At the start of this project we have focused on the production of a set of proteins that are modified by six different post-translational modifications (PTM) and on the biochemical characterization of the interaction of complement proteins with the PTM proteins. We have now successfully generated six PTM versions of two proteins to be used with human serum and also a mouse protein with all six modifications. The presence of the modifications has now been verified by biochemical methods and by mass-spectrometry.
The modified proteins, and their controls have now been used to study the interaction with complement. Importantly, we could replicate the preliminary data used in the proposal, that carbamylated proteins, one of the PTM, indeed bind complement proteins. In addition, we observed that this is occurring for more, but not all, of the modifications. We have now confirmed that not only complement proteins from human serum are binding PTM proteins but that also mouse complement proteins can do so in a similar way. This is highly encouraging as it indicates that the mouse is indeed an appropriate animal model to use for the proposed studies.
We next analyzed if anti-PTM antibodies occur in major human autoimmune diseases. Here we focused on Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE) and Auto-Immune Hepatitis (AIH). In all cohorts tested, we could readily identify anti-PTM autoantibodies targeting all 6 PTMs, but with clear differences in the frequencies and in the combinations of the anti-PTM antibodies. In each of the cohorts, the presence of anti-PTM antibodies was associated with disease severity and or response to therapy.
Combined the data clearly indicate: 4 of the 6 PTMs bind and activate complement directly, without prior binding of anti-PTM antibodies.
To study the impact on clearance we performed in vitro phagocytosis experiments, based on beads coated with PTM proteins and control proteins incubated with serum. We observed again that 4 of the 6 PTMs triggered complement activation and that this resulted in enhanced phagocytosis.
We have performed immunization experiments with PTM protein in the presence or absence of exogenous adjuvant and observed strong antibody responses against 4 of the 6 PTMs as well as a break of tolerance against the mouse carrier protein for some of the PTMs. Comparing the observed effects in different strains of mice revealed that the relative contribution of complement on the production of anti-PTM antibodies is limited, but complex and multifactorial.
In currently still ongoing experiments we analyze the impact of immunizing with PTM modified proteins on the development of not just autoimmunity but also on autoimmune diseases such as in this case the development of diabetes.
The results of these studies have been presented at several (international) conferences as well has have now been published in several peer-reviewed publications. Other manuscripts are still pending, and two PhD students will defend a thesis based on these results.
