Carbamylation of immunoglobulin abrogates activation of the classical complement pathway.
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.
Affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway.Show MeSH
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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.
Affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway.