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