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Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins.

Miyashita H, Chikazawa M, Otaki N, Hioki Y, Shimozu Y, Nakashima F, Shibata T, Hagihara Y, Maruyama S, Matsumi N, Uchida K - Sci Rep (2014)

Bottom Line: Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences.This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators.Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

ABSTRACT
Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences. We now report the identification and verification of an electrically-active form of modified proteins recognized by a group of small molecules commonly used to interact with DNA. This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators. Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct. Unexpectedly, the pyrrolation conferred an electronegativity and electronic properties on the proteins that potentially constitute an electrical mimic to the DNA. In addition, we found that the pyrrolated proteins indeed triggered an autoimmune response and that the production of specific antibodies against the pyrrolated proteins was accelerated in human systemic lupus erythematosus. These findings and the apparent high abundance of N(ε)-pyrrolelysine in vivo suggest that protein pyrrolation could be an endogenous source of DNA mimic proteins, providing a possible link connecting protein turnover and immune disorders.

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Related in: MedlinePlus

Pyrrolation transforms self-molecules into autoantigens.(a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.
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f6: Pyrrolation transforms self-molecules into autoantigens.(a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.

Mentions: To evaluate the biological significance of the protein pyrrolation as a source of DNA mimic proteins, we examined the autoantigenic property of the pyrrolated self-proteins. Balb/c mice were immunized every two weeks with the BDA-modified mouse serum albumin (MSA) emulsified with complete Freund's adjuvant, and the Ab responses to the DNA and pyrrolated BSA were examined. The Ab titers to both antigens were enhanced by the immunization with the pyrrolated MSA (Fig. 6a). We then isolated the hybridoma clones, producing mAbs specific for the DNA and/or pyrrolated proteins, from the mice and characterized their specificity in detail. Spleen cells from the balb/c mice immunized with the pyrrolated MSA were fused with P3/U1 murine myeloma cells and, after screening based on a specific binding to the pyrrolated proteins, we established one hybridoma clone, PSB, producing the mAb specific for the pyrrolated protein. The mAb PSB established from the mice immunized with the pyrrolated protein was found to be IgM. Of interest, the specificity study showed that the mAb cross-reacted with multiple antigens, including dsDNA and aldehyde-modified proteins (Fig. 6b).


Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins.

Miyashita H, Chikazawa M, Otaki N, Hioki Y, Shimozu Y, Nakashima F, Shibata T, Hagihara Y, Maruyama S, Matsumi N, Uchida K - Sci Rep (2014)

Pyrrolation transforms self-molecules into autoantigens.(a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4061549&req=5

f6: Pyrrolation transforms self-molecules into autoantigens.(a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.
Mentions: To evaluate the biological significance of the protein pyrrolation as a source of DNA mimic proteins, we examined the autoantigenic property of the pyrrolated self-proteins. Balb/c mice were immunized every two weeks with the BDA-modified mouse serum albumin (MSA) emulsified with complete Freund's adjuvant, and the Ab responses to the DNA and pyrrolated BSA were examined. The Ab titers to both antigens were enhanced by the immunization with the pyrrolated MSA (Fig. 6a). We then isolated the hybridoma clones, producing mAbs specific for the DNA and/or pyrrolated proteins, from the mice and characterized their specificity in detail. Spleen cells from the balb/c mice immunized with the pyrrolated MSA were fused with P3/U1 murine myeloma cells and, after screening based on a specific binding to the pyrrolated proteins, we established one hybridoma clone, PSB, producing the mAb specific for the pyrrolated protein. The mAb PSB established from the mice immunized with the pyrrolated protein was found to be IgM. Of interest, the specificity study showed that the mAb cross-reacted with multiple antigens, including dsDNA and aldehyde-modified proteins (Fig. 6b).

Bottom Line: Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences.This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators.Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

ABSTRACT
Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences. We now report the identification and verification of an electrically-active form of modified proteins recognized by a group of small molecules commonly used to interact with DNA. This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators. Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct. Unexpectedly, the pyrrolation conferred an electronegativity and electronic properties on the proteins that potentially constitute an electrical mimic to the DNA. In addition, we found that the pyrrolated proteins indeed triggered an autoimmune response and that the production of specific antibodies against the pyrrolated proteins was accelerated in human systemic lupus erythematosus. These findings and the apparent high abundance of N(ε)-pyrrolelysine in vivo suggest that protein pyrrolation could be an endogenous source of DNA mimic proteins, providing a possible link connecting protein turnover and immune disorders.

Show MeSH
Related in: MedlinePlus