Rheumatoid Arthritis (RA) is an autoimmune disease characterised by auto-antibodies generated to antigens that do not under healthy physiological conditions trigger an immune response. The resultant “auto-antigens” trigger and sustain a chronic inflammatory state within the joints of RA patients.
The auto-antigens that result in the sustained onset of chronic inflammation in RA are unknown however one common feature is the posttranslational conversion of peptidyl arginine to peptidyl citrulline on the target proteins.
Many pre-clinical studies support the role of anti-citrullinated protein antibodies (ACPA’s) in exaggerating the disease symptoms in RA, making citrullinated proteins a strong candidate for driving the immune response in the disease.
Citrullination of proteins:
Citrullination of proteins is present in both healthy and disease states. Under normal physiological conditions citrullinated proteins play a role in the generation of structural tissue, however in patients with RA tolerance to citrullinated proteins is selectively lost resulting in auto antibodies being generated.
Evidence indicates that ACPA’s target epitopes on citrullinated fibrinogen, vimentin, type II collagen and citrullinated α enolase. Alpha enolase, a candidate auto-antigen for RA, is abundantly expressed in the joints and synovial fluid of RA patients, more over antibodies targeting the citrullinated form of the protein are specific to the disease. Kinloch A, et al. 2008. “Antibodies to citrullinated α-enolase peptide 1 are specific for rheumatoid arthritis and cross react with bacterial enolase”. Arthritis and Rheumatism, 58 (10) , Pages 3009-3019.
The role of citrullinated α enolase:
To examine the role of citrullinated α enolase in RA Cambridge Research Biochemicals (CRB) synthesized fifteen cyclic 15 – 23mer peptides , corresponding to the human α enolase (Swiss-Prot accession no. P06733) or P gingivalis enolase (Swiss-Prot accession no. AAQ66821). The sequences were modified to add a terminal cysteine residue for conjugation and the exchange of arginine for citrulline residues at certain positions, Table 1 details the sequences below.
The peptides synthesized in table 1 were used to screen 3 groups – 102 RA patient, 110 control disease patient and 92 control patient samples to determine reactivity against each peptide. The results demonstrated significant cross reactivity to one and in most cases multiple peptides from the RA group, unlike the disease and healthy control groups which displayed limited reactivity to the peptides and significantly lower levels of IgG antibodies than the RA group.
Many naturally occurring peptides have one or more intra-molecular disulphide bridges between cysteine side chains forming cyclic peptides. Peptides with a single disulphide bridge are readily synthesized using trityl (Trt) protecting groups on the cysteine side chains which are removed on cleavage from the resin. The resulting linear peptide is cyclised by air oxidation in dilute aqueous solution at pH8-9. The reaction can be followed by HPLC and MALDI TOF mass spectrometry, the linear peptide losing two mass units (2H) on cyclisation. Peptides with two or more disulphide bridges require selective protection of the cysteine side chains to ensure that the correct disulphide bridges are formed.
Cyclic peptides can also be formed by linking the amino (N) terminus of the peptide to the carboxy (C) terminus via an amide bond. The amino side chains of Lys and Orn and the carboxy side chains of Asp and Glu can also be used to construct cyclic peptides via an amide bond. Cyclisation in this manner may be useful in the synthesis of bioactive peptide analogues.
Custom rabbit polyclonal antibody:
Cambridge Research Biochemicals raised a custom rabbit polyclonal antibody to peptide 1A (CEP-1). Once a suitable antibody titre was achieved the crude anti-serum was depleted using a thiopropyl sepharose affinity depletion column constructed using peptide 1D to remove antibodies generated to the arginine form of the protein. The depleted anti-serum was then affinity purified using a thiopropyl Sepharose affinity column derivatised with the peptide antigen 1A (CEP-1).
The custom anti-CEP-1 antibody generated was then used to show preferential binding in Western blotting to the citrullinated form of the protein target over the non-citrullinated target. The peptides and antibody generated have identified an immunodominant epitope in citrullinated α enolase, to which antibodies are characteristic of Rheumatoid Arthritis.