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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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Carbamylation of CH2 and the hinge region sites of IgG1. (A) Schematic representation of the IgG1 sites observed to be preferentially carbamylated in our experiments. The positions of the highly modified lysine residues are indicated in red. (B) An example of the tandem mass spectrometry assignment of carbamylation of the CH2 site K326. The MS/MS spectrum displays the VSN-Hcit-ALPAPIEK peptide fully sequenced from the C-terminus (y-ions) and nearly fully sequenced from the N-terminus (b-ions). In the spectrum, additional evidence of ions formed by collision-induced neutral loss of water, ammonia, and isocyanic acid ([MH+-CONH]+2) are marked; the latter is a characteristic feature of carbamylation. Results shown are representative of 50 MS/MS spectra assigned to the VSN-Hcit-ALPAPIEK modification. (C) Mass spectrometric evaluation of the time-dependent carbamylation of detected putative carbamylation sites in IgG1 performed using spectral counting at the indicated time points. Each bar represents the spectral count of a single carbamylation experiment and is representative of two independent experiments.
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fig02: Carbamylation of CH2 and the hinge region sites of IgG1. (A) Schematic representation of the IgG1 sites observed to be preferentially carbamylated in our experiments. The positions of the highly modified lysine residues are indicated in red. (B) An example of the tandem mass spectrometry assignment of carbamylation of the CH2 site K326. The MS/MS spectrum displays the VSN-Hcit-ALPAPIEK peptide fully sequenced from the C-terminus (y-ions) and nearly fully sequenced from the N-terminus (b-ions). In the spectrum, additional evidence of ions formed by collision-induced neutral loss of water, ammonia, and isocyanic acid ([MH+-CONH]+2) are marked; the latter is a characteristic feature of carbamylation. Results shown are representative of 50 MS/MS spectra assigned to the VSN-Hcit-ALPAPIEK modification. (C) Mass spectrometric evaluation of the time-dependent carbamylation of detected putative carbamylation sites in IgG1 performed using spectral counting at the indicated time points. Each bar represents the spectral count of a single carbamylation experiment and is representative of two independent experiments.

Mentions: To determine the pattern and extent of IgG1 carbamylation, we employed mass spectrometry. As expected, we detected a time-dependent increase in the amount of carbamylation 25,26. Intriguingly, the modifications were not random and only a few of the 26 available lysines within the IgG1 heavy chain underwent modification (Fig.2). After 1 h of incubation with KCNO, we observed only two lysine residues that were efficiently modified with spectral counts above 10 (Fig.2B and C). These residues were identified as K326 and K334, located at the N-terminus of the polypeptide chain folding into the CH2 domain (Fig.2B). Consequently, the degree of modification of these two residues increased with time of incubation, suggesting that they are exposed and easily accessible for modification. After 6- and 12-h incubations, in addition to increased modification of K326 and K334 (Fig.2A), our analysis revealed a significant amount of carbamylation of K322 on the CH2 domain and K222 and K246 within the hinge region and in its proximity, respectively. This fully corroborates our findings from the Kgp cleavage experiment. Increasing the time of exposure of IgG1 to cyanate up to 24 h did not change the overall pattern of modification. Carbamylation of K322, K326, and K334 remained comparable to that observed at the 6- and 12-h time points. By contrast, carbamylation of hinge region lysine K222 and neighboring K246 progressed to very high levels after 24 h.


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)

Carbamylation of CH2 and the hinge region sites of IgG1. (A) Schematic representation of the IgG1 sites observed to be preferentially carbamylated in our experiments. The positions of the highly modified lysine residues are indicated in red. (B) An example of the tandem mass spectrometry assignment of carbamylation of the CH2 site K326. The MS/MS spectrum displays the VSN-Hcit-ALPAPIEK peptide fully sequenced from the C-terminus (y-ions) and nearly fully sequenced from the N-terminus (b-ions). In the spectrum, additional evidence of ions formed by collision-induced neutral loss of water, ammonia, and isocyanic acid ([MH+-CONH]+2) are marked; the latter is a characteristic feature of carbamylation. Results shown are representative of 50 MS/MS spectra assigned to the VSN-Hcit-ALPAPIEK modification. (C) Mass spectrometric evaluation of the time-dependent carbamylation of detected putative carbamylation sites in IgG1 performed using spectral counting at the indicated time points. Each bar represents the spectral count of a single carbamylation experiment and is representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Carbamylation of CH2 and the hinge region sites of IgG1. (A) Schematic representation of the IgG1 sites observed to be preferentially carbamylated in our experiments. The positions of the highly modified lysine residues are indicated in red. (B) An example of the tandem mass spectrometry assignment of carbamylation of the CH2 site K326. The MS/MS spectrum displays the VSN-Hcit-ALPAPIEK peptide fully sequenced from the C-terminus (y-ions) and nearly fully sequenced from the N-terminus (b-ions). In the spectrum, additional evidence of ions formed by collision-induced neutral loss of water, ammonia, and isocyanic acid ([MH+-CONH]+2) are marked; the latter is a characteristic feature of carbamylation. Results shown are representative of 50 MS/MS spectra assigned to the VSN-Hcit-ALPAPIEK modification. (C) Mass spectrometric evaluation of the time-dependent carbamylation of detected putative carbamylation sites in IgG1 performed using spectral counting at the indicated time points. Each bar represents the spectral count of a single carbamylation experiment and is representative of two independent experiments.
Mentions: To determine the pattern and extent of IgG1 carbamylation, we employed mass spectrometry. As expected, we detected a time-dependent increase in the amount of carbamylation 25,26. Intriguingly, the modifications were not random and only a few of the 26 available lysines within the IgG1 heavy chain underwent modification (Fig.2). After 1 h of incubation with KCNO, we observed only two lysine residues that were efficiently modified with spectral counts above 10 (Fig.2B and C). These residues were identified as K326 and K334, located at the N-terminus of the polypeptide chain folding into the CH2 domain (Fig.2B). Consequently, the degree of modification of these two residues increased with time of incubation, suggesting that they are exposed and easily accessible for modification. After 6- and 12-h incubations, in addition to increased modification of K326 and K334 (Fig.2A), our analysis revealed a significant amount of carbamylation of K322 on the CH2 domain and K222 and K246 within the hinge region and in its proximity, respectively. This fully corroborates our findings from the Kgp cleavage experiment. Increasing the time of exposure of IgG1 to cyanate up to 24 h did not change the overall pattern of modification. Carbamylation of K322, K326, and K334 remained comparable to that observed at the 6- and 12-h time points. By contrast, carbamylation of hinge region lysine K222 and neighboring K246 progressed to very high levels after 24 h.

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