Background While the association between ACPA and RA has been established, the impact of citrullination at the level of the protein is not well-defined. PAD enzymes remove an imine moiety from arginine to form citrulline. This enzymatic process results in the loss of a positive charge which can lead to a change in protein structure, consequential function or susceptibility to proteolytic processing.1 In an analogous fashion to the phosphorylome (phosphoproteins), to fully elucidate the role of citrullination for applied basic and clinical research, the field requires a laboratory developed pan reactive citrullination specific antibody (Ab) that identifies members of the “citrullinome” and site-specific citrullination antibodies that are generated to each target epitope since individual citrullination sites within a protein might elicit a gain or loss in function for the protein. These types of tools have clear advantages over other identification approaches like mass spectrometry, in that they are highly reproducible, can easily be integrated into multiple assay formats and are cost effective.
Objectives The objective of this study was to 1) develop a pan reactive citrullination specific antibody and 2) an assay that can detect citrullinated 14-3-3η in patient serum.
Methods 5 cit peptides and their corresponding arg peptides were synthesized and conjugated to BSA. 4 of the 5 peptides corresponded to putative 14-3-3η cit sites. One of the peptides, Peptide 4, was modelled on a PAD consensus motif. Polyclonal Abs to Peptide 4 were reproducibly raised in two independent species; twice in rabbits and once in goats with 3 animals per study. Reactivity was assessed by examining titres to both the citrullinated and arginylated forms of Peptide 4. Sera selected for purification underwent a two-step purification process utilizing the arg and cit peptide. Purified Cit-Ab was tested by examining reactivity to the other 4 cit-peptides and the corresponding arg forms. The Cit-Ab was tested by immunoblot analysis using cit 14-3-3η and cit vimetin that was generated using recombinant PAD4 as well as by 14-3-3η ELISA in seven 14-3-3η positive rheumatoid arthritis patients with levels >20 ng/ml.
Results Five of the 6 rabbits and 2 of the 3 goats elicited immune responses that preferentially reacted with the cit form of Peptide 4. In a serial dilution study (1:50,000 to 1:250,000) the goat sera selected for purification exhibited 20X greater reactivity for the cit versus arg form of peptide 4. Purified Ab preferentially reacted with 3 of the 5 peptides based on a 2X ratio, and detected cit 14-3-3η and vimentin over the corresponding non-modified forms of these 2 proteins. As demonstrated in the table below, the burden of citrullination varied amongst patients despite having serum 14-3-3η levels >20ng/ml.
Conclusions The pan reactive cit-specific Ab can be reliably reproduced and binds to multiple cit sites within a protein and across different proteins. The ELISA data reveals that the burden of citrullination varies amongst RA patients despite having similar levels of 14-3-3η protein. Citrullination of the “14-3-3η protein” is being clinically investigated in the context of disease outcomes.
International Journal of Biochemistry and Cell Biology
Disclosure of Interest Y. Gui Employee of: Augurex Life Sciences Corp, M. Murphy Employee of: Augurex Life Sciences Corp, W. Maksymowych Consultant for: Augurex Life Sciences Corp, (Co-inventor of 14-3-3η), A. Marotta Employee of: Augurex Life Sciences Corp, (Co-inventor of 14-3-3η)