Midway evaluation - Catalin Koro
Nearly all newly translated proteins undergo post-translational modifications that affect their range of function. Recently, the chemical conversion of lysine into uncharged homocitrulline (i.e., carbamylation) within the polypeptide chain has raised considerable interest in the context of inflammatory and autoimmune diseases. In vivo carbamylation occurs mainly through cyanate formation generated by myeloperoxidase, an enzyme released by activated neutrophils. Thus, the amount of carbamylated residues is relatively high in the inflammatory milieu. Loss of charge due to carbamylation may induce conformational changes and subsequently result in the loss of function of a protein.
In our first project, we investigated the pattern of IgG1 carbamylation under inflammatory conditions and the effect that this modification has on the ability to trigger complement activation via the classical pathway. Our results showed that only a few lysine residues within the hinge (K222 and K246) and the N-terminal of the CH2 domain (K322, K326 and K334) of IgG1 were modified after a brief exposure of cyanate. Importantly, we were able to confirm our in vitro findings using synovial fluid from rheumatoid arthritis patients. In addition, we observed that carbamylation for more than 3 hours rendered IgG incapable of complement activation, with the levels of C1q equivalent to the negative control.
In our second project, we studied the effects of carbamylation on the immune modulatory and antimicrobial peptide LL-37. Our results showed that carbamylation decreases bactericidal capacity of LL-37 against B. subtilis and E. coli. Interestingly, LL-37 carbamylated at position 12 and 15 was strongly lytic to PMN, but had no effect on erythrocytes. Modification at these positions did also induce a strong chemoattractant ability of LL-37 for PMNs. Moreover, PMNs stimulated with carbamylated LL-37 became phagocyted by macrophages to a higher degree, as compared to native peptide suggesting its pro-apoptotic activity. Taken together, carbamylated LL-37 exerted strong pro-inflammatory activity that we suggest may lead to exacerbation of inflammation with concomitant host tissue damage.