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Carbamylation of immunoglobulin abrogates activation of the classical complement pathway.

Koro C, Bielecka E, Dahl-Knudsen A, Enghild JJ, Scavenius C, Brun JG, Binder V, Hellvard A, Bergum B, Jonsson R, Potempa J, Blom AM, Mydel P - Eur. J. Immunol. (2014)

Bottom Line: We found that the lysine residues of IgG1 are rapidly modified after brief exposure to CNO- .The reaction can apparently occur in vivo, as we found carbamylated antibodies in synovial fluid from rheumatoid arthritis patients.Taken together, our data suggest that carbamylation has a profound impact on the complement-activating ability of IgG1 and reveals a pivotal role for previously uncharacterized lysine residues in this process.

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Affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway.

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Human IgG1 undergoes rapid carbamylation, which protects it from Kgp cleavage. (A) Western blot analysis of 2 μg of IgG1 carbamylated with KCNO in a time-dependent manner. The level of carbamylation was detected with homocitrulline-specific antibodies (upper panel) together with loading controls (Coomassie staining; lower panel). Data are representative of three independent experiments. (B) The time-dependent effect of KCNO incubation on carbamylation of IgG1. Carbamylated IgG1 was degraded with proteinase K and samples were incubated with urea nitrogen reagent and 3% w/v 2,3-butanedione monoxime. The amount of homocitrulline/mg IgG1 was quantified by measurement of the absorbance at 530 nm. Each point represents the mean of duplicate samples and data are from a single experiment representative of four independent experiments using IgG1 and RTX. (C) 2.25 μg of carbamylated and control IgG1 were incubated for the indicated times with 10 nM Kgp and SDS-PAGE was performed to assess the cleavage of IgG. All samples were separated on 4–15% gradient gels under reducing conditions. Data shown are representative of three independent experiments.
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fig01: Human IgG1 undergoes rapid carbamylation, which protects it from Kgp cleavage. (A) Western blot analysis of 2 μg of IgG1 carbamylated with KCNO in a time-dependent manner. The level of carbamylation was detected with homocitrulline-specific antibodies (upper panel) together with loading controls (Coomassie staining; lower panel). Data are representative of three independent experiments. (B) The time-dependent effect of KCNO incubation on carbamylation of IgG1. Carbamylated IgG1 was degraded with proteinase K and samples were incubated with urea nitrogen reagent and 3% w/v 2,3-butanedione monoxime. The amount of homocitrulline/mg IgG1 was quantified by measurement of the absorbance at 530 nm. Each point represents the mean of duplicate samples and data are from a single experiment representative of four independent experiments using IgG1 and RTX. (C) 2.25 μg of carbamylated and control IgG1 were incubated for the indicated times with 10 nM Kgp and SDS-PAGE was performed to assess the cleavage of IgG. All samples were separated on 4–15% gradient gels under reducing conditions. Data shown are representative of three independent experiments.

Mentions: The unique domain architecture at the hinge and within the Fc fragment is crucial for activation of the classical complement pathway and interaction with the Fc receptor on immune cells. Immunoglobulins present in the inflammatory environment are exposed to thiocyanate and MPO released by activated neutrophils. Therefore, we evaluated the process of carbamylation of human IgG1 and the potential downstream effects of this modification on immunoglobulin function. To this end, IgG1 was incubated with 0.1M potassium cyanate (KCNO) for up to 24 h at 37°C and the carbamylation of Lys residues was assessed by western blotting using antibodies designed to detect homocitrulline residues. The amount of homocitrulline increased significantly within the first 3 h. Prolonged incubation (6 and 12 h) resulted in only a moderate increase in band density (Fig.1A). Using a colorimetric method we estimated the total amount of formed homocitrulline residues on IgG1 treated with 0.1M KCNO. The number of modifications increased rapidly within first 3 h of incubation reaching 80 nmol/mg of protein. Further time points showed only moderate increase of the amount of the homocitrulline within the molecule reaching 95–130 nmol/mg after 24 h incubation. That indicates that the majority of lysines susceptible to carbamylation are modified very rapidly in presence of KCNO (Fig.1B).


Carbamylation of immunoglobulin abrogates activation of the classical complement pathway.

Koro C, Bielecka E, Dahl-Knudsen A, Enghild JJ, Scavenius C, Brun JG, Binder V, Hellvard A, Bergum B, Jonsson R, Potempa J, Blom AM, Mydel P - Eur. J. Immunol. (2014)

Human IgG1 undergoes rapid carbamylation, which protects it from Kgp cleavage. (A) Western blot analysis of 2 μg of IgG1 carbamylated with KCNO in a time-dependent manner. The level of carbamylation was detected with homocitrulline-specific antibodies (upper panel) together with loading controls (Coomassie staining; lower panel). Data are representative of three independent experiments. (B) The time-dependent effect of KCNO incubation on carbamylation of IgG1. Carbamylated IgG1 was degraded with proteinase K and samples were incubated with urea nitrogen reagent and 3% w/v 2,3-butanedione monoxime. The amount of homocitrulline/mg IgG1 was quantified by measurement of the absorbance at 530 nm. Each point represents the mean of duplicate samples and data are from a single experiment representative of four independent experiments using IgG1 and RTX. (C) 2.25 μg of carbamylated and control IgG1 were incubated for the indicated times with 10 nM Kgp and SDS-PAGE was performed to assess the cleavage of IgG. All samples were separated on 4–15% gradient gels under reducing conditions. Data shown are representative of three independent experiments.
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Related In: Results  -  Collection

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fig01: Human IgG1 undergoes rapid carbamylation, which protects it from Kgp cleavage. (A) Western blot analysis of 2 μg of IgG1 carbamylated with KCNO in a time-dependent manner. The level of carbamylation was detected with homocitrulline-specific antibodies (upper panel) together with loading controls (Coomassie staining; lower panel). Data are representative of three independent experiments. (B) The time-dependent effect of KCNO incubation on carbamylation of IgG1. Carbamylated IgG1 was degraded with proteinase K and samples were incubated with urea nitrogen reagent and 3% w/v 2,3-butanedione monoxime. The amount of homocitrulline/mg IgG1 was quantified by measurement of the absorbance at 530 nm. Each point represents the mean of duplicate samples and data are from a single experiment representative of four independent experiments using IgG1 and RTX. (C) 2.25 μg of carbamylated and control IgG1 were incubated for the indicated times with 10 nM Kgp and SDS-PAGE was performed to assess the cleavage of IgG. All samples were separated on 4–15% gradient gels under reducing conditions. Data shown are representative of three independent experiments.
Mentions: The unique domain architecture at the hinge and within the Fc fragment is crucial for activation of the classical complement pathway and interaction with the Fc receptor on immune cells. Immunoglobulins present in the inflammatory environment are exposed to thiocyanate and MPO released by activated neutrophils. Therefore, we evaluated the process of carbamylation of human IgG1 and the potential downstream effects of this modification on immunoglobulin function. To this end, IgG1 was incubated with 0.1M potassium cyanate (KCNO) for up to 24 h at 37°C and the carbamylation of Lys residues was assessed by western blotting using antibodies designed to detect homocitrulline residues. The amount of homocitrulline increased significantly within the first 3 h. Prolonged incubation (6 and 12 h) resulted in only a moderate increase in band density (Fig.1A). Using a colorimetric method we estimated the total amount of formed homocitrulline residues on IgG1 treated with 0.1M KCNO. The number of modifications increased rapidly within first 3 h of incubation reaching 80 nmol/mg of protein. Further time points showed only moderate increase of the amount of the homocitrulline within the molecule reaching 95–130 nmol/mg after 24 h incubation. That indicates that the majority of lysines susceptible to carbamylation are modified very rapidly in presence of KCNO (Fig.1B).

Bottom Line: We found that the lysine residues of IgG1 are rapidly modified after brief exposure to CNO- .The reaction can apparently occur in vivo, as we found carbamylated antibodies in synovial fluid from rheumatoid arthritis patients.Taken together, our data suggest that carbamylation has a profound impact on the complement-activating ability of IgG1 and reveals a pivotal role for previously uncharacterized lysine residues in this process.

View Article: PubMed Central - PubMed

Affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway.

Show MeSH
Related in: MedlinePlus