HCl). For deamidation, mild acid treatment of glycinin was performed at 70 °C for 1.5-9 h. Deamidation levels (1.7-25.0%) increased linearly with time of heating. These results were compared to those obtained by alkaline deamidation. The reducing thermal treatment led to both soluble and insoluble polypeptides. Greater soluble protein yields were obtained when this treatment was applied to previously deamidated glycinins. The structural properties and surface activity of native and modified glycinin were studied. Dissociation, deamidation, and reduction resulted in structural changes on glycinin (increased surface hydrophobicity, decreased molecular size, and isoelectric pH) which enhanced adsorption of proteins at the air-water interface. In contrast to native glycinin, the influence of ionic strength on surface behaviors is rather limited when the protein is both deamidated and reduced.

GLIADIN MODIFICATIONS CATALYZED BY GUINEA PIG LIVER TRANSGLUTAMINASE

Wheat gliadins, that are glutamine rich and lysine poor proteins, are good substrates for transglutaminases reactions. This study was conducted to determine the efficiency with which guinea pig liver transglutaminase catalyzes transfer and hydrolysis reactions of native and acylated gliadins. In all reactions, 35% of the total glutaminyl residues were modified. Neutral pH simultaneously enhanced glutaminyl residue hydrolysis and protein cross-linking, while acidic pH reduced the cross-linking reaction. Functional properties of two enzymatically modified gliadins and a chemically deamidated one were tested at neutral pH. A deamidation level of 25-27% appeared to be an optimum for the emulsification properties. Enzymatically modified gliadin showed better resistance to coalescence than the chemically deamidated one; a result that probably is related to the presence of high molecular weight polymers.

Effect of temperature, feed moisture, and pH on protein deamidation in an extruded wheat flour

The effect of three parameters on deamidation levels during twin-screw extrusion of wheat flour was studied: temperature, feed moisture content, and pH. This was accomplished by generating three sets of extrudates. The first set varied temperature (130, 155, and 170 o C), the second set varied feed moisture content (20%, 25%, and 30%), and the third set varied pH of the liquid feed (3.0, 7.0, and 12.0). In each set all other parameters were held constant. It was found that increases in temperature and feed moisture enhanced deamidation and that deamidation was also favored at extremes of pH