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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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Pyrrolated proteins as a molecular target of autoimmunity.(a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left, immunoblot analysis of the modified proteins using the sera from MRL-lpr mice. Right, immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL-lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL-lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left, IgG response. Right, IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL-lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA (left), pyrrolated BSA (middle), and dsDNA (right) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL-lpr mice are indicated by asterisks (*, P < 0.05; ***, P < 0.005). (d) Elevation of immune response to dsDNA (left panel) and pyrrolated proteins (right panel) in autoimmune diseases. The plasma samples were prepared from 5 healthy individuals, 20 patients with IgA nephropathy (IgA-N), and 26 patients with SLE. The levels of the IgG Abs against the dsDNA and pyrrolated proteins in the plasma samples were measured by ELISA using calf-thymus dsDNA and pyrrolated BSA, respectively, as the coating antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between control and SLE and between SLE and IgA-N are indicated by asterisks (**, P < 0.01; ***, P < 0.005).
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f7: Pyrrolated proteins as a molecular target of autoimmunity.(a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left, immunoblot analysis of the modified proteins using the sera from MRL-lpr mice. Right, immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL-lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL-lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left, IgG response. Right, IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL-lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA (left), pyrrolated BSA (middle), and dsDNA (right) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL-lpr mice are indicated by asterisks (*, P < 0.05; ***, P < 0.005). (d) Elevation of immune response to dsDNA (left panel) and pyrrolated proteins (right panel) in autoimmune diseases. The plasma samples were prepared from 5 healthy individuals, 20 patients with IgA nephropathy (IgA-N), and 26 patients with SLE. The levels of the IgG Abs against the dsDNA and pyrrolated proteins in the plasma samples were measured by ELISA using calf-thymus dsDNA and pyrrolated BSA, respectively, as the coating antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between control and SLE and between SLE and IgA-N are indicated by asterisks (**, P < 0.01; ***, P < 0.005).

Mentions: The observations that the pyrrolated proteins showed a significant cross-reactivity with the SLE sera and the anti-DNA mAb (Fig. 7a) suggested that the production of specific antibodies against the pyrrolated proteins might be accelerated in autoimmune diseases, such as SLE. Hence, we evaluated the Ab titers to the pyrrolated proteins in the SLE-prone MRL-lpr mice. When the age-dependent change in the Ab titers was measured in the sera from the MRL-lpr mice and control MRL-MpJ mice, only the MRL-lpr mice displayed a spontaneous age-dependent elevation of the IgG and IgM responses to both DNA and the pyrrolated proteins (Fig. 7b). We established the IgM mAb PSL from the MRL-lpr mice, which showed specificities toward multiple antigens, including dsDNA and the pyrrolated proteins (Fig. S13). Of interest, the mAb PSL mainly cross-reacted with the modified proteins that showed binding potentials with SG.


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)

Pyrrolated proteins as a molecular target of autoimmunity.(a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left, immunoblot analysis of the modified proteins using the sera from MRL-lpr mice. Right, immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL-lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL-lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left, IgG response. Right, IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL-lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA (left), pyrrolated BSA (middle), and dsDNA (right) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL-lpr mice are indicated by asterisks (*, P < 0.05; ***, P < 0.005). (d) Elevation of immune response to dsDNA (left panel) and pyrrolated proteins (right panel) in autoimmune diseases. The plasma samples were prepared from 5 healthy individuals, 20 patients with IgA nephropathy (IgA-N), and 26 patients with SLE. The levels of the IgG Abs against the dsDNA and pyrrolated proteins in the plasma samples were measured by ELISA using calf-thymus dsDNA and pyrrolated BSA, respectively, as the coating antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between control and SLE and between SLE and IgA-N are indicated by asterisks (**, P < 0.01; ***, P < 0.005).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: Pyrrolated proteins as a molecular target of autoimmunity.(a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left, immunoblot analysis of the modified proteins using the sera from MRL-lpr mice. Right, immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL-lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL-lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left, IgG response. Right, IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle, anti-BSA titer; closed circle, anti-pyrrolated BSA titer; closed triangle, anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL-lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA (left), pyrrolated BSA (middle), and dsDNA (right) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL-lpr mice are indicated by asterisks (*, P < 0.05; ***, P < 0.005). (d) Elevation of immune response to dsDNA (left panel) and pyrrolated proteins (right panel) in autoimmune diseases. The plasma samples were prepared from 5 healthy individuals, 20 patients with IgA nephropathy (IgA-N), and 26 patients with SLE. The levels of the IgG Abs against the dsDNA and pyrrolated proteins in the plasma samples were measured by ELISA using calf-thymus dsDNA and pyrrolated BSA, respectively, as the coating antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between control and SLE and between SLE and IgA-N are indicated by asterisks (**, P < 0.01; ***, P < 0.005).
Mentions: The observations that the pyrrolated proteins showed a significant cross-reactivity with the SLE sera and the anti-DNA mAb (Fig. 7a) suggested that the production of specific antibodies against the pyrrolated proteins might be accelerated in autoimmune diseases, such as SLE. Hence, we evaluated the Ab titers to the pyrrolated proteins in the SLE-prone MRL-lpr mice. When the age-dependent change in the Ab titers was measured in the sera from the MRL-lpr mice and control MRL-MpJ mice, only the MRL-lpr mice displayed a spontaneous age-dependent elevation of the IgG and IgM responses to both DNA and the pyrrolated proteins (Fig. 7b). We established the IgM mAb PSL from the MRL-lpr mice, which showed specificities toward multiple antigens, including dsDNA and the pyrrolated proteins (Fig. S13). Of interest, the mAb PSL mainly cross-reacted with the modified proteins that showed binding potentials with SG.

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